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1
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Mandala VS, MacKinnon R. Electric field-induced pore constriction in the human K v2.1 channel. Proc Natl Acad Sci U S A 2025; 122:e2426744122. [PMID: 40366685 DOI: 10.1073/pnas.2426744122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2024] [Accepted: 04/10/2025] [Indexed: 05/15/2025] Open
Abstract
Gating in voltage-dependent ion channels is regulated by the transmembrane voltage. This form of regulation is enabled by voltage-sensing domains (VSDs) that respond to transmembrane voltage differences by changing their conformation and exerting force on the pore to open or close it. Here, we use cryogenic electron microscopy to study the neuronal Kv2.1 channel in lipid vesicles with and without a voltage difference across the membrane. Hyperpolarizing voltage differences displace the positively charged S4 helix in the voltage sensor by one helical turn (~5 Å). When this displacement occurs, the S4 helix changes its contact with the pore at two different interfaces. When these changes are observed in fewer than four voltage sensors, the pore remains open, but when they are observed in all four voltage sensors, the pore constricts. The constriction occurs because the S4 helix, as it displaces inward, squeezes the right-handed helical bundle of pore-lining S6 helices. A similar conformational change occurs upon hyperpolarization of the EAG1 channel but with two helical turns displaced instead of one. Therefore, while Kv2.1 and EAG1 are from distinct architectural classes of voltage-dependent ion channels, called domain-swapped and non-domain-swapped, the way the voltage sensors gate their pores is very similar.
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Affiliation(s)
- Venkata Shiva Mandala
- Laboratory of Molecular Neurobiology and Biophysics, HHMI, The Rockefeller University, New York, NY 10065
| | - Roderick MacKinnon
- Laboratory of Molecular Neurobiology and Biophysics, HHMI, The Rockefeller University, New York, NY 10065
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2
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Serano M, Perni S, Pierantozzi E, Laurino A, Sorrentino V, Rossi D. Intracellular Membrane Contact Sites in Skeletal Muscle Cells. MEMBRANES 2025; 15:29. [PMID: 39852269 PMCID: PMC11767089 DOI: 10.3390/membranes15010029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/20/2024] [Revised: 01/09/2025] [Accepted: 01/10/2025] [Indexed: 01/26/2025]
Abstract
Intracellular organelles are common to eukaryotic cells and provide physical support for the assembly of specialized compartments. In skeletal muscle fibers, the largest intracellular organelle is the sarcoplasmic reticulum, a specialized form of the endoplasmic reticulum primarily devoted to Ca2+ storage and release for muscle contraction. Occupying about 10% of the total cell volume, the sarcoplasmic reticulum forms multiple membrane contact sites, some of which are unique to skeletal muscle. These contact sites primarily involve the plasma membrane; among these, specialized membrane contact sites between the transverse tubules and the terminal cisternae of the sarcoplasmic reticulum form triads. Triads are skeletal muscle-specific contact sites where Ca2+ channels and regulatory proteins assemble to form the so-called calcium release complex. Additionally, the sarcoplasmic reticulum contacts mitochondria to enable a more precise regulation of Ca2+ homeostasis and energy metabolism. The sarcoplasmic reticulum and the plasma membrane also undergo dynamic remodeling to allow Ca2+ entry from the extracellular space and replenish the stores. This process involves the formation of dynamic membrane contact sites called Ca2+ Entry Units. This review explores the key processes in biogenesis and assembly of intracellular membrane contact sites as well as the membrane remodeling that occurs in response to muscle fatigue.
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Affiliation(s)
- Matteo Serano
- Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; (M.S.); (S.P.); (E.P.); (A.L.); (V.S.)
| | - Stefano Perni
- Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; (M.S.); (S.P.); (E.P.); (A.L.); (V.S.)
| | - Enrico Pierantozzi
- Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; (M.S.); (S.P.); (E.P.); (A.L.); (V.S.)
| | - Annunziatina Laurino
- Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; (M.S.); (S.P.); (E.P.); (A.L.); (V.S.)
| | - Vincenzo Sorrentino
- Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; (M.S.); (S.P.); (E.P.); (A.L.); (V.S.)
- Program of Molecular Diagnosis of Rare Genetic Diseases, Azienda Ospedaliera Universitaria Senese, 53100 Siena, Italy
| | - Daniela Rossi
- Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; (M.S.); (S.P.); (E.P.); (A.L.); (V.S.)
- Program of Molecular Diagnosis of Rare Genetic Diseases, Azienda Ospedaliera Universitaria Senese, 53100 Siena, Italy
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3
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Rall JA. The foundation of excitation-contraction coupling in skeletal muscle: communication between the transverse tubules and sarcoplasmic reticulum. ADVANCES IN PHYSIOLOGY EDUCATION 2024; 48:759-769. [PMID: 39116389 DOI: 10.1152/advan.00086.2024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Revised: 07/26/2024] [Accepted: 08/02/2024] [Indexed: 08/10/2024]
Abstract
The expression excitation-contraction (EC) coupling in skeletal muscle was coined in 1952 (Sandow A. Yale J Biol Med 25: 176-201, 1952). The term evolved narrowly to include only the processes at the triad that intervene between depolarization of the transverse tubular (T-tubular) membrane and Ca2+ release from the sarcoplasmic reticulum (SR). From 1970 to 1988, the foundation of EC coupling was elucidated. The channel through which Ca2+ was released during activation was located in the SR by its specific binding to the plant insecticide ryanodine. This channel was called the ryanodine receptor (RyR). The RyR contained four subunits that together constituted the "SR foot" structure that traversed the gap between the SR and the T-tubular membrane. Ca2+ channels, also called dihydropyridine receptors (DHPRs), were located in the T-tubular membrane at the triadic junction and shown to be essential for EC coupling. There was a precise relationship between the two channels. Four DHPRs, organized as tetrads, were superimposed on alternate RyRs. This structure was consistent with the proposal that EC coupling was mediated via a movement of intramembrane charge in the T-tubular system. The speculation was that the DHPR acted as a voltage sensor transferring information to the RyRs of the SR by protein-protein interaction causing the release of Ca2+ from the SR. A great deal of progress was made by 1988 toward understanding EC coupling. However, the ultimate question of how voltage sensing is coupled to the opening of the SR Ca2+ release channel remains unresolved.NEW & NOTEWORTHY The least understood part of the series of events in excitation-contraction coupling in skeletal muscle was how information was transmitted from the transverse tubules to the sarcoplasmic (SR) and how Ca2+ was released from the SR. Through an explosion of technical approaches including physiological, biochemical, structural, pharmacological, and molecular genetics, much was discovered between 1970 and 1988. By the end of 1988, the foundation of EC coupling in skeletal muscle was established.
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Affiliation(s)
- Jack A Rall
- Department of Physiology and Cell Biology, College of MedicineOhio State University, Columbus, Ohio, United States
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4
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Endo Y, Groom L, Wang SM, Pannia E, Griffiths NW, Van Gennip JLM, Ciruna B, Laporte J, Dirksen RT, Dowling JJ. Two zebrafish cacna1s loss-of-function variants provide models of mild and severe CACNA1S-related myopathy. Hum Mol Genet 2024; 33:254-269. [PMID: 37930228 PMCID: PMC10800018 DOI: 10.1093/hmg/ddad178] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 09/25/2023] [Accepted: 10/09/2023] [Indexed: 11/07/2023] Open
Abstract
CACNA1S-related myopathy, due to pathogenic variants in the CACNA1S gene, is a recently described congenital muscle disease. Disease associated variants result in loss of gene expression and/or reduction of Cav1.1 protein stability. There is an incomplete understanding of the underlying disease pathomechanisms and no effective therapies are currently available. A barrier to the study of this myopathy is the lack of a suitable animal model that phenocopies key aspects of the disease. To address this barrier, we generated knockouts of the two zebrafish CACNA1S paralogs, cacna1sa and cacna1sb. Double knockout fish exhibit severe weakness and early death, and are characterized by the absence of Cav1.1 α1 subunit expression, abnormal triad structure, and impaired excitation-contraction coupling, thus mirroring the severe form of human CACNA1S-related myopathy. A double mutant (cacna1sa homozygous, cacna1sb heterozygote) exhibits normal development, but displays reduced body size, abnormal facial structure, and cores on muscle pathologic examination, thus phenocopying the mild form of human CACNA1S-related myopathy. In summary, we generated and characterized the first cacna1s zebrafish loss-of-function mutants, and show them to be faithful models of severe and mild forms of human CACNA1S-related myopathy suitable for future mechanistic studies and therapy development.
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Affiliation(s)
- Yukari Endo
- Program for Genetics and Genome Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Linda Groom
- Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642, United States
| | - Sabrina M Wang
- Program for Genetics and Genome Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Emanuela Pannia
- Program for Genetics and Genome Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
- Zebrafish Genetics and Disease Models Core Facility, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Nigel W Griffiths
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Jenica L M Van Gennip
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Brian Ciruna
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
- Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Jocelyn Laporte
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Inserm U1258, Cnrs UMR7104, Université de Strasbourg, 1 Rue Laurent Fries, Illkirch 67400, France
| | - Robert T Dirksen
- Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642, United States
| | - James J Dowling
- Program for Genetics and Genome Biology, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
- Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
- Division of Neurology, Hospital for Sick Children, 555 University Ave, Toronto, ON M5G 1X8, Canada
- Department of Paediatrics, University of Toronto, 555 University Ave, Toronto, ON M5G 1X8, Canada
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5
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Bibollet H, Kramer A, Bannister RA, Hernández-Ochoa EO. Advances in Ca V1.1 gating: New insights into permeation and voltage-sensing mechanisms. Channels (Austin) 2023; 17:2167569. [PMID: 36642864 PMCID: PMC9851209 DOI: 10.1080/19336950.2023.2167569] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Accepted: 01/09/2023] [Indexed: 01/17/2023] Open
Abstract
The CaV1.1 voltage-gated Ca2+ channel carries L-type Ca2+ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, CaV1.1 was the first voltage-gated Ca2+ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how CaV1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca2+ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.
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Affiliation(s)
- Hugo Bibollet
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Audra Kramer
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Roger A. Bannister
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Erick O. Hernández-Ochoa
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA
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6
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Campiglio M, Dyrda A, Tuinte WE, Török E. Ca V1.1 Calcium Channel Signaling Complexes in Excitation-Contraction Coupling: Insights from Channelopathies. Handb Exp Pharmacol 2023; 279:3-39. [PMID: 36592225 DOI: 10.1007/164_2022_627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
In skeletal muscle, excitation-contraction (EC) coupling relies on the mechanical coupling between two ion channels: the L-type voltage-gated calcium channel (CaV1.1), located in the sarcolemma and functioning as the voltage sensor of EC coupling, and the ryanodine receptor 1 (RyR1), located on the sarcoplasmic reticulum serving as the calcium release channel. To this day, the molecular mechanism by which these two ion channels are linked remains elusive. However, recently, skeletal muscle EC coupling could be reconstituted in heterologous cells, revealing that only four proteins are essential for this process: CaV1.1, RyR1, and the cytosolic proteins CaVβ1a and STAC3. Due to the crucial role of these proteins in skeletal muscle EC coupling, any mutation that affects any one of these proteins can have devastating consequences, resulting in congenital myopathies and other pathologies.Here, we summarize the current knowledge concerning these four essential proteins and discuss the pathophysiology of the CaV1.1, RyR1, and STAC3-related skeletal muscle diseases with an emphasis on the molecular mechanisms. Being part of the same signalosome, mutations in different proteins often result in congenital myopathies with similar symptoms or even in the same disease.
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Affiliation(s)
- Marta Campiglio
- Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria.
| | - Agnieszka Dyrda
- Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria
| | - Wietske E Tuinte
- Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria
| | - Enikő Török
- Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria
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7
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Gu XY, Jin B, Qi ZD, Yin XF. MicroRNA is a potential target for therapies to improve the physiological function of skeletal muscle after trauma. Neural Regen Res 2021; 17:1617-1622. [PMID: 34916449 PMCID: PMC8771090 DOI: 10.4103/1673-5374.330620] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
MicroRNAs can regulate the function of ion channels in many organs. Based on our previous study we propose that miR-142a-39, which is highly expressed in denervated skeletal muscle, might affect cell excitability through similar mechanisms. In this study, we overexpressed or knocked down miR-142a-3p in C2C12 cells using a lentivirus method. After 7 days of differentiation culture, whole-cell currents were recorded. The results showed that overexpression of miR-142a-3p reduced the cell membrane capacitance, increased potassium current density and decreased calcium current density. Knockdown of miR-142a-3p reduced sodium ion channel current density. The results showed that change in miR-142a-3p expression affected the ion channel currents in C2C12 cells, suggesting its possible roles in muscle cell electrophysiology. This study was approved by the Animal Ethics Committee of Peking University in July 2020 (approval No. LA2017128).
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Affiliation(s)
- Xin-Yi Gu
- Department of Orthopedics and Traumatology, Peking University People's Hospital; Key Laboratory of Trauma and Neural Regeneration (Peking University), Beijing, China
| | - Bo Jin
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, Jiangsu Province, China
| | - Zhi-Dan Qi
- Department of Orthopedics and Traumatology, Peking University People's Hospital; Key Laboratory of Trauma and Neural Regeneration (Peking University), Beijing, China
| | - Xiao-Feng Yin
- Department of Orthopedics and Traumatology, Peking University People's Hospital; Key Laboratory of Trauma and Neural Regeneration (Peking University), Beijing, China
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8
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Miranda DR, Voss AA, Bannister RA. Into the spotlight: RGK proteins in skeletal muscle. Cell Calcium 2021; 98:102439. [PMID: 34261001 DOI: 10.1016/j.ceca.2021.102439] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Revised: 06/24/2021] [Accepted: 06/26/2021] [Indexed: 10/20/2022]
Abstract
The RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTPases are potent endogenous inhibitors of voltage-gated Ca2+ channels (VGCCs). While the impact of RGK proteins on cardiac physiology has been investigated extensively, much less is known regarding their influence on skeletal muscle biology. Thus, the purpose of this article is to establish a basis for future investigation into the role of RGK proteins in regulating the skeletal muscle excitation-contraction (EC) coupling complex via modulation of the L-type CaV1.1 VGCC. The pathological consequences of elevated muscle RGK protein expression in Type II Diabetes, Amyotrophic Lateral Sclerosis (ALS), Duchenne's Muscular Dystrophy and traumatic nerve injury are also discussed.
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Affiliation(s)
- Daniel R Miranda
- Department of Biological Sciences, College of Science and Mathematics, Wright State University, 235A Biological Sciences, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA
| | - Andrew A Voss
- Department of Biological Sciences, College of Science and Mathematics, Wright State University, 235A Biological Sciences, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
| | - Roger A Bannister
- Departments of Pathology and Biochemistry & Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.
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9
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Balderas-Villalobos J, Steele TWE, Eltit JM. Physiological and Pathological Relevance of Selective and Nonselective Ca 2+ Channels in Skeletal and Cardiac Muscle. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1349:225-247. [PMID: 35138617 PMCID: PMC10683374 DOI: 10.1007/978-981-16-4254-8_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Contraction of the striated muscle is fundamental for human existence. The action of voluntary skeletal muscle enables activities such as breathing, establishing body posture, and diverse body movements. Additionally, highly precise motion empowers communication, artistic expression, and other activities that define everyday human life. The involuntary contraction of striated muscle is the core function of the heart and is essential for blood flow. Several ion channels are important in the transduction of action potentials to cytosolic Ca2+ signals that enable muscle contraction; however, other ion channels are involved in the progression of muscle pathologies that can impair normal life or threaten it. This chapter describes types of selective and nonselective Ca2+ permeable ion channels expressed in the striated muscle, their participation in different aspects of muscle excitation and contraction, and their relevance to the progression of some pathological states.
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Affiliation(s)
- Jaime Balderas-Villalobos
- Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA
| | - Tyler W E Steele
- Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA
| | - Jose M Eltit
- Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA.
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10
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Jin B, Gu X, Li D, Qi Z, Jiang B, Yin X. Effects of miR-34c-5p on Sodium, Potassium, and Calcium Channel Currents in C2C12 Myotubes. Cell Mol Neurobiol 2020; 40:1223-1230. [PMID: 32100187 PMCID: PMC11448787 DOI: 10.1007/s10571-020-00810-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 02/04/2020] [Indexed: 10/24/2022]
Abstract
The aim of this study was to investigate the effects of miR-34c-5p on the main voltage-dependent ion channels in skeletal muscle cells. This study focused on the effects of miR-34c-5p on sodium, potassium, and calcium currents in C2C12 myoblasts. The miR-34c-5p overexpression group, knockdown group, and control group were differentiated for 7 days, fused into myotubes, and used for the whole-cell patch clamp recording. Compared with the control group, the whole-cell sodium current density of the other two groups had no significant changes. In the knockdown group, the delayed rectifier potassium current density was increased (statistically significant), and the whole-cell calcium channel current density did not change. In the overexpression group, the change of rectifier potassium current density was not obvious, while the peak calcium channel current density increased (- 9.23 ± 0.95 pA/pF, n = 6 cells for the overexpression group vs. - 6.48 ± 0.64 pA/pF, n = 7 cells for the control; p < 0.05). Changes in the expression of miR-34c-5p can affect the electrophysiological characteristics of calcium and potassium voltage-gated channels in C2C12 myotubes. Overexpression of miR-34c-5p increased whole-cell L-type calcium channel current (ICa,L), while miR-34c-5p knockdown increased whole-cell delayed rectifier potassium current (IKd).
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Affiliation(s)
- Bo Jin
- Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China
| | - Xinyi Gu
- Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China
| | - Dongdong Li
- Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China
- Department of Surgery, The 517th Hospital of the People's Liberation Army, Xinzhou, Shanxi Province, China
| | - Zhidan Qi
- Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China
| | - Baoguo Jiang
- Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China.
| | - Xiaofeng Yin
- Department of Orthopedics and Traumatology, Peking University People's Hospital, Beijing, China.
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11
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Allard B, Fuster C. When muscle Ca 2+ channels carry monovalent cations through gating pores: insights into the pathophysiology of type 1 hypokalaemic periodic paralysis. J Physiol 2018; 596:2019-2027. [PMID: 29572832 DOI: 10.1113/jp274955] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2017] [Accepted: 03/12/2018] [Indexed: 12/22/2022] Open
Abstract
Patients suffering from type 1 hypokalaemic periodic paralysis (HypoPP1) experience attacks of muscle paralysis associated with hypokalaemia. The disease arises from missense mutations in the gene encoding the α1 subunit of the dihydropyridine receptor (DHPR), a protein complex anchored in the tubular membrane of skeletal muscle fibres which controls the release of Ca2+ from sarcoplasmic reticulum and also functions as a Ca2+ channel. The vast majority of mutations consist of the replacement of one of the outer arginines in S4 segments of the α1 subunit by neutral residues. Early studies have shown that muscle fibres from HypoPP1 patients are abnormally depolarized at rest in low K+ to the point of inducing muscle inexcitability. The relationship between HypoPP1 mutations and depolarization has long remained unknown. More recent investigations conducted in the closely structurally related voltage-gated Na+ and K+ channels have shown that comparable S4 arginine substitutions gave rise to elevated inward currents at negative potentials called gating pore currents. Experiments performed in muscle fibres from different models revealed such an inward resting current through HypoPP1 mutated Ca2+ channels. In mouse fibres transfected with HypoPP1 mutated channels, the elevated resting current was found to carry H+ for the R1239H arginine-to-histidine mutation in a S4 segment and Na+ for the V876E HypoPP1 mutation, which has the peculiarity of not being located in S4 segments. Muscle paralysis probably results from the presence of a gating pore current associated with hypokalaemia for both mutations, possibly aggravated by external acidosis for the R1239H mutation.
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Affiliation(s)
- Bruno Allard
- Institut NeuroMyoGene, Université de Lyon, Université Lyon 1, UMR CNRS 5310, Inserm U1217, 43 bd du 11 Novembre 1918, 69622 Villeurbanne, France
| | - Clarisse Fuster
- Institut NeuroMyoGene, Université de Lyon, Université Lyon 1, UMR CNRS 5310, Inserm U1217, 43 bd du 11 Novembre 1918, 69622 Villeurbanne, France
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12
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Disturbed Ca 2+ Homeostasis in Muscle-Wasting Disorders. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2018; 1088:307-326. [PMID: 30390258 DOI: 10.1007/978-981-13-1435-3_14] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Ca2+ is essential for proper structure and function of skeletal muscle. It not only activates contraction and force development but also participates in multiple signaling pathways. Low levels of Ca2+ restrain muscle regeneration by limiting the fusion of satellite cells. Ironically, sustained elevations of Ca2+ also result in muscle degeneration as this ion promotes high rates of protein breakdown. Moreover, transforming growth factors (TGFs) which are well known for controlling muscle growth also regulate Ca2+ channels. Thus, therapies focused on changing levels of Ca2+ and TGFs are promising for treating muscle-wasting disorders. Three principal systems govern the homeostasis of Ca2+, namely, excitation-contraction (EC) coupling, excitation-coupled Ca2+ entry (ECCE), and store-operated Ca2+ entry (SOCE). Accordingly, alterations in these systems can lead to weakness and atrophy in many hereditary diseases, such as Brody disease, central core disease (CCD), tubular aggregate myopathy (TAM), myotonic dystrophy type 1 (MD1), oculopharyngeal muscular dystrophy (OPMD), and Duchenne muscular dystrophy (DMD). Here, the interrelationship between all these molecules and processes is reviewed.
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13
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Meissner G. The structural basis of ryanodine receptor ion channel function. J Gen Physiol 2017; 149:1065-1089. [PMID: 29122978 PMCID: PMC5715910 DOI: 10.1085/jgp.201711878] [Citation(s) in RCA: 168] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Accepted: 10/12/2017] [Indexed: 01/25/2023] Open
Abstract
Large-conductance Ca2+ release channels known as ryanodine receptors (RyRs) mediate the release of Ca2+ from an intracellular membrane compartment, the endo/sarcoplasmic reticulum. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RyR3 is expressed at low levels in many tissues including brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four ∼12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases. RyRs share ∼70% sequence identity, with the greatest sequence similarity in the C-terminal region that forms the transmembrane, ion-conducting domain comprising ∼500 amino acids. The remaining ∼4,500 amino acids form the large regulatory cytoplasmic "foot" structure. Experimental evidence for Ca2+, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca2+ releasing agents caffeine and ryanodine. Recent work describing the near atomic structures of mammalian skeletal and cardiac muscle RyRs provides a structural basis for the regulation of the RyRs by their multiple effectors.
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Affiliation(s)
- Gerhard Meissner
- Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, NC
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14
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Abstract
Skeletal muscle is the largest tissue in the body and loss of its function or its regenerative properties results in debilitating musculoskeletal disorders. Understanding the mechanisms that drive skeletal muscle formation will not only help to unravel the molecular basis of skeletal muscle diseases, but also provide a roadmap for recapitulating skeletal myogenesis in vitro from pluripotent stem cells (PSCs). PSCs have become an important tool for probing developmental questions, while differentiated cell types allow the development of novel therapeutic strategies. In this Review, we provide a comprehensive overview of skeletal myogenesis from the earliest premyogenic progenitor stage to terminally differentiated myofibers, and discuss how this knowledge has been applied to differentiate PSCs into muscle fibers and their progenitors in vitro.
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Affiliation(s)
- Jérome Chal
- Department of Pathology, Brigham and Women's Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.,Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.,Harvard Stem Cell Institute, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
| | - Olivier Pourquié
- Department of Pathology, Brigham and Women's Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115, USA .,Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.,Harvard Stem Cell Institute, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.,Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), Inserm U964, Université de Strasbourg, 67400 Illkirch-Graffenstaden, France
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15
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Bannister RA. Bridging the myoplasmic gap II: more recent advances in skeletal muscle excitation-contraction coupling. ACTA ACUST UNITED AC 2016; 219:175-82. [PMID: 26792328 DOI: 10.1242/jeb.124123] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
In skeletal muscle, excitation-contraction (EC) coupling relies on the transmission of an intermolecular signal from the voltage-sensing regions of the L-type Ca(2+) channel (Ca(V)1.1) in the plasma membrane to the channel pore of the type 1 ryanodine receptor (RyR1) nearly 10 nm away in the membrane of the sarcoplasmic reticulum (SR). Even though the roles of Ca(V)1.1 and RyR1 as voltage sensor and SR Ca(2+) release channel, respectively, have been established for nearly 25 years, the mechanism underlying communication between these two channels remains undefined. In the course of this article, I will review current viewpoints on this topic with particular emphasis on recent studies.
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Affiliation(s)
- Roger A Bannister
- Department of Medicine-Cardiology Division, University of Colorado Denver-Anschutz Medical Campus, 12700 East 19th Avenue, Room 8006, B-139, Aurora, CO 80045, USA
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16
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Samsó M. A guide to the 3D structure of the ryanodine receptor type 1 by cryoEM. Protein Sci 2016; 26:52-68. [PMID: 27671094 DOI: 10.1002/pro.3052] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2016] [Revised: 09/21/2016] [Accepted: 09/22/2016] [Indexed: 01/04/2023]
Abstract
Signal transduction by the ryanodine receptor (RyR) is essential in many excitable cells including all striated contractile cells and some types of neurons. While its transmembrane domain is a classic tetrameric, six-transmembrane cation channel, the cytoplasmic domain is uniquely large and complex, hosting a multiplicity of specialized domains. The overall outline and substructure readily recognizable by electron microscopy make RyR a geometrically well-behaved specimen. Hence, for the last two decades, the 3D structural study of the RyR has tracked closely the technological advances in electron microscopy, cryo-electron microscopy (cryoEM), and computerized 3D reconstruction. This review summarizes the progress in the structural determination of RyR by cryoEM and, bearing in mind the leap in resolution provided by the recent implementation of direct electron detection, analyzes the first near-atomic structures of RyR. These reveal a complex orchestration of domains controlling the channel's function, and help to understand how this could break down as a consequence of disease-causing mutations.
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Affiliation(s)
- Montserrat Samsó
- Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia
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17
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Lipscombe D, Andrade A. Calcium Channel CaVα₁ Splice Isoforms - Tissue Specificity and Drug Action. Curr Mol Pharmacol 2016; 8:22-31. [PMID: 25966698 DOI: 10.2174/1874467208666150507103215] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2014] [Revised: 01/20/2015] [Accepted: 04/20/2015] [Indexed: 12/11/2022]
Abstract
Voltage-gated calcium ion channels are essential for numerous biological functions of excitable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique CaVα1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cellspecific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains can differ substantially among CaV isoforms expressed in different tissues, and such differences may be greater than those that exist across CaV channel homologs of closely related species. Here we highlight examples of CaV isoforms that have unique expression patterns and that exhibit different pharmacological sensitivities. Knowledge of expression patterns of CaV isoforms in different human tissues, cell populations, ages, and disease states should inform strategies aimed at developing the next generation of CaV channel inhibitors and agonists with improved tissue-specificity.
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Affiliation(s)
- Diane Lipscombe
- Department of Neuroscience, Brown University. Providence, RI, USA.
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Beqollari D, Romberg CF, Dobrowolny G, Martini M, Voss AA, Musarò A, Bannister RA. Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis. Skelet Muscle 2016; 6:24. [PMID: 27340545 PMCID: PMC4918102 DOI: 10.1186/s13395-016-0094-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 06/03/2016] [Indexed: 11/24/2022] Open
Abstract
Background Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3–5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca2+ channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation. Methods We recorded L-type currents, charge movements, and myoplasmic Ca2+ transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1G93A). Results In FDB fibers obtained from “symptomatic” global SOD1G93A mice, we observed a substantial reduction of SR Ca2+ release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1G93A fibers when compared to control fibers. Ca2+ transients were not significantly reduced in similar experiments performed with FDB fibers obtained from “early-symptomatic” SOD1G93A mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca2+ release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1G93A expression was restricted to skeletal muscle. Conclusions We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1G93A mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1G93A and skeletal muscle autonomous MLC/SOD1G93A mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status. Electronic supplementary material The online version of this article (doi:10.1186/s13395-016-0094-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Donald Beqollari
- Department of Medicine-Cardiology Division, University of Colorado School of Medicine, 12700 East 19th Avenue, B-139, Aurora, CO 80045 USA
| | - Christin F Romberg
- Department of Medicine-Cardiology Division, University of Colorado School of Medicine, 12700 East 19th Avenue, B-139, Aurora, CO 80045 USA
| | - Gabriella Dobrowolny
- Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, La Sapienza University, Via A. Scarpa, 14, 00161 Rome, Italy ; Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy
| | - Martina Martini
- Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, La Sapienza University, Via A. Scarpa, 14, 00161 Rome, Italy ; Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy
| | - Andrew A Voss
- Department of Biological Sciences, College of Science and Mathematics, Wright State University, 235A Biological Sciences, 3640 Colonel Glenn Highway, Dayton, OH 45435 USA
| | - Antonio Musarò
- Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, La Sapienza University, Via A. Scarpa, 14, 00161 Rome, Italy ; Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy
| | - Roger A Bannister
- Department of Medicine-Cardiology Division, University of Colorado School of Medicine, 12700 East 19th Avenue, B-139, Aurora, CO 80045 USA
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Mahalingam M, Perez CF, Fessenden JD. Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit. J Biol Chem 2016; 291:13762-70. [PMID: 27129199 DOI: 10.1074/jbc.m115.704049] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2015] [Indexed: 11/06/2022] Open
Abstract
The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling.
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Affiliation(s)
- Mohana Mahalingam
- From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
| | - Claudio F Perez
- From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
| | - James D Fessenden
- From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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20
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Beqollari D, Romberg CF, Meza U, Papadopoulos S, Bannister RA. Differential effects of RGK proteins on L-type channel function in adult mouse skeletal muscle. Biophys J 2014; 106:1950-7. [PMID: 24806927 DOI: 10.1016/j.bpj.2014.03.033] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2013] [Revised: 02/24/2014] [Accepted: 03/25/2014] [Indexed: 11/19/2022] Open
Abstract
Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small GTP-binding proteins profoundly inhibit L-type Ca(2+) channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of Po without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca(2+) channel (CaV1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of CaV1.1's voltage-sensors, whereas Rad utilizes at least one mode that limits voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of CaV1.1.
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Affiliation(s)
- D Beqollari
- Department of Medicine-Cardiology Division, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado
| | - C F Romberg
- Department of Medicine-Cardiology Division, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado
| | - U Meza
- Department of Medicine-Cardiology Division, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado; Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
| | - S Papadopoulos
- Institute of Vegetative Physiology, University Hospital of Cologne, Cologne, Germany
| | - R A Bannister
- Department of Medicine-Cardiology Division, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.
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21
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Eltit JM, Franzini-Armstrong C, Perez CF. Amino acid residues 489-503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor. J Biol Chem 2014; 289:36116-24. [PMID: 25384984 DOI: 10.1074/jbc.m114.615526] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The β1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca(2+) release channel (RyR1). Progressive truncation of the β1a C terminus showed that deletion of amino acid residues Gln(489) to Trp(503) resulted in a loss of depolarization-induced Ca(2+) release, a severe reduction of L-type Ca(2+) currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu(496), Leu(500), and Trp(503), which are thought to mediate direct β1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence (489)QVQVLTSLRRNLSFW(503) of β1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu(496)-Leu(500)-Trp(503) within the β1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling.
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Affiliation(s)
- Jose M Eltit
- the Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virgina 23298, and
| | - Clara Franzini-Armstrong
- the Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
| | - Claudio F Perez
- From the Department of Anesthesiology Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115,
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Calderón JC, Bolaños P, Caputo C. The excitation-contraction coupling mechanism in skeletal muscle. Biophys Rev 2014; 6:133-160. [PMID: 28509964 PMCID: PMC5425715 DOI: 10.1007/s12551-013-0135-x] [Citation(s) in RCA: 114] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2013] [Accepted: 12/06/2013] [Indexed: 12/27/2022] Open
Abstract
First coined by Alexander Sandow in 1952, the term excitation-contraction coupling (ECC) describes the rapid communication between electrical events occurring in the plasma membrane of skeletal muscle fibres and Ca2+ release from the SR, which leads to contraction. The sequence of events in twitch skeletal muscle involves: (1) initiation and propagation of an action potential along the plasma membrane, (2) spread of the potential throughout the transverse tubule system (T-tubule system), (3) dihydropyridine receptors (DHPR)-mediated detection of changes in membrane potential, (4) allosteric interaction between DHPR and sarcoplasmic reticulum (SR) ryanodine receptors (RyR), (5) release of Ca2+ from the SR and transient increase of Ca2+ concentration in the myoplasm, (6) activation of the myoplasmic Ca2+ buffering system and the contractile apparatus, followed by (7) Ca2+ disappearance from the myoplasm mediated mainly by its reuptake by the SR through the SR Ca2+ adenosine triphosphatase (SERCA), and under several conditions movement to the mitochondria and extrusion by the Na+/Ca2+ exchanger (NCX). In this text, we review the basics of ECC in skeletal muscle and the techniques used to study it. Moreover, we highlight some recent advances and point out gaps in knowledge on particular issues related to ECC such as (1) DHPR-RyR molecular interaction, (2) differences regarding fibre types, (3) its alteration during muscle fatigue, (4) the role of mitochondria and store-operated Ca2+ entry in the general ECC sequence, (5) contractile potentiators, and (6) Ca2+ sparks.
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Affiliation(s)
- Juan C Calderón
- Physiology and Biochemistry Research Group-Physis, Department of Physiology and Biochemistry, Faculty of Medicine, University of Antioquia UdeA, Calle 70 No 52-21, Medellín, Colombia.
- Laboratory of Cellular Physiology, Centre of Biophysics and Biochemistry, Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela.
- Departamento de Fisiología y Bioquímica, Grupo de Investigación en Fisiología y Bioquímica-Physis, Facultad de Medicina, Universidad de Antioquia, Calle 70 No 52-21, Medellín, Colombia.
| | - Pura Bolaños
- Laboratory of Cellular Physiology, Centre of Biophysics and Biochemistry, Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela
| | - Carlo Caputo
- Laboratory of Cellular Physiology, Centre of Biophysics and Biochemistry, Venezuelan Institute for Scientific Research (IVIC), Caracas, Venezuela
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Bannister RA, Beam KG. Ca(V)1.1: The atypical prototypical voltage-gated Ca²⁺ channel. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2012; 1828:1587-97. [PMID: 22982493 DOI: 10.1016/j.bbamem.2012.09.007] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2012] [Revised: 09/04/2012] [Accepted: 09/05/2012] [Indexed: 11/28/2022]
Abstract
Ca(V)1.1 is the prototype for the other nine known Ca(V) channel isoforms, yet it has functional properties that make it truly atypical of this group. Specifically, Ca(V)1.1 is expressed solely in skeletal muscle where it serves multiple purposes; it is the voltage sensor for excitation-contraction coupling and it is an L-type Ca²⁺ channel which contributes to a form of activity-dependent Ca²⁺ entry that has been termed Excitation-coupled Ca²⁺ entry. The ability of Ca(V)1.1 to serve as voltage-sensor for excitation-contraction coupling appears to be unique among Ca(V) channels, whereas the physiological role of its more conventional function as a Ca²⁺ channel has been a matter of uncertainty for nearly 50 years. In this chapter, we discuss how Ca(V)1.1 supports excitation-contraction coupling, the possible relevance of Ca²⁺ entry through Ca(V)1.1 and how alterations of Ca(V)1.1 function can have pathophysiological consequences. This article is part of a Special Issue entitled: Calcium channels.
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Affiliation(s)
- Roger A Bannister
- Department of Medicine, Cardiology Division, University of Colorado Denver-Anschutz Medical Campus, Aurora, CO 80045, USA.
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24
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Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor. Proc Natl Acad Sci U S A 2012; 109:7923-8. [PMID: 22547813 DOI: 10.1073/pnas.1119207109] [Citation(s) in RCA: 72] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.
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25
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Hernández-Ochoa EO, Schneider MF. Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2012; 108:98-118. [PMID: 22306655 DOI: 10.1016/j.pbiomolbio.2012.01.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/07/2011] [Revised: 01/14/2012] [Accepted: 01/17/2012] [Indexed: 01/03/2023]
Abstract
Skeletal muscle excitation-contraction (E-C)(1) coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)(2) Ca(2+) release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca(2+), due to depolarization-initiated SR Ca(2+) release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca(2+) signalling properties of mouse skeletal muscle membranes are being investigated.
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Affiliation(s)
- Erick O Hernández-Ochoa
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA.
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Huang CLH, Pedersen TH, Fraser JA. Reciprocal dihydropyridine and ryanodine receptor interactions in skeletal muscle activation. J Muscle Res Cell Motil 2011; 32:171-202. [PMID: 21993921 DOI: 10.1007/s10974-011-9262-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2011] [Accepted: 09/12/2011] [Indexed: 11/25/2022]
Abstract
Dihydropyridine (DHPR) and ryanodine receptors (RyRs) are central to transduction of transverse (T) tubular membrane depolarisation initiated by surface action potentials into release of sarcoplasmic reticular (SR) Ca2+ in skeletal muscle excitation-contraction coupling. Electronmicroscopic methods demonstrate an orderly positioning of such tubular DHPRs relative to RyRs in the SR at triad junctions where their membranes come into close proximity. Biochemical and genetic studies associated expression of specific, DHPR and RyR, isoforms with the particular excitation-contraction coupling processes and related elementary Ca2+ release events found respectively in skeletal and cardiac muscle. Physiological studies of intramembrane charge movements potentially related to voltage triggering of Ca2+ release demonstrated a particular qγ charging species identifiable with DHPRs through its T-tubular localization, pharmacological properties, and steep voltage-dependence paralleling Ca2+ release. Its nonlinear kinetics implicated highly co-operative conformational events in its transitions in response to voltage change. The effects of DHPR and RyR agonists and antagonists upon this intramembrane charge in turn implicated reciprocal rather than merely unidirectional DHPR-RyR interactions in these complex reactions. Thus, following membrane potential depolarization, an orthograde qγ-DHPR-RyR signaling likely initiates conformational alterations in the RyR with which it makes contact. The latter changes could then retrogradely promote further qγ-DHPR transitions through reciprocal co-operative allosteric interactions between receptors. These would relieve the resting constraints on both further, delayed, nonlinear qγ-DHPR charge transfers and on RyR-mediated Ca2+ release. They would also explain the more rapid charging and recovery qγ transients following larger depolarizations and membrane potential repolarization to the resting level.
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Affiliation(s)
- Christopher L-H Huang
- Physiological Laboratory, Department of Biochemistry, University of Cambridge, Cambridge, CB2 3EG, UK.
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Perez CF. On the footsteps of Triadin and its role in skeletal muscle. World J Biol Chem 2011; 2:177-83. [PMID: 21909459 PMCID: PMC3165967 DOI: 10.4331/wjbc.v2.i8.177] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2011] [Revised: 07/29/2011] [Accepted: 08/05/2011] [Indexed: 02/05/2023] Open
Abstract
Calcium is a crucial element for striated muscle function. As such, myoplasmic free Ca2+ concentration is delicately regulated through the concerted action of multiple Ca2+ pathways that relay excitation of the plasma membrane to the intracellular contractile machinery. In skeletal muscle, one of these major Ca2+ pathways is Ca2+ release from intracellular Ca2+ stores through type-1 ryanodine receptor/Ca2+ release channels (RyR1), which positions RyR1 in a strategic cross point to regulate Ca2+ homeostasis. This major Ca2+ traffic point appears to be highly sensitive to the intracellular environment, which senses through a plethora of chemical and protein-protein interactions. Among these modulators, perhaps one of the most elusive is Triadin, a muscle-specific protein that is involved in many crucial aspect of muscle function. This family of proteins mediates complex interactions with various Ca2+ modulators and seems poised to be a relevant modulator of Ca2+ signaling in cardiac and skeletal muscles. The purpose of this review is to examine the most recent evidence and current understanding of the role of Triadin in muscle function, in general, with particular emphasis on its contribution to Ca2+ homeostasis.
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Affiliation(s)
- Claudio F Perez
- Claudio F Perez, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, United States
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Local calcium signals induced by hyper-osmotic stress in mammalian skeletal muscle cells. J Muscle Res Cell Motil 2009; 30:97-109. [PMID: 19437123 DOI: 10.1007/s10974-009-9179-8] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2009] [Accepted: 04/27/2009] [Indexed: 10/20/2022]
Abstract
Strenuous activitiy of skeletal muscle leads to temporary osmotic dysbalance and isolated skeletal muscle fibers exposed to osmotic stress respond with characteristic micro-domain calcium signals. It has been suggested that osmotic stress targets transverse tubular (TT) dihydropyridine receptors (DHPRs) which normally serve as voltage-dependent activators of Ca release via ryanodine receptor (RyR1s) of the sarcoplasmic reticulum (SR). Here, we pursued this hypothesis by imaging the response to hyperosmotic solutions in both mouse skeletal muscle fibers and myotubes. Ca fluctuations in the cell periphery of fibers exposed to osmotic stress were accompanied by a substantial dilation of the peripheral TT. The Ca signals were completely inhibited by a conditioning depolarization that inactivates the DHPR. Dysgenic myotubes, lacking the DHP-receptor-alpha1-subunit, showed strongly reduced, yet not completely inhibited activity when stimulated with solutions of elevated tonicity. The results point to a modulatory, even though not essential, role of the DHP receptor for osmotic stress-induced Ca signals in skeletal muscle.
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Bannister RA, Pessah IN, Beam KG. The skeletal L-type Ca(2+) current is a major contributor to excitation-coupled Ca(2+) entry. ACTA ACUST UNITED AC 2009; 133:79-91. [PMID: 19114636 PMCID: PMC2606935 DOI: 10.1085/jgp.200810105] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The term excitation-coupled Ca(2+) entry (ECCE) designates the entry of extracellular Ca(2+) into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca(2+) entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 microM), and is permeable to Mn(2+). Here, we have examined the effects of these agents on the L-type Ca(2+) current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La(3+), and Gd(3+)) and dantrolene all inhibited the L-type Ca(2+) current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 microM. Like ECCE, the L-type Ca(2+) channel displays permeability to Mn(2+) in the absence of external Ca(2+) and produces a Ca(2+) current that persists during prolonged ( approximately 10-second) depolarization. This current appears to contribute to the Ca(2+) transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca(2+); (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR alpha(1S) pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca(2+)-free solution and were unaffected by Ca(2+) removal; and (3) after block of SR Ca(2+) release by 200 microM ryanodine, normal myotubes still displayed a large Ca(2+) transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca(2+) entry attributed to ECCE.
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Affiliation(s)
- Roger A Bannister
- Department of Physiology and Biophysics, School of Medicine, University of Colorado Denver, Aurora, CO 80045, USA
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Schredelseker J, Dayal A, Schwerte T, Franzini-Armstrong C, Grabner M. Proper restoration of excitation-contraction coupling in the dihydropyridine receptor beta1-null zebrafish relaxed is an exclusive function of the beta1a subunit. J Biol Chem 2009; 284:1242-51. [PMID: 19008220 PMCID: PMC2613631 DOI: 10.1074/jbc.m807767200] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2008] [Indexed: 11/06/2022] Open
Abstract
The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) beta(1a) subunit. Lack of beta(1a) results in (i) reduced membrane expression of the pore forming DHPR alpha(1S) subunit, (ii) elimination of alpha(1S) charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of beta(1a) from rather general functions of beta isoforms. Zebrafish and mammalian beta(1a) subunits quantitatively restored alpha(1S) triad targeting and charge movement as well as intracellular Ca(2+) release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal beta(2a) as the phylogenetically closest, and the ancestral housefly beta(M) as the most distant isoform to beta(1a) also completely recovered alpha(1S) triad expression and charge movement. However, both revealed drastically impaired intracellular Ca(2+) transients and very limited tetrad formation compared with beta(1a). Consequently, larval motility was either only partially restored (beta(2a)-injected larvae) or not restored at all (beta(M)). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested beta subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the beta(1a) isoform. Consequently, we postulate a model that presents beta(1a) as an allosteric modifier of alpha(1S) conformation enabling skeletal muscle-type EC coupling.
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Affiliation(s)
- Johann Schredelseker
- Department of Medical Genetics, Clinical and Molecular Pharmacology, Division of Biochemical Pharmacology, Innsbruck Medical University, A-6020 Innsbruck, Austria
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Bannister RA, Grabner M, Beam KG. The alpha(1S) III-IV loop influences 1,4-dihydropyridine receptor gating but is not directly involved in excitation-contraction coupling interactions with the type 1 ryanodine receptor. J Biol Chem 2008; 283:23217-23. [PMID: 18556650 PMCID: PMC2516988 DOI: 10.1074/jbc.m804312200] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2008] [Revised: 06/13/2008] [Indexed: 11/06/2022] Open
Abstract
In skeletal muscle, coupling between the 1,4-dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) underlies excitation-contraction (EC) coupling. The III-IV loop of the DHPR alpha(1S) subunit binds to a segment of RyR1 in vitro, and mutations in the III-IV loop alter the voltage dependence of EC coupling, raising the possibility that this loop is directly involved in signal transmission from the DHPR to RyR1. To clarify the role of the alpha(1S) III-IV loop in EC coupling, we examined the functional properties of a chimera (GFP-alpha(1S)[III-IVa]) in which the III-IV loop of the divergent alpha(1A) isoform replaced that of alpha(1S). Dysgenic myotubes expressing GFP-alpha(1S)[III-IVa] yielded myoplasmic Ca(2+) transients that activated at approximately 10 mV more hyperpolarized potentials and that were approximately 65% smaller than those of GFP-alpha(1S). A similar reduction was observed in voltage-dependent charge movements for GFP-alpha(1S)[III-IVa], indicating that the chimeric channels trafficked less well to the membrane but that those that were in the membrane functioned as efficiently in EC coupling as GFP-alpha(1S). Relative to GFP-alpha(1S), L-type currents mediated by GFP-alpha(1S)[III-IVa] were approximately 40% smaller and activated at approximately 5 mV more hyperpolarized potentials. The altered gating of GFP-alpha(1S)[III-IVa] was accentuated by exposure to +/-Bay K 8644, which caused a much larger hyperpolarizing shift in activation compared with its effect on GFP-alpha(1S). Taken together, our observations indicate that the alpha(1S) III-IV loop is not directly involved in EC coupling but does influence DHPR gating transitions important both for EC coupling and activation of L-type conductance.
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Affiliation(s)
- Roger A Bannister
- Department of Physiology and Biophysics, University of Colorado-Denver, Aurora, Colorado 80045, USA
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[Human calcium channelopathies. Voltage-gated Ca(2+) channels in etiology, pathogenesis, and pharmacotherapy of neurologic disorders]. DER NERVENARZT 2008; 79:426-36. [PMID: 18210049 DOI: 10.1007/s00115-007-2398-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Voltage-gated calcium channels are key components in a variety of physiological processes. Within the last decade an increasing number of voltage-gated Ca(2+) channelopathies in both humans and animal models has been described, most of which are related to the neurologic and muscular system. In humans, mutations were found in L-type Ca(v)1.2 and Ca(v)1.4 Ca(2+) channels as well as the non-L-type Ca(v)2.1 and T-type Ca(v)3.2 channels, resulting in altered electrophysiologic properties. Based on their widespread distribution within the CNS, voltage-gated calcium channels are of particular importance in the etiology and pathogenesis of various forms of epilepsy and neuropsychiatric disorders. In this review we characterise the different human Ca(2+) channelopathies known so far, further illuminating basic pathophysiologic mechanisms and clinical aspects.
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Abstract
1. Excitation-contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum. 2. We now know that excitation-contraction coupling depends on a macromolecular protein complex or 'calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3. The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a 'voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca(2+) load within the calcium store. 4. The molecular components of the 'calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation-contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca(2+) through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5. Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years.
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Affiliation(s)
- A F Dulhunty
- Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, Australian Capital Territory, Australia.
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McKeown L, Robinson P, Jones OT. Molecular basis of inherited calcium channelopathies: role of mutations in pore-forming subunits. Acta Pharmacol Sin 2006; 27:799-812. [PMID: 16787562 DOI: 10.1111/j.1745-7254.2006.00394.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The pore-forming alpha subunits of voltage-gated calcium channels contain the essential biophysical machinery that underlies calcium influx in response to cell depolarization. In combination with requisite auxiliary subunits, these pore subunits form calcium channel complexes that are pivotal to the physiology and pharmacology of diverse cells ranging from sperm to neurons. Not surprisingly, mutations in the pore subunits generate diverse pathologies, termed channelopathies, that range from failures in excitation-contraction coupling to night blindness. Over the last decade, major insights into the mechanisms of pathogenesis have been derived from animals showing spontaneous or induced mutations. In parallel, there has been considerable growth in our understanding of the workings of voltage-gated ion channels from a structure-function, regulation and cell biology perspective. Here we document our current understanding of the mutations underlying channelopathies involving the voltage-gated calcium channel alpha subunits in humans and other species.
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Affiliation(s)
- Lynn McKeown
- Faculty of Life Sciences, the University of Manchester, Manchester, UK
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35
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Affiliation(s)
- H Glossmann
- Institut für Biochemische Pharmakologie der Leopold-Franzens-Universität Innsbruck, Austria
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36
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Porzig H. Pharmacological modulation of voltage-dependent calcium channels in intact cells. Rev Physiol Biochem Pharmacol 2006; 114:209-62. [PMID: 2155471 DOI: 10.1007/bfb0031020] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- H Porzig
- Pharmakologisches Institut, Universität Bern, Switzerland
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37
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Ursu D, Schuhmeier RP, Melzer W. Voltage-controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse. J Physiol 2004; 562:347-65. [PMID: 15528246 PMCID: PMC1665514 DOI: 10.1113/jphysiol.2004.073882] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
The voltage-activated fluxes of Ca(2+) from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 mm EGTA and 0.2 mm of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca(2+) inward current exhibiting half-maximal activation at -7.60 +/- 1.29 and 3.0 +/- 1.44 mV, respectively. The rate of Ca(2+) release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca(2+) removal. Results obtained with the two indicators were similar. Ca(2+) release was 2-3 orders of magnitude larger than the flux carried by the L-type Ca(2+) current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca(2+) content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation-contraction coupling in future studies of genetically altered mice.
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Affiliation(s)
- D Ursu
- University of Ulm, Department of Applied Physiology, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
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38
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Cherednichenko G, Hurne AM, Fessenden JD, Lee EH, Allen PD, Beam KG, Pessah IN. Conformational activation of Ca2+ entry by depolarization of skeletal myotubes. Proc Natl Acad Sci U S A 2004; 101:15793-8. [PMID: 15505226 PMCID: PMC524834 DOI: 10.1073/pnas.0403485101] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Store-operated Ca(2+) entry (SOCE) occurs in diverse cell types in response to depletion of Ca(2+) within the endoplasmic/sarcoplasmic reticulum and functions both to refill these stores and to shape cytoplasmic Ca(2+) transients. Here we report that in addition to conventional SOCE, skeletal myotubes display a physiological mechanism that we term excitation-coupled Ca(2+) entry (ECCE). ECCE is rapidly initiated by membrane depolarization. Like excitation-contraction coupling, ECCE is absent in both dyspedic myotubes that lack the skeletal muscle-type ryanodine receptor 1 and dysgenic myotubes that lack the dihydropyridine receptor (DHPR), and is independent of the DHPR l-type Ca(2+) current. Unlike classic SOCE, ECCE does not depend on sarcoplasmic reticulum Ca(2+) release. Indeed, ECCE produces a large Ca(2+) entry in response to physiological stimuli that do not produce substantial store depletion and depends on interactions among three different Ca(2+) channels: the DHPR, ryanodine receptor 1, and a Ca(2+) entry channel with properties corresponding to those of store-operated Ca(2+) channels. ECCE may provide a fundamental means to rapidly maintain Ca(2+) stores and control important aspects of Ca(2+) signaling in both muscle and nonmuscle cells.
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Affiliation(s)
- Gennady Cherednichenko
- Department of Molecular Biosciences and Center for Children's Environmental Health and Disease Prevention, University of California, Davis, CA 95616, USA
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Spangenburg EE, Bowles DK, Booth FW. Insulin-like growth factor-induced transcriptional activity of the skeletal alpha-actin gene is regulated by signaling mechanisms linked to voltage-gated calcium channels during myoblast differentiation. Endocrinology 2004; 145:2054-63. [PMID: 14684598 DOI: 10.1210/en.2003-1476] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
IGF-I activates signaling pathways that increase the expression of muscle-specific genes in differentiating myoblasts. Induction of skeletal alpha-actin expression occurs during differentiation through unknown mechanisms. The purpose of this investigation was to examine the mechanisms that IGF-I uses to induce skeletal alpha-actin gene expression in C2C12 myoblasts. IGF-I increased skeletal alpha-actin promoter activity by 107% compared with the control condition. Ni(+) [T-type voltage-gated Ca(2+) channel (VGCC) inhibitor] reduced basal-induced activation of the skeletal alpha-actin promoter by approximately 84%, and nifedipine (L-type VGCC inhibitor) inhibited IGF-I-induced activation of the skeletal alpha-actin promoter by 29-48%. IGF-I failed to increase skeletal alpha-actin promoter activity in differentiating dysgenic (lack functional L-type VGCC) myoblasts; 30 mm K(+) and 30 mm K(+)+IGF-I increased skeletal alpha-actin promoter activity by 162% and 76% compared with non-IGF-I or IGF-I-only conditions, respectively. IGF-I increased calcineurin activity, which was inhibited by cyclosporine A. Further, cyclosporine A inhibited K(+)+IGF-I-induced activation of the skeletal alpha-actin promoter. Constitutively active calcineurin increased skeletal alpha-actin promoter activity by 154% and rescued the nifedipine-induced inhibition of L-type VGCC but failed to rescue the Ni(+)-inhibition of T-type VGCC. IGF-I-induced nuclear factor of activated T-cells transcriptional activity was not inhibited by nifedipine or Ni(+). IGF-I failed to increase serum response factor transcriptional activity; however, serum response factor activity was reduced in the presence of Ni(+). These data suggest that IGF-I-induced activation of the skeletal alpha-actin promoter is regulated by the L-type VGCC and calcineurin but independent of nuclear factor of activated T-cell transcriptional activity as C2C12 myoblasts differentiate into myotubes.
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Affiliation(s)
- Espen E Spangenburg
- Department of Biomedical Sciences, University of Missouri, Columbia 65211, USA.
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40
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Ingalls CP, Warren GL, Zhang JZ, Hamilton SL, Armstrong RB. Dihydropyridine and ryanodine receptor binding after eccentric contractions in mouse skeletal muscle. J Appl Physiol (1985) 2003; 96:1619-25. [PMID: 14672973 DOI: 10.1152/japplphysiol.00084.2003] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The purpose of this study was to determine whether there are alterations in the dihydropyridine and/or ryanodine receptors that might explain the excitation-contraction uncoupling associated with eccentric contraction-induced skeletal muscle injury. The left anterior crural muscles (i.e., tibialis anterior, extensor digitorum longus, and extensor hallucis longus) of mice were injured in vivo by 150 eccentric contractions. Peak isometric tetanic torque of the anterior crural muscles was reduced approximately 45% immediately and 3 days after the eccentric contractions. Partial restoration of peak isometric tetanic and subtetanic forces of injured extensor digitorum longus muscles by 10 mM caffeine indicated the presence of excitation-contraction uncoupling. Scatchard analysis of [3H]ryanodine binding indicated that the number of ryanodine receptor binding sites was not altered immediately postinjury but decreased 16% 3 days later. Dihydropyridine receptor binding sites increased approximately 20% immediately after and were elevated to the same extent 3 days after the injury protocol. Muscle injury did not alter the sensitivity of either receptor. These data suggest that a loss or altered sensitivity of the dihydropyridine and ryanodine receptors does not contribute to the excitation-contraction uncoupling immediately after contraction-induced muscle injury. We also concluded that the loss in ryanodine receptors 3 days after injury is not the primary cause of excitation-contraction uncoupling at that time.
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Affiliation(s)
- Christopher P Ingalls
- Muscle Biology Laboratory, Department of Kinesiology and Health, Georgia State University, Atlanta, GA 30303, USA.
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41
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Lee EH, Lopez JR, Li J, Protasi F, Pessah IN, Kim DH, Allen PD. Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module. Am J Physiol Cell Physiol 2003; 286:C179-89. [PMID: 13679303 DOI: 10.1152/ajpcell.00176.2003] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca2+ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La3+ and Cd2+ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La3+ and Cd2+ or nifedipine in the external medium at concentrations known to block Ca2+ entry through the DHPRs significantly decreased the caffeine EC50 of wtRyR1 (2.80 +/- 0.12 to 0.83 +/- 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC50 values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC50 values of chimeras R4 and R9 were significantly increased (1.27 +/- 0.05 to 2.60 +/- 0.16 mM, and 1.15 +/- 0.03 to 2.11 +/- 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca2+ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [3H]ryanodine regardless of Ca2+ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca2+ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism.
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Affiliation(s)
- Eun Hui Lee
- Department of Anesthesia Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
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Chun LG, Ward CW, Schneider MF. Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally. Am J Physiol Cell Physiol 2003; 285:C686-97. [PMID: 12724135 DOI: 10.1152/ajpcell.00072.2003] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
"Spontaneous" Ca2+ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca2+ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca2+ indicator dye fluo 4 to show that the frequency of spontaneous Ca2+ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca2+ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca2+ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl2 (5 mM) or nifedipine (30 microM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca2+ sparks may be triggered by locally elevated [Ca2+] due to Ca2+ influx via dihydropyridine receptor L-type Ca2+ channels in embryonic mammalian skeletal muscle.
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Affiliation(s)
- Lois G Chun
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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43
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Abstract
T-type Ca2+ channels were originally called low-voltage-activated (LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neurons Ca2+ influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentials mediated by Na+ channels. Burst firing is thought to play an important role in the synchronized activity of the thalamus observed in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. In addition to a pacemaker role, Ca2+ entry via T-type channels can directly regulate intracellular Ca2+ concentrations, which is an important second messenger for a variety of cellular processes. Molecular cloning revealed the existence of three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that found in high-voltage-activated (HVA) Ca2+ channels, and Na+ channels, indicating that they are evolutionarily related. Hence, the alpha1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes are expressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. The electrophysiological activities of recombinant Cav3 channels are very similar to native T-type currents and can be differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, and smaller conductance of Ba2+. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block by Ni2+. The goal of this review is to provide a comprehensive description of T-type currents, their distribution, regulation, pharmacology, and cloning.
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Affiliation(s)
- Edward Perez-Reyes
- Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908-0735, USA.
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44
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Jacobson D, Herson PS, Neelands TR, Maylie J, Adelman JP. SK channels are necessary but not sufficient for denervation-induced hyperexcitability. Muscle Nerve 2002; 26:817-22. [PMID: 12451607 DOI: 10.1002/mus.10280] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Skeletal muscle hyperexcitability is characteristically associated with denervation. Expression of SK3, a small conductance Ca(2+)-activated K(+) channel (SK channel) in skeletal muscle is induced by denervation, and direct application of apamin, a peptide blocker of SK channels, dramatically reduces hyperexcitability. To investigate the role of SK3 channels in denervation- induced hyperexcitability, SK3 expression was manipulated using a transgenic mouse that harbors a tetracycline-regulated SK3 gene. Electromyographic (EMG) recordings from anterior tibial (AT) muscle showed that denervated muscle from transgenic or wild-type animals had equivalent hyperexcitability that was blocked by apamin. In contrast, denervated skeletal muscle from SK3tTA mice lacking SK3 channels showed little or no hyperexcitability, similar to results from wild-type innervated skeletal muscle. However, innervated skeletal muscle from SK3tTA mice containing SK3 channels did not show hyperexcitability. The results demonstrate that SK3 channels are necessary but not sufficient for denervation-induced skeletal muscle hyperexcitability.
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Affiliation(s)
- David Jacobson
- Department of Molecular and Medical Genetics, Oregon Health and Sciences University, Portland, Oregon, USA
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45
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Protasi F, Paolini C, Nakai J, Beam KG, Franzini-Armstrong C, Allen PD. Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle. Biophys J 2002; 83:3230-44. [PMID: 12496092 PMCID: PMC1302400 DOI: 10.1016/s0006-3495(02)75325-3] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
Excitation-contraction (e-c) coupling in muscle relies on the interaction between dihydropyridine receptors (DHPRs) and RyRs within Ca(2+) release units (CRUs). In skeletal muscle this interaction is bidirectional: alpha(1S)DHPRs trigger RyR1 (the skeletal form of the ryanodine receptor) to release Ca(2+) in the absence of Ca(2+) permeation through the DHPR, and RyR1s, in turn, affect the open probability of alpha(1S)DHPRs. alpha(1S)DHPR and RyR1 are linked to each other, organizing alpha(1S)-DHPRs into groups of four, or tetrads. In cardiac muscle, however, alpha(1C)DHPR Ca(2+) current is important for activation of RyR2 (the cardiac isoform of the ryanodine receptor) and alpha(1C)-DHPRs are not organized into tetrads. We expressed RyR1, RyR2, and four different RyR1/RyR2 chimeras (R4: Sk1635-3720, R9: Sk2659-3720, R10: Sk1635-2559, R16: Sk1837-2154) in 1B5 dyspedic myotubes to test their ability to restore skeletal-type e-c coupling and DHPR tetrads. The rank-order for restoring skeletal e-c coupling, indicated by Ca(2+) transients in the absence of extracellular Ca(2+), is RyR1 > R4 > R10 >> R16 > R9 >> RyR2. The rank-order for restoration of DHPR tetrads is RyR1 > R4 = R9 > R10 = R16 >> RyR2. Because the skeletal segment in R9 does not overlap with that in either R10 or R16, our results indicate that multiple regions of RyR1 may interact with alpha(1S)DHPRs and that the regions responsible for tetrad formation do not correspond exactly to the ones required for functional coupling.
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MESH Headings
- Animals
- Caffeine/pharmacology
- Calcium/metabolism
- Calcium Channels/drug effects
- Calcium Channels/physiology
- Calcium Channels/ultrastructure
- Calcium Channels, L-Type/drug effects
- Calcium Channels, L-Type/physiology
- Calcium Channels, L-Type/ultrastructure
- Cell Line
- Freeze Fracturing
- Mice
- Microscopy, Electron
- Muscle Contraction/drug effects
- Muscle Contraction/physiology
- Muscle Fibers, Skeletal/drug effects
- Muscle Fibers, Skeletal/physiology
- Muscle Fibers, Skeletal/ultrastructure
- Muscle, Skeletal/drug effects
- Muscle, Skeletal/physiology
- Muscle, Skeletal/ultrastructure
- Ryanodine Receptor Calcium Release Channel/drug effects
- Ryanodine Receptor Calcium Release Channel/physiology
- Ryanodine Receptor Calcium Release Channel/ultrastructure
- Structure-Activity Relationship
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Affiliation(s)
- Feliciano Protasi
- Department of Anesthesia Research, Brigham and Women's Hospital, Boston, MA 02115, USA
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46
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Hong CS, Cho MC, Kwak YG, Song CH, Lee YH, Lim JS, Kwon YK, Chae SW, Kim DH. Cardiac remodeling and atrial fibrillation in transgenic mice overexpressing junctin. FASEB J 2002; 16:1310-2. [PMID: 12154005 DOI: 10.1096/fj.01-0908fje] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Junctin is a 26-kDa integral membrane protein, colocalized with the ryanodine receptor (RyR) and calsequestrin at the junctional sarcoplasmic reticulum (SR) membrane in cardiac and skeletal muscles. To elucidate the functional role of junctin in heart, transgenic (TG) mice overexpressing canine junctin (24-29 folds) under the control of mouse a-myosin heavy chain promoter were generated. Overexpression of the junctin in mouse heart was associated with heart enlargements, bradycardia, atrial fibrillation, and increased fibrosis. Many ultrastructural alterations were observed in TG atria. The junctional SR cisternae facing transverse-tubules contained a dense matrix of calsequestrin in TG heart. According to echocardiography, TG mice showed enlarged left ventricles, dilated right atriums, and ventricles with paradoxical septal motion and impaired left ventricular systolic function. Overexpression of junctin led to down-regulation of triadin and RyR but to up-regulation of dihydropyridine receptor. The L-type Ca2+ current density and action potential durations increased, which could be the cause for the bradycardia in TG heart. This study provides an important example of pathogenesis leading to substantial cardiac remodeling and atrial fibrillation, which was caused by overexpression of junctin in heart.
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Affiliation(s)
- Chang-Soo Hong
- Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), Kwangju, Korea
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Felder E, Protasi F, Hirsch R, Franzini-Armstrong C, Allen PD. Morphology and molecular composition of sarcoplasmic reticulum surface junctions in the absence of DHPR and RyR in mouse skeletal muscle. Biophys J 2002; 82:3144-9. [PMID: 12023238 PMCID: PMC1302103 DOI: 10.1016/s0006-3495(02)75656-7] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Calcium release during excitation-contraction coupling of skeletal muscle cells is initiated by the functional interaction of the exterior membrane and the sarcoplasmic reticulum (SR), mediated by the "mechanical" coupling of ryanodine receptors (RyR) and dihydropyridine receptors (DHPR). RyR is the sarcoplasmic reticulum Ca(2+) release channel and DHPR is an L-type calcium channel of exterior membranes (surface membrane and T tubules), which acts as the voltage sensor of excitation-contraction coupling. The two proteins communicate with each other at junctions between SR and exterior membranes called calcium release units and are associated with several proteins of which triadin and calsequestrin are the best characterized. Calcium release units are present in diaphragm muscles and hind limb derived primary cultures of double knock out mice lacking both DHPR and RyR. The junctions show coupling between exterior membranes and SR, and an apparently normal content and disposition of triadin and calsequestrin. Therefore SR-surface docking, targeting of triadin and calsequestrin to the junctional SR domains and the structural organization of the two latter proteins are not affected by lack of DHPR and RyR. Interestingly, simultaneous lack of the two major excitation-contraction coupling proteins results in decrease of calcium release units frequency in the diaphragm, compared with either single knockout mutation.
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Affiliation(s)
- Edward Felder
- Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058, USA.
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48
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Dulhunty AF, Haarmann CS, Green D, Laver DR, Board PG, Casarotto MG. Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2002; 79:45-75. [PMID: 12225776 DOI: 10.1016/s0079-6107(02)00013-5] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca(2+) channels in the surface/transverse tubular membrane and ryanodine receptor Ca(2+) release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation-contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein-protein interaction remain unknown. The trigger for Ca(2+) release through ryanodine receptors in cardiac muscle is a Ca(2+) influx through the L-type Ca(2+) channel. The Ca(2+) entering through the surface membrane Ca(2+) channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.
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Affiliation(s)
- A F Dulhunty
- John Curtin School of Medical Research, Australian National University, P.O. Box 334 2601 Canberra, Australia.
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49
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Hong CS, Kwak YG, Ji JH, Chae SW, Kim DH. Molecular cloning and characterization of mouse cardiac junctate isoforms. Biochem Biophys Res Commun 2001; 289:882-7. [PMID: 11735129 DOI: 10.1006/bbrc.2001.6056] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Junctate is a newly identified integral ER/SR membrane calcium binding protein, which is an alternative splicing form of the same gene generating aspartyl beta-hydroxylase and junctin. Screening a mouse heart cDNA library using canine junctin cDNA as a probe yielded three complete mouse heart cDNAs. One of the cDNAs is homologous to the previously reported human junctate. The three mouse junctate proteins are composed of 270, 259, and 215 amino acids (we named them junctate-1, -2, and -3). The apparent molecular masses of the mouse junctates in SDS-PAGE were in the range between 40 and 53 kDa. Northern and Western blot analyses indicate that mouse junctates are expressed in heart, brain, spleen, lung, liver, kidney, and stomach, but not in skeletal muscle. The apparent molecular weights of junctates from heart and brain were somewhat different from those from the other tissues tested, suggesting that there are tissue-specific expression patterns of the different junctate isoforms. Immunohistochemical studies showed that junctates were expressed both in ventricular and atrial tissues. This is the first study that shows the presence of 3 distinct cardiac junctate isoforms expressed in various mammalian tissues.
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Affiliation(s)
- C S Hong
- Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, 500-712, Korea
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50
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Ursu D, Sebille S, Dietze B, Freise D, Flockerzi V, Melzer W. Excitation-contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit gamma1. J Physiol 2001; 533:367-77. [PMID: 11389198 PMCID: PMC2278637 DOI: 10.1111/j.1469-7793.2001.0367a.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
1. In skeletal muscle, dihydropyridine (DHP) receptors control both Ca(2+) entry (L-type current) and internal Ca(2+) release in a voltage-dependent manner. Here we investigated the question of whether elimination of the skeletal muscle-specific DHP receptor subunit gamma1 affects excitation-contraction (E-C) coupling. We studied intracellular Ca(2+) release and force production in muscle preparations of a mouse deficient in the gamma1 subunit (gamma-/-). 2. The rate of internal Ca(2+) release at large depolarization (+20 mV) was determined in voltage-clamped primary-cultured myotubes derived from satellite cells of adult mice by analysing fura-2 fluorescence signals and estimating the concentration of free and bound Ca(2+). On average, gamma-/- cells showed an increase in release of about one-third of the control value and no alterations in the time course. 3. Voltage of half-maximal activation (V(1/2)) and voltage sensitivity (k) were not significantly different in gamma-/- myotubes, either for internal Ca(2+) release activation or for the simultaneously measured L-type Ca(2+) conductance. The same was true for maximal Ca(2+) inward current and conductance. 4. Contractions evoked by electrical stimuli were recorded in isolated extensor digitorum longus (EDL; fast, glycolytic) and soleus (slow, oxidative) muscles under normal conditions and during fatigue induced by repetitive tetanic stimulation. Neither time course nor amplitudes of twitches and tetani nor force-frequency relations showed significant alterations in the gamma1-deficient muscles. 5. In conclusion, the overall results show that the gamma1 subunit is not essential for voltage-controlled Ca(2+) release and force production.
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MESH Headings
- Action Potentials/physiology
- Animals
- Calcium/metabolism
- Calcium Channels, L-Type/genetics
- Calcium Channels, L-Type/metabolism
- Cells, Cultured
- Ion Channel Gating/physiology
- Mice
- Mice, Mutant Strains
- Muscle Contraction/physiology
- Muscle Fatigue/physiology
- Muscle Fibers, Fast-Twitch/cytology
- Muscle Fibers, Fast-Twitch/physiology
- Muscle Fibers, Slow-Twitch/cytology
- Muscle Fibers, Slow-Twitch/physiology
- Muscle, Skeletal/cytology
- Muscle, Skeletal/physiology
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Affiliation(s)
- D Ursu
- Universität Ulm, Abteilung für Angewandte Physiologie, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
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