|
1
|
Li Z, Wu Q, Yan N. A structural atlas of druggable sites on Na v channels. Channels (Austin) 2024; 18:2287832. [PMID: 38033122 PMCID: PMC10732651 DOI: 10.1080/19336950.2023.2287832] [Citation(s) in RCA: 21] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Accepted: 11/20/2023] [Indexed: 12/02/2023] Open
Abstract
Voltage-gated sodium (Nav) channels govern membrane excitability by initiating and propagating action potentials. Consistent with their physiological significance, dysfunction, or mutations in these channels are associated with various channelopathies. Nav channels are thereby major targets for various clinical and investigational drugs. In addition, a large number of natural toxins, both small molecules and peptides, can bind to Nav channels and modulate their functions. Technological breakthrough in cryo-electron microscopy (cryo-EM) has enabled the determination of high-resolution structures of eukaryotic and eventually human Nav channels, alone or in complex with auxiliary subunits, toxins, and drugs. These studies have not only advanced our comprehension of channel architecture and working mechanisms but also afforded unprecedented clarity to the molecular basis for the binding and mechanism of action (MOA) of prototypical drugs and toxins. In this review, we will provide an overview of the recent advances in structural pharmacology of Nav channels, encompassing the structural map for ligand binding on Nav channels. These findings have established a vital groundwork for future drug development.
Collapse
Affiliation(s)
- Zhangqiang Li
- Beijing Frontier Research Center for Biological Structures, State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China
| | - Qiurong Wu
- Beijing Frontier Research Center for Biological Structures, State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China
| | - Nieng Yan
- Beijing Frontier Research Center for Biological Structures, State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China
- Shenzhen Medical Academy of Research and Translation, Shenzhen, Guangdong Province, China
| |
Collapse
|
|
2
|
Huang J, Pan X, Yan N. Structural biology and molecular pharmacology of voltage-gated ion channels. Nat Rev Mol Cell Biol 2024; 25:904-925. [PMID: 39103479 DOI: 10.1038/s41580-024-00763-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/26/2024] [Indexed: 08/07/2024]
Abstract
Voltage-gated ion channels (VGICs), including those for Na+, Ca2+ and K+, selectively permeate ions across the cell membrane in response to changes in membrane potential, thus participating in physiological processes involving electrical signalling, such as neurotransmission, muscle contraction and hormone secretion. Aberrant function or dysregulation of VGICs is associated with a diversity of neurological, psychiatric, cardiovascular and muscular disorders, and approximately 10% of FDA-approved drugs directly target VGICs. Understanding the structure-function relationship of VGICs is crucial for our comprehension of their working mechanisms and role in diseases. In this Review, we discuss how advances in single-particle cryo-electron microscopy have afforded unprecedented structural insights into VGICs, especially on their interactions with clinical and investigational drugs. We present a comprehensive overview of the recent advances in the structural biology of VGICs, with a focus on how prototypical drugs and toxins modulate VGIC activities. We explore how these structures elucidate the molecular basis for drug actions, reveal novel pharmacological sites, and provide critical clues to future drug discovery.
Collapse
Affiliation(s)
- Jian Huang
- Department of Molecular Biology, Princeton University, Princeton, NJ, USA
| | - Xiaojing Pan
- Institute of Bio-Architecture and Bio-Interactions (IBABI), Shenzhen Medical Academy of Research and Translation (SMART), Shenzhen, Guangdong, China.
| | - Nieng Yan
- Institute of Bio-Architecture and Bio-Interactions (IBABI), Shenzhen Medical Academy of Research and Translation (SMART), Shenzhen, Guangdong, China.
- Beijing Frontier Research Center for Biological Structure, Tsinghua-Peking Joint Center for Life Sciences, State Key Laboratory of Membrane Biology, Tsinghua University, Beijing, China.
| |
Collapse
|
|
3
|
Waheed S, Ramzan K, Ahmad S, Khan MS, Wajid M, Ullah H, Umar A, Iqbal R, Ullah R, Bari A. Identification and In-Silico study of non-synonymous functional SNPs in the human SCN9A gene. PLoS One 2024; 19:e0297367. [PMID: 38394191 PMCID: PMC10889873 DOI: 10.1371/journal.pone.0297367] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2023] [Accepted: 12/29/2023] [Indexed: 02/25/2024] Open
Abstract
Single nucleotide polymorphisms are the most common form of DNA alterations at the level of a single nucleotide in the genomic sequence. Genome-wide association studies (GWAS) were carried to identify potential risk genes or genomic regions by screening for SNPs associated with disease. Recent studies have shown that SCN9A comprises the NaV1.7 subunit, Na+ channels have a gene encoding of 1988 amino acids arranged into 4 domains, all with 6 transmembrane regions, and are mainly found in dorsal root ganglion (DRG) neurons and sympathetic ganglion neurons. Multiple forms of acute hypersensitivity conditions, such as primary erythermalgia, congenital analgesia, and paroxysmal pain syndrome have been linked to polymorphisms in the SCN9A gene. Under this study, we utilized a variety of computational tools to explore out nsSNPs that are potentially damaging to heath by modifying the structure or activity of the SCN9A protein. Over 14 potentially damaging and disease-causing nsSNPs (E1889D, L1802P, F1782V, D1778N, C1370Y, V1311M, Y1248H, F1237L, M936V, I929T, V877E, D743Y, C710W, D623H) were identified by a variety of algorithms, including SNPnexus, SNAP-2, PANTHER, PhD-SNP, SNP & GO, I-Mutant, and ConSurf. Homology modeling, structure validation, and protein-ligand interactions also were performed to confirm 5 notable substitutions (L1802P, F1782V, D1778N, V1311M, and M936V). Such nsSNPs may become the center of further studies into a variety of disorders brought by SCN9A dysfunction. Using in-silico strategies for assessing SCN9A genetic variations will aid in organizing large-scale investigations and developing targeted therapeutics for disorders linked to these variations.
Collapse
Affiliation(s)
- Sana Waheed
- Faculty of Life Science, Department of Zoology, University of Okara, Okara, Pakistan
| | - Kainat Ramzan
- Faculty of Life Science, Department of Biochemistry, University of Okara, Okara, Pakistan
| | - Sibtain Ahmad
- Faculty of Animal Husbandry, Institute of Animal and Dairy Sciences, University of Agriculture, Faisalabad, Pakistan
| | - Muhammad Saleem Khan
- Faculty of Life Science, Department of Zoology, University of Okara, Okara, Pakistan
| | - Muhammad Wajid
- Faculty of Life Science, Department of Zoology, University of Okara, Okara, Pakistan
| | - Hayat Ullah
- Department of Chemistry, University of Okara, Okara, Pakistan
| | - Ali Umar
- Faculty of Life Science, Department of Zoology, University of Okara, Okara, Pakistan
| | - Rashid Iqbal
- Faculty of Agriculture and Environment, Department of Agronomy, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
| | - Riaz Ullah
- Department of Pharmacognosy College of Pharmacy King Saud University, Riyadh, Saudi Arabia
| | - Ahmed Bari
- Department of Pharmaceutical Chemistry, College of Pharmacy King Saud University, Riyadh, Saudi Arabia
| |
Collapse
|
|
4
|
Bunduc S, Varzaru B, Iacob RA, Sorop A, Manea I, Spiridon A, Chelaru R, Croitoru AE, Becheanu G, Dumbrava M, Dima S, Popescu I, Gheorghe C. Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing: A cohort analysis. World J Gastroenterol 2023; 29:2864-2874. [PMID: 37274073 PMCID: PMC10237110 DOI: 10.3748/wjg.v29.i18.2864] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 03/14/2023] [Accepted: 04/18/2023] [Indexed: 05/11/2023] Open
Abstract
BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer. For genotyping aimed samples current guidelines recommend using core specimens, although based on moderate quality evidence. However, in clinical practice among the endoscopic ultrasound (EUS) guided tissue acquisition methods, fine needle aspiration (FNA) is the most widely performed.
AIM To assess the adequacy for next generation sequencing (NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma (PDAC) samples.
METHODS Between November 2018 and December 2021, 105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center. Either 22 gauge (G) or 19G FNA needles were used. One pass was dedicated to DNA extraction. DNA concentration and purity (A260/280, A260/230) were assessed by spectrophotometry. We assessed the differences in DNA parameters according to needle size and tumor characteristics (size, location) and the adequacy of the extracted DNA for NGS (defined as A260/280 ≥ 1.7, and DNA yield: ≥ 10 ng for amplicon based NGS, ≥ 50 ng for whole exome sequencing [WES], ≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and t-test respectively. Moreover, we compared DNA purity parameters across the different DNA yield categories.
RESULTS Our cohort included 49% male patients, aged 67.02 ± 8.38 years. The 22G needle was used in 71% of the cases. The DNA parameters across our samples varied as follows: DNA yield: 1289 ng (inter quartile range: 534.75-3101), A260/280 = 1.85 (1.79-1.86), A260/230 = 2.2 (1.72-2.36). DNA yield was > 10 ng in all samples and > 100 ng in 93% of them (one sample < 50 ng). There were no significant differences in the concentration and A260/280 between samples by needle size. Needle size was the only independent predictor of A260/230 which was higher in the 22G samples (P = 0.038). NGS adequacy rate was 90% for 19G samples regardless of NGS type, and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS. Samples with DNA yield > 100 ng had significantly higher A260/280 (1.89 ± 0.32 vs 1.34 ± 0.42, P = 0.013). Tumor characteristics were not corelated with the DNA parameters.
CONCLUSION EUS-FNA PDAC samples yield DNA adequate for subsequent NGS. DNA amount was similar between 22G and 19G FNA needles. DNA purity parameters may vary indirectly with needle size.
Collapse
Affiliation(s)
- Stefania Bunduc
- Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest 020021, Romania
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Bianca Varzaru
- Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest 020021, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Razvan Andrei Iacob
- Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest 020021, Romania
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Andrei Sorop
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Ioana Manea
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Andreea Spiridon
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Raluca Chelaru
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Adina Emilia Croitoru
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Gabriel Becheanu
- Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest 020021, Romania
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Mona Dumbrava
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Simona Dima
- Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest 020021, Romania
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Irinel Popescu
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| | - Cristian Gheorghe
- Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest 020021, Romania
- Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest 022328, Romania
- Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest 022328, Romania
| |
Collapse
|
|
5
|
Huang G, Liu D, Wang W, Wu Q, Chen J, Pan X, Shen H, Yan N. High-resolution structures of human Na v1.7 reveal gating modulation through α-π helical transition of S6 IV. Cell Rep 2022; 39:110735. [PMID: 35476982 DOI: 10.1016/j.celrep.2022.110735] [Citation(s) in RCA: 49] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 02/28/2022] [Accepted: 04/02/2022] [Indexed: 11/26/2022] Open
Abstract
Nav1.7 represents a preeminent target for next-generation analgesics for its critical role in pain sensation. Here we report a 2.2-Å resolution cryo-EM structure of wild-type (WT) Nav1.7 complexed with the β1 and β2 subunits that reveals several previously indiscernible cytosolic segments. Reprocessing of the cryo-EM data for our reported structures of Nav1.7(E406K) bound to various toxins identifies two distinct conformations of S6IV, one composed of α helical turns only and the other containing a π helical turn in the middle. The structure of ligand-free Nav1.7(E406K), determined at 3.5-Å resolution, is identical to the WT channel, confirming that binding of Huwentoxin IV or Protoxin II to VSDII allosterically induces the α → π transition of S6IV. The local secondary structural shift leads to contraction of the intracellular gate, closure of the fenestration on the interface of repeats I and IV, and rearrangement of the binding site for the fast inactivation motif.
Collapse
Affiliation(s)
- Gaoxingyu Huang
- Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 18 Shilongshan Road, Hangzhou, Zhejiang Province 310024, China; Institute of Biology, Westlake Institute for Advanced Study, 18 Shilongshan Road, Hangzhou, Zhejiang Province 310024, China
| | - Dongliang Liu
- Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 18 Shilongshan Road, Hangzhou, Zhejiang Province 310024, China; Institute of Biology, Westlake Institute for Advanced Study, 18 Shilongshan Road, Hangzhou, Zhejiang Province 310024, China
| | - Weipeng Wang
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Qiurong Wu
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Jiaofeng Chen
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Xiaojing Pan
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Huaizong Shen
- Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 18 Shilongshan Road, Hangzhou, Zhejiang Province 310024, China; Institute of Biology, Westlake Institute for Advanced Study, 18 Shilongshan Road, Hangzhou, Zhejiang Province 310024, China.
| | - Nieng Yan
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
| |
Collapse
|
|
6
|
Kowalska M, Nowaczyk J, Nowaczyk A. K V11.1, Na V1.5, and Ca V1.2 Transporter Proteins as Antitarget for Drug Cardiotoxicity. Int J Mol Sci 2020; 21:E8099. [PMID: 33143033 PMCID: PMC7663169 DOI: 10.3390/ijms21218099] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Revised: 10/24/2020] [Accepted: 10/27/2020] [Indexed: 02/07/2023] Open
Abstract
Safety assessment of pharmaceuticals is a rapidly developing area of pharmacy and medicine. The new advanced guidelines for testing the toxicity of compounds require specialized tools that provide information on the tested drug in a quick and reliable way. Ion channels represent the third-largest target. As mentioned in the literature, ion channels are an indispensable part of the heart's work. In this paper the most important information concerning the guidelines for cardiotoxicity testing and the way the tests are conducted has been collected. Attention has been focused on the role of selected ion channels in this process.
Collapse
Affiliation(s)
- Magdalena Kowalska
- Department of Organic Chemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, 85-094 Bydgoszcz, Poland;
| | - Jacek Nowaczyk
- Faculty of Chemistry, Nicolaus Copernicus University, 87-100 Toruń, Poland;
| | - Alicja Nowaczyk
- Department of Organic Chemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, 85-094 Bydgoszcz, Poland;
| |
Collapse
|
|
7
|
Shen H, Li Z, Jiang Y, Pan X, Wu J, Cristofori-Armstrong B, Smith JJ, Chin YKY, Lei J, Zhou Q, King GF, Yan N. Structural basis for the modulation of voltage-gated sodium channels by animal toxins. Science 2018; 362:science.aau2596. [DOI: 10.1126/science.aau2596] [Citation(s) in RCA: 150] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Accepted: 07/17/2018] [Indexed: 12/31/2022]
Abstract
Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories—pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Navchannel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDIIand the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Navchannel drugs.
Collapse
|
|
8
|
Yan Z, Zhou Q, Wang L, Wu J, Zhao Y, Huang G, Peng W, Shen H, Lei J, Yan N. Structure of the Na v1.4-β1 Complex from Electric Eel. Cell 2017; 170:470-482.e11. [PMID: 28735751 DOI: 10.1016/j.cell.2017.06.039] [Citation(s) in RCA: 245] [Impact Index Per Article: 30.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Revised: 05/26/2017] [Accepted: 06/23/2017] [Indexed: 02/07/2023]
Abstract
Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Nav channel from electric eel, in complex with the β1 subunit at 4.0 Å resolution. The immunoglobulin domain of β1 docks onto the extracellular L5I and L6IV loops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit "up" conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.
Collapse
Affiliation(s)
- Zhen Yan
- State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Qiang Zhou
- State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Lin Wang
- State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Jianping Wu
- State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Yanyu Zhao
- Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Gaoxingyu Huang
- State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Wei Peng
- Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Huaizong Shen
- Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Jianlin Lei
- Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China; Technology Center for Protein Sciences, Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China
| | - Nieng Yan
- State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China; Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
| |
Collapse
|
|
9
|
Goldschen-Ohm MP, Chanda B. Probing gating mechanisms of sodium channels using pore blockers. Handb Exp Pharmacol 2014; 221:183-201. [PMID: 24737237 DOI: 10.1007/978-3-642-41588-3_9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Several classes of small molecules and peptides bind at the central pore of voltage-gated sodium channels either from the extracellular or intracellular side of the membrane and block ion conduction through the pore. Biophysical studies that shed light on the chemical nature, accessibility, and kinetics of binding of these naturally occurring and synthetic compounds reveal a wealth of information about how these channels gate. Here, we discuss insights into the structural underpinnings of gating of the channel pore and its coupling to the voltage sensors obtained from pore blockers including site 1 neurotoxins and local anesthetics.
Collapse
|
|
10
|
Huang CJ, Schild L, Moczydlowski EG. Use-dependent block of the voltage-gated Na(+) channel by tetrodotoxin and saxitoxin: effect of pore mutations that change ionic selectivity. ACTA ACUST UNITED AC 2013; 140:435-54. [PMID: 23008436 PMCID: PMC3457692 DOI: 10.1085/jgp.201210853] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.
Collapse
|
|
11
|
Human ScFv that block sodium ion channel activity of tetrodotoxin. Toxicon 2012; 59:272-82. [DOI: 10.1016/j.toxicon.2011.11.012] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2011] [Revised: 11/11/2011] [Accepted: 11/15/2011] [Indexed: 12/31/2022]
|
|
12
|
The tetrodotoxin binding site is within the outer vestibule of the sodium channel. Mar Drugs 2010; 8:219-34. [PMID: 20390102 PMCID: PMC2852835 DOI: 10.3390/md8020219] [Citation(s) in RCA: 94] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2009] [Revised: 01/10/2010] [Accepted: 01/28/2010] [Indexed: 12/21/2022] Open
Abstract
Tetrodotoxin and saxitoxin are small, compact asymmetrical marine toxins that block voltage-gated Na channels with high affinity and specificity. They enter the channel pore’s outer vestibule and bind to multiple residues that control permeation. Radiolabeled toxins were key contributors to channel protein purification and subsequent cloning. They also helped identify critical structural elements called P loops. Spacial organization of their mutation-identified interaction sites in molecular models has generated a molecular image of the TTX binding site in the outer vestibule and the critical permeation and selectivity features of this region. One site in the channel’s domain I P loop determines affinity differences in mammalian isoforms.
Collapse
|
|
13
|
Kaufmann SG, Westenbroek RE, Zechner C, Maass AH, Bischoff S, Muck J, Wischmeyer E, Scheuer T, Maier SKG. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes. J Mol Cell Cardiol 2009; 48:261-9. [PMID: 19426735 DOI: 10.1016/j.yjmcc.2009.04.017] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2009] [Revised: 04/14/2009] [Accepted: 04/29/2009] [Indexed: 11/29/2022]
Abstract
Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.
Collapse
Affiliation(s)
- Susann G Kaufmann
- Medizinische Klinik und Poliklinik I, Universität Würzburg, Josef-Schneider-Strabetae 2, 97080 Würzburg, Germany
| | | | | | | | | | | | | | | | | |
Collapse
|
|
14
|
Scornik FS, Desai M, Brugada R, Guerchicoff A, Pollevick GD, Antzelevitch C, Pérez GJ. Functional expression of "cardiac-type" Nav1.5 sodium channel in canine intracardiac ganglia. Heart Rhythm 2006; 3:842-50. [PMID: 16818219 PMCID: PMC1989775 DOI: 10.1016/j.hrthm.2006.03.021] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/15/2006] [Accepted: 03/14/2006] [Indexed: 10/24/2022]
Abstract
BACKGROUND The autonomic nervous system has been implicated in several arrhythmogenic diseases, including long QT syndrome type 3 (LQT3) and Brugada syndrome. Scarce information on the cellular components of the intrinsic cardiac ganglia from higher mammals has limited our understanding of the role of the autonomic nervous system in such diseases. OBJECTIVES The purpose of this study was to isolate and characterize the electrophysiologic properties of canine intracardiac neurons. METHODS Action potentials (APs) and ionic currents were studied in enzymatically dissociated canine intracardiac neurons under current and voltage clamp conditions. Immunohistochemical and reverse transcription-polymerase chain reaction analysis was performed using freshly isolated intracardiac ganglia. RESULTS APs recorded from intracardiac neurons displayed a tetrodotoxin-resistant (TTX-R) component. TTX-R APs were abolished in the absence of sodium but persisted in the absence of external calcium. Immunohistochemical studies showed the presence of TTX-R sodium channels in these ganglia. Sodium currents were characterized by two components with different affinities for TTX: a tetrodotoxin-sensitive (TTX-S) component and a TTX-R component. TTX-S current inactivation was characteristic of neuronal sodium currents, whereas TTX-R current inactivation time constants were similar to those previously reported for Na(v)1.5 channels. TTX sensitivity (IC(50) = 1.17 microM) of the TTX-R component was in the range reported for Na(v)1.5 channels. Expression of Na(v)1.5 channels in intracardiac ganglia was confirmed by PCR analysis and sequencing. CONCLUSION Our results suggest that canine intracardiac neurons functionally express Na(v)1.5 channels. These findings open an exciting new door to our understanding of autonomically modulated arrhythmogenic diseases linked to mutations in Na(v)1.5 channels, including Brugada syndrome and LQT3.
Collapse
|
|
15
|
Brette F, Orchard CH. No Apparent Requirement for Neuronal Sodium Channels in Excitation-Contraction Coupling in Rat Ventricular Myocytes. Circ Res 2006; 98:667-74. [PMID: 16484618 DOI: 10.1161/01.res.0000209963.02720.70] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The majority of Na channels in the heart are composed of the tetrodotoxin (TTX)-resistant (K
D
, 2 to 6 μmol/L) “cardiac” Na
V
1.5 isoform; however, TTX-sensitive (K
D
, 1 to 25 nmol/L) “neuronal” Na channel isoforms have recently been detected in several cardiac preparations. In the present study, we determined the functional subcellular localization of Na channel isoforms (according to their TTX sensitivity) in rat ventricular myocytes by recording
I
Na
in control and detubulated myocytes. We found that TTX-sensitive
I
Na
(K
D
, &8.8 nmol/L) makes up 14±3% of total
I
Na
in control and ≤4% in detubulated myocytes and calculated that &80% of TTX-sensitive
I
Na
is located in the t-tubules, where it generates &1/3 of t-tubular
I
Na
. In contrast, TTX-resistant
I
Na
is located predominantly (&78%) at the surface membrane. We also investigated the possible contribution of TTX-sensitive
I
Na
to excitation-contraction coupling, using 200 nmol/L TTX to selectively block TTX-sensitive
I
Na
. TTX decreased the rate of depolarization of the action potential by 10% but did not delay the rise of systolic Ca
2+
in the center of the cell (transverse confocal line scan), suggesting that TTX-sensitive
I
Na
does not play a role in synchronizing Ca
2+
release at the t-tubules; the amplitude of the Ca
2+
transient and contraction were also unchanged by 200 nmol/L TTX. The quantity of charge entering via
I
Ca
elicited by control or TTX action potential waveforms was similar, suggesting that the trigger for Ca
2+
release is not altered by blocking TTX-sensitive
I
Na
. We conclude that neuronal
I
Na
is concentrated at the t-tubules, but there is no evidence of a requirement for these channels in normal excitation-contraction coupling in ventricular myocytes.
Collapse
Affiliation(s)
- Fabien Brette
- Department of Physiology, University of Bristol, United Kingdom.
| | | |
Collapse
|
|
16
|
French RJ, Zamponi GW. Voltage-gated sodium and calcium channels in nerve, muscle, and heart. IEEE Trans Nanobioscience 2005; 4:58-69. [PMID: 15816172 DOI: 10.1109/tnb.2004.842500] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Voltage-gated ion channels are membrane proteins which underlie rapid electrical signals among neurons and the spread of excitation in skeletal muscle and heart. We outline some recent advances in the study of voltage-sensitive sodium and calcium channels. Investigations are providing insight into the changes in molecular conformation associated with open-closed gating of the channels, the mechanisms by which they allow only specific ion species to pass through and carry an electric current, and the pathological consequences of small perturbations in channel structure which result from genetic mutations. Determination of three-dimensional structures, coupled with molecular manipulations by site-directed mutagenesis, and parallel electrophysiological analyses of currents through the ion channels, are providing an understanding of the roles and function of these channels at an unprecedented level of molecular detail. Crucial to these advances are studies of bacterial homologues of ion channels from man and other eukaryotes, and the use of naturally occurring peptide toxins which target different ion channel types with exquisite specificity.
Collapse
Affiliation(s)
- Robert J French
- Department of Physiology and Biophysics, University of Calgary, Calgary, AB T2N 4N1, Canada.
| | | |
Collapse
|
|
17
|
Pauron D, Barhanin J, Amichot M, Pralavorio M, Berge JB, Lazdunski M. Pyrethroid receptor in the insect sodium channel: alteration of its properties in pyrethroid-resistant flies. Biochemistry 2002. [DOI: 10.1021/bi00430a037] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
|
|
18
|
Maier SKG, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T, Catterall WA. An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc Natl Acad Sci U S A 2002; 99:4073-8. [PMID: 11891345 PMCID: PMC122650 DOI: 10.1073/pnas.261705699] [Citation(s) in RCA: 210] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Voltage-gated sodium channels composed of pore-forming alpha and auxiliary beta subunits are responsible for the rising phase of the action potential in cardiac muscle, but the functional roles of distinct sodium channel subtypes have not been clearly defined. Immunocytochemical studies show that the principal cardiac pore-forming alpha subunit isoform Na(v)1.5 is preferentially localized in intercalated disks, whereas the brain alpha subunit isoforms Na(v)1.1, Na(v)1.3, and Na(v)1.6 are localized in the transverse tubules. Sodium currents due to the highly tetrodotoxin (TTX)-sensitive brain isoforms in the transverse tubules are small and are detectable only after activation with beta scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface membrane to contraction, because low TTX concentrations reduce left ventricular function. Our results suggest that the principal cardiac isoform in the intercalated disks is primarily responsible for action potential conduction between cells and reveal an unexpected role for brain sodium channel isoforms in the transverse tubules in coupling electrical excitation to contraction in cardiac muscle.
Collapse
Affiliation(s)
- Sebastian K G Maier
- Department of Pharmacology, University of Washington, Seattle, WA 98195, USA
| | | | | | | | | | | |
Collapse
|
|
19
|
Doucette GJ, Powell CL, Do EU, Byon CY, Cleves F, McClain SG. Evaluation of 11-[3H]-tetrodotoxin use in a heterologous receptor binding assay for PSP toxins. Toxicon 2000; 38:1465-74. [PMID: 10775748 DOI: 10.1016/s0041-0101(99)00240-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant(K(d))=4.77+/-1.54nM; maximum binding(B(max))=1. 62+/-0.24pmol/mg of synaptosomal protein). The inhibition constant (K(i)) for the assay was 1.46+/-0.28 nM STX equiv. (n=6), with an estimated detection limit of ca. 2-4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [3H]-STX as the radioligand.
Collapse
Affiliation(s)
- G J Doucette
- Marine Biotoxins Program, NOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412, USA.
| | | | | | | | | | | |
Collapse
|
|
20
|
Rosso JP, Vargas-Rosso O, Gutiérrez JM, Rochat H, Bougis PE. Characterization of alpha-neurotoxin and phospholipase A2 activities from Micrurus venoms. Determination of the amino acid sequence and receptor-binding ability of the major alpha-neurotoxin from Micrurus nigrocinctus nigrocinctus. EUROPEAN JOURNAL OF BIOCHEMISTRY 1996; 238:231-9. [PMID: 8665942 DOI: 10.1111/j.1432-1033.1996.0231q.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse-phase high-performance liquid chromatography and specific assays for alpha-neurotoxin and phospholipase A2 activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A2 activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven alpha-neurotoxins, five phospholipases A2 and four toxin homologs. The amino acid sequence of the major alpha-neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short-chain alpha-neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.
Collapse
Affiliation(s)
- J P Rosso
- Laboratoire de Biochimie, Unité de Recherche Associée 1455 du Centre National de la Recherche Scientifique, Institut Fédératif Jean Roche, Université de la Méditérranée, Faculté de Médecine Secteur Nord, Marseille, France
| | | | | | | | | |
Collapse
|
|
21
|
Strichartz GR, Hall S, Magnani B, Hong CY, Kishi Y, Debin JA. The potencies of synthetic analogues of saxitoxin and the absolute stereoselectivity of decarbamoyl saxitoxin. Toxicon 1995; 33:723-37. [PMID: 7676464 DOI: 10.1016/0041-0101(95)00031-g] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The potencies of synthetic saxitoxin (+/- STX) and six of its synthetic analogues, including the enantioselectively synthesized unnatural (-)enantiomer of decarbamoyl saxitoxin (dcSTX), were measured and compared to those of natural saxitoxin [(+)STX]. The analogues, all of which were racemic (+/-) mixtures except for dcSTX, varied in the substituents at the C6 position, the carbamoyl 'moeity', and the C12 position, the hydrated ketone. The ability of the toxins to inhibit the compound action potential (AP) and to displace radiolabeled natural saxitoxin (3H-STX) from nerve membranes at equilibrium were both used as potency assays. Biological activity of both (+)- and (-)dcSTX was analyzed by the kinetics of block of single Na+ channels reconstituted in planar lipid bilayer membranes, where it was demonstrated that only (+)dcSTX had biological activity. The potency of STX analogues fell markedly as the substituent at the C6 position became smaller; Ki values from the binding competition assay (at 4 degrees C) are: (+/-)6-methanolic-STX, 5 x 10(-10) M; (+/-)6-methyl-STX, 1 x 10(-6) M; (+/-)6-dihydro-STX, 3.5 x 10(-5) M. Replacement of the ketone at the C12 position by a methylene group was accomplished in two derivatives, although both also had substituents at the C6 position. The compound (+/-)6-methyl-12-deoxy-STX was about 0.03 as potent as (+/-)6-methyl-STX and only 10(-5) as potent as racemic (+/-)STX. In synthetic compounds where the benzyloxymethyl (-CH2OCH2C6H5) substituent occurred at the C6 position, the C12-methylene derivative still displayed some binding activity (Ki = 6 x 10(-4) M). However, when the same C6 derivatized compounds also contained a 6-membered heterocyclic group (-C3H8S2-) conjugated to carbon 12, the measured binding affinity was even further decreased (Ki = 2 x 10(-3) M). The findings show that substitutions on the carbon 6 position of STX have stronger effects on STX potency than previously believed, and that the toxin may form a hydrogen bond with the sodium channel at this site. Furthermore, the total removal of oxygen from the C12 position does not completely abolish the binding activity of the molecule.
Collapse
Affiliation(s)
- G R Strichartz
- Anesthesia Research Laboratories, Brigham and Women's Hospital, Boston, MA, USA
| | | | | | | | | | | |
Collapse
|
|
22
|
Llewellyn LE, Moczydlowski EG. Characterization of saxitoxin binding to saxiphilin, a relative of the transferrin family that displays pH-dependent ligand binding. Biochemistry 1994; 33:12312-22. [PMID: 7918453 DOI: 10.1021/bi00206a039] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Saxiphilin is a 91 kDa saxitoxin-binding protein that is homologous to members of the transferrin family of Fe(3+)-binding proteins noted for pH-dependent release of Fe3+. The mechanism of toxin binding to purified native saxiphilin from the bullfrog (Rana catesbeiana) was studied using [3H]saxitoxin. At pH 7.4 and 0 degrees C [3H]saxitoxin binds to a single site on saxiphilin with a KD of approximately 0.2 nM. The pH dependence of [3H]saxitoxin binding follows a one-site titration curve in the range of pH 9-4 with maximal binding from pH 9 to 7 and half-inhibition at pH 5.7. Inhibition of toxin binding at low pH is the combined result of a decrease in the rate of toxin association and an increase in the rate of toxin dissociation. The dependence of the apparent rate constants for [3H]saxitoxin association and dissociation on [H+] can be accounted for by a four-state model of allosteric interaction between the toxin-binding site and a single titratable residue of saxiphilin with a pKa of 7.2 in the toxin-free form and 4.3 in the toxin-bound form. From 0 to 25 degrees C, the temperature dependence of [3H]saxitoxin binding to saxiphilin is characterized by delta H degrees = -8.3 kcal mol-1, delta S degrees = 13.8 cal mol-1 K-1, and activation energies of 22.5 kcal mol-1 for dissociation and 11.1 kcal mol-1 for association. Binding of [3H]saxitoxin to saxiphilin is competitively inhibited with low affinity by a variety of divalent metal and lanthanide cations. Inhibition of toxin binding by the carboxyl-methylating reagent trimethyloxonium is prevented by pre-equilibration with [3H]saxitoxin, implicating the presence of one or more carboxyl groups in the binding site. Functional similarities suggest that the saxitoxin-binding site of saxiphilin is located in an interdomain cleft analogous to the location of one of the two homologous Fe(3+)-binding sites of transferrins. On the basis of residue substitutions between saxiphilin and transferrins, it is proposed that the saxitoxin-binding site is located in the carboxy terminal lobe of saxiphilin and that binding is modulated by protonation of a conserved histidine residue.
Collapse
Affiliation(s)
- L E Llewellyn
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066
| | | |
Collapse
|
|
23
|
Kallen RG, Cohen SA, Barchi RL. Structure, function and expression of voltage-dependent sodium channels. Mol Neurobiol 1993; 7:383-428. [PMID: 8179845 DOI: 10.1007/bf02769184] [Citation(s) in RCA: 56] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Voltage-dependent sodium channels control the transient inward current responsible for the action potential in most excitable cells. Members of this multigene family have been cloned, sequenced, and functionally expressed from various tissues and species, and common features of their structure have clearly emerged. Site-directed mutagenesis coupled with in vitro expression has provided additional insight into the relationship between structure and function. Subtle differences between sodium channel isoforms are also important, and aspects of the regulation of sodium channel gene expression and the modulation of channel function are becoming topics of increasing importance. Finally, sodium channel mutations have been directly linked to human disease, yielding insight into both disease pathophysiology and normal channel function. After a brief discussion of previous work, this review will focus on recent advances in each of these areas.
Collapse
Affiliation(s)
- R G Kallen
- Mahoney Institute of Neurological Sciences, University of Pennsylvania School of Medicine, Philadelphia
| | | | | |
Collapse
|
|
24
|
Dudley SC, Baumgarten CM. Modification of cardiac sodium channels by carboxyl reagents. Trimethyloxonium and water-soluble carbodiimide. J Gen Physiol 1993; 101:651-71. [PMID: 8393064 PMCID: PMC2216784 DOI: 10.1085/jgp.101.5.651] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.
Collapse
Affiliation(s)
- S C Dudley
- Department of Physiology, Medical College of Virginia, Richmond 23298-0551
| | | |
Collapse
|
|
25
|
Doyle DD, Guo Y, Lustig SL, Satin J, Rogart RB, Fozzard HA. Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction. J Gen Physiol 1993; 101:153-82. [PMID: 8384241 PMCID: PMC2216764 DOI: 10.1085/jgp.101.2.153] [Citation(s) in RCA: 51] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)
Collapse
Affiliation(s)
- D D Doyle
- University of Chicago, Department of Medicine, Illinois 60637
| | | | | | | | | | | |
Collapse
|
|
26
|
Satin J, Kyle JW, Chen M, Bell P, Cribbs LL, Fozzard HA, Rogart RB. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 1992; 256:1202-5. [PMID: 1375397 DOI: 10.1126/science.256.5060.1202] [Citation(s) in RCA: 284] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.
Collapse
Affiliation(s)
- J Satin
- Department of Medicine, University of Chicago, IL 60637
| | | | | | | | | | | | | |
Collapse
|
|
27
|
Hansen G, Ulbricht W. Influence of Na+ and Li+ ions on the kinetics of sodium channel block by tetrodotoxin and saxitoxin. Pflugers Arch 1991; 419:588-95. [PMID: 1664935 DOI: 10.1007/bf00370300] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Voltage clamp experiments were done on single myelinated frog nerve fibres. The rate of block of Na+ channels by tetrodotoxin (TTX) was obtained from changes in peak Na+ current during 1-Hz trains of depolarizing impulses. In hypertonic Na(+)-rich solution (216 mM) the stationary block was reduced compared with Na(+)-poor solutions (54 mM or less; tetramethylammonium ions substituting for Na+). Washout in 216 mM Na+ was faster than in 54 mM Na+. Concentration of Na+ [( Na+]) little affected onset of block. After equilibration in Na(+)-poor TTX solution, a sudden application of Na(+)-rich toxin solution led to a partial relief from block that proceeded faster than the onset in the latter solution. Comparable results were obtained with saxitoxin (STX) and in analogous Li+ solutions. Most of the observed phenomena could be quantitatively fitted by a cyclic model in which cations favour the transition of channels (unblocked and blocked) from a high- to a low-affinity state from which toxin dissociates faster.
Collapse
Affiliation(s)
- G Hansen
- Physiologisches Institut, Universität Kiel, Federal Republic of Germany
| | | |
Collapse
|
|
28
|
Terlau H, Heinemann SH, Stühmer W, Pusch M, Conti F, Imoto K, Numa S. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett 1991; 293:93-6. [PMID: 1660007 DOI: 10.1016/0014-5793(91)81159-6] [Citation(s) in RCA: 346] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA-derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single-channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single-channel conductance.
Collapse
Affiliation(s)
- H Terlau
- Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany
| | | | | | | | | | | | | |
Collapse
|
|
29
|
Patton DE, Goldin AL. A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes. Neuron 1991; 7:637-47. [PMID: 1657057 DOI: 10.1016/0896-6273(91)90376-b] [Citation(s) in RCA: 61] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
We have utilized molecular biological techniques to demonstrate that rat IIA sodium channels expressed in Xenopus oocytes were blocked by tetrodotoxin (TTX) in a use-dependent manner. This use dependence was the result of an increased affinity of the channels for TTX upon depolarization, most likely due to a conformational change in the channel. Using a mutant with a slower macroscopic rate of inactivation, we have demonstrated that this conformational change is not the transition into the fast-inactivated state. The transition is probably one occurring during activation of the channel, as suggested by the fact that one sodium channel mutant demonstrated comparable depolarizing shifts in the voltage dependence of both activation and use-dependent block by TTX. The transition occurred at potentials more negative than those resulting in channel conductance, suggesting that the conformational change that causes use-dependent block by TTX is a closed-state voltage-dependent gating transition.
Collapse
Affiliation(s)
- D E Patton
- Department of Microbiology and Molecular Genetics, University of California, Irvine 92717
| | | |
Collapse
|
|
30
|
Schild L, Moczydlowski E. Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site. Biophys J 1991; 59:523-37. [PMID: 1646656 PMCID: PMC1281218 DOI: 10.1016/s0006-3495(91)82269-x] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.
Collapse
Affiliation(s)
- L Schild
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
| | | |
Collapse
|
|
31
|
Lönnendonker U, Neumcke B, Stämpfli R. Interaction of monovalent cations with tetrodotoxin and saxitoxin binding at sodium channels of frog myelinated nerve. Pflugers Arch 1990; 416:750-7. [PMID: 2174148 DOI: 10.1007/bf00370625] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Na currents and Na-current fluctuations were measured in myelinated frog nerve fibres to study interactions between monovalent externally applied cations and the binding of the Na-channel blockers tetrodotoxin (TTX) or saxitoxin (STX). Adding 110 mM NaCl to Ringer's solution increased the maximum peak Na conductance by a factor of 2.51 in the presence of 12 nM TTX and by a factor of 2.43 in the presence of 4 nM STX. According to the analysis of Na-current fluctuations this increase of the Na conductance is mainly caused by an increase of the number N of unblocked Na channels per node, while the conductance of a single channel saturates in the hyperosmolar solutions. The increase of N is interpreted by displacement of TTX or STX from Na channels by external Na+. Relief of TTX blockage was also observed by adding 110 mM chloride salts of Li+, hydrazine+, guanidine+ and K+ to Ringer, but not in Ringer + 110 mM tetramethylammonium chloride or 250 mM sucrose. The increase of N by the external cations is a saturating function of the permeability of the Na channel to these ions. The results are interpreted by a toxin receptor in a superficial prefilter to the Na channel, which contributes to cation discrimination at the outer channel region.
Collapse
Affiliation(s)
- U Lönnendonker
- I. Physiologisches Institut der Universität des Saarlandes, Hamburg, Federal Republic of Germany
| | | | | |
Collapse
|
|
32
|
Levinson SR, Thornhill WB, Duch DS, Recio-Pinto E, Urban BW. The role of nonprotein domains in the function and synthesis of voltage-gated sodium channels. ION CHANNELS 1990; 2:33-64. [PMID: 1966381 DOI: 10.1007/978-1-4615-7305-0_2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Affiliation(s)
- S R Levinson
- Department of Physiology, University of Colorado Medical School, Denver 80262
| | | | | | | | | |
Collapse
|
|
33
|
Noda M, Suzuki H, Numa S, Stühmer W. A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. FEBS Lett 1989; 259:213-6. [PMID: 2557243 DOI: 10.1016/0014-5793(89)81531-5] [Citation(s) in RCA: 248] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage-clamp in Xenopus oocytes injected with the corresponding cDNA-derived mRNA.
Collapse
Affiliation(s)
- M Noda
- Department of Medical Chemistry, Kyoto University Faculty of Medicine, Japan
| | | | | | | |
Collapse
|
|
34
|
Emerick MC, Agnew WS. Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus. Biochemistry 1989; 28:8367-80. [PMID: 2557902 DOI: 10.1021/bi00447a016] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.
Collapse
Affiliation(s)
- M C Emerick
- Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
| | | |
Collapse
|
|
35
|
Ravindran A, Moczydlowski E. Influence of negative surface charge on toxin binding to canine heart Na channels in planar bilayers. Biophys J 1989; 55:359-65. [PMID: 2540849 PMCID: PMC1330479 DOI: 10.1016/s0006-3495(89)82813-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The presence of negative surface charge near the tetrodotoxin/saxitoxin binding site of canine heart Na channels was revealed by analysis of the kinetics of toxin block of single batrachotoxin-activated Na channels in planar bilayers as a function of [NaCl]. The voltage-dependence of toxin binding and the toxin dissociation rate are nearly constant as [NaCl] is varied from 0.05 to 3 M. In contrast, the association rate constant of the toxins is inversely dependent on [NaCl], with the rate for the divalent toxin, saxitoxin2+, affected more steeply than that of the monovalent toxin, tetrodotoxin1+. These results for toxin-insensitive Na channels from canine heart parallel previous findings for toxin-sensitive Na channels from canine brain. The model of Green et al. (Green, W. N., L. B. Weiss, and O. S. Anderson. 1987. J. Gen. Physiol. 89:873-903), which includes Na+ competition and Gouy-Chapman screening of surface charge, provided an excellent fit to the data. The results suggest that the two canine Na channel subtypes have a similar density of negative surface charge (1 e-/400 A2) and a similar dissociation constant for Na+ competition (0.5 M) at the toxin binding site. Thus, negative surface charge is a conserved feature of channel function of these two subtypes. The difference in toxin binding affinities arises from small differences in intrinsic association and dissociation rates.
Collapse
Affiliation(s)
- A Ravindran
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
| | | |
Collapse
|
|
36
|
Marchot P, Frachon P, Bougis PE. Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration. EUROPEAN JOURNAL OF BIOCHEMISTRY 1988; 174:537-42. [PMID: 3391171 DOI: 10.1111/j.1432-1033.1988.tb14132.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.
Collapse
Affiliation(s)
- P Marchot
- Laboratoire de Biochimie, Faculté de Médecine, Université d'Aix-Marseille, France
| | | | | |
Collapse
|
|
37
|
Ritchie JM. Sodium-channel turnover in rabbit cultured Schwann cells. PROCEEDINGS OF THE ROYAL SOCIETY OF LONDON. SERIES B, BIOLOGICAL SCIENCES 1988; 233:423-30. [PMID: 2456584 DOI: 10.1098/rspb.1988.0031] [Citation(s) in RCA: 31] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Radiolabelled saxitoxin has been used as a chemical marker for the voltage-dependent sodium channels expressed in the plasmalemma of rabbit Schwann cells in culture. Proteolytic enzymes destroy this saxitoxin-binding capacity, which gradually reappears with an exponential time constant of about 3.1 days. Exposure of cultured Schwann cells to tunicamycin, an inhibitor of glycosylation, leads to a progressive exponential fall in saxitoxin-binding capacity, again with a time constant of about 3.1 days. The assumption that the steady-state density of Schwann cell sodium channels is maintained by a constant synthesis of channels in the face of a rate of loss from the membrane proportional to the amount of channel already present, leads to the conclusion that these channels have an average lifetime of about 3.1 days. The metabolic consequences of this rapid turnover of Schwann cell sodium channels is discussed.
Collapse
Affiliation(s)
- J M Ritchie
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510-8066
| |
Collapse
|
|
38
|
Rack M. Periodate treatment reduces the tetrodotoxin-sensitivity of voltage-gated Na+ channels. BIOCHIMICA ET BIOPHYSICA ACTA 1988; 939:47-51. [PMID: 2450584 DOI: 10.1016/0005-2736(88)90045-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with periodate in the extracellular solution. (2) Periodate treatment irreversibly reduced the effect of tetrodotoxin (TTX) on the Na+ currents. (3) The effect of saxitoxin (STX) was also reduced but less than that of TTX. (4) The presence of STX during the application of periodate to the nerve fibre almost completely prevented the effect of the chemical reagent on the TTX sensitivity of the Na+ channels. (5) The reduction of the TTX effect is not due to the reaction of small amounts of periodate with the diol group of this toxin, because the effect was seen after prolonged washing with reagent-free Ringer solution with or without high amounts of ribose. (6) Carboxyl groups present in the Na+ channel seem to be quite important for the binding of TTX and STX. Periodate modifies several amino acid side chains, however, it does not attack carboxyl groups in a peptide chain. Thus, these results suggest that periodate modifies a further group critically involved in the binding of TTX and STX.
Collapse
Affiliation(s)
- M Rack
- Institut für Physiologische Chemie, Universität des Saarlandes, Homburg/Saar F.R.G
| |
Collapse
|
|
39
|
Chapter 16 Sodium Channels in Lipid Bilayers: Have We Learned Anything Yet? ACTA ACUST UNITED AC 1988. [DOI: 10.1016/s0070-2161(08)60906-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
|
|
40
|
Sorokina ZA, Chizhmakov IV. Mechanism underlying the action of blocking toxins on voltage-operated sodium channels: Recent advances. NEUROPHYSIOLOGY+ 1988. [DOI: 10.1007/bf01058052] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
|
|
41
|
Green WN, Weiss LB, Andersen OS. Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures. J Gen Physiol 1987; 89:873-903. [PMID: 2440978 PMCID: PMC2215969 DOI: 10.1085/jgp.89.6.873] [Citation(s) in RCA: 58] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.
Collapse
|
|
42
|
Barnola FV, Sevcik C, Delgado D. Isolation of membrane fractions with sodium channels sensitive to veratridine and tetrodotoxin from the electric organ of the eel Electrophorus electricus. J Neurochem 1987; 48:846-52. [PMID: 2433399 DOI: 10.1111/j.1471-4159.1987.tb05594.x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The veratridine/tetrodotoxin-sensitive sodium influx was measured in membrane fractions isolated from the electric organ of Electrophorus electricus. The fractions were characterized, and the main biochemical markers and their acetylcholine receptor content were determined. The innervated and noninnervated faces of the electroplax were separated. The different biochemical criteria used indicate that the pre- and postsynaptic membranes of the innervated face were isolated. Sodium influx increased by veratridine and blocked by tetrodotoxin was found in fractions from the presynaptic membrane. Because some of the vesicles in this fraction are in the inside-out conformation, tetrodotoxin had to be applied to both faces of the vesicles so that sodium influx was blocked completely. The fractions from the innervated face of the electroplax contained sodium channels with sensitivities to tetrodotoxin and veratridine similar to those of fractions from other nerve membrane preparations.
Collapse
|
|
43
|
Duch DS, Levinson SR. Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles. J Membr Biol 1987; 98:43-55. [PMID: 2444706 DOI: 10.1007/bf01871044] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Using the dialysable detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), the tetrodotoxin-binding protein from the electroplax of the electric eel has been purified to a high degree of both chemical homogeneity and toxin-binding activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the best preparations showed only a single microheterogeneous band at Mr approximately 260,000, despite attempts to visualize smaller bands by sample overloading. Upon dialysis, this material became incorporated into the membranes of small unilamellar vesicles, and in this form the purified protein exhibited tetrodotoxin-binding properties similar to the component in the original electroplax membrane. Furthermore, in the presence of activator neurotoxins the vesicles were able to accumulate isotopic sodium in a manner similar to that previously described for less active or less pure preparations of vesicles containing either mammalian or eel electroplax toxin-binding proteins. Quantitative consideration of the isotopic transport activity of this pure material, along with the high degree of purity of the protein, strongly suggests that the 260-kDa glycopeptide from electroplax is necessary and sufficient to account for the sodium channel function seen in these studies, and eliminates the possible involvement of smaller peptides in the channel phenomena observed.
Collapse
Affiliation(s)
- D S Duch
- Department of Physiology, University of Colorado Medical School, Denver 80262
| | | |
Collapse
|
|
44
|
Krueger BK, Worley JF, French RJ. Block of sodium channels in planar lipid bilayers by guanidium toxins and calcium. Are the mechanisms of voltage dependence the same? Ann N Y Acad Sci 1986; 479:257-68. [PMID: 2433995 DOI: 10.1111/j.1749-6632.1986.tb15574.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The block of single, batrachotoxin-activated sodium channels by saxitoxin (STX), tetrodotoxin (TTX), and Ca2+ has been investigated in planar bilayers. All three substances block in a voltage-dependent manner with hyperpolarizing potentials favoring block. Extracellular Ca2+ competitively inhibits binding of STX and relieves STX block. Trimethyloxonium, a carboxyl-methylating agent, eliminates block by STX and TTX and dramatically reduces block by Ca2+. These results suggest that STX, TTX, and Ca2+ compete for a negative site on the outside of the channel. The voltage dependence of block by STX (divalent cation) and TTX (monovalent) was similar (40 mV/e-fold), suggesting that voltage dependence is due to a conformational change in the channel rather than to the toxins entering the membrane electric field to block. A physical model, with an external binding site for toxins and Ca2+ and another site deeper within the electric field (associated with the "selectivity filter") that is accessible to Ca2+ but not toxins, predicts voltage-dependence Ca2+ block without invoking the conformational change needed to explain the voltage dependence of block by TTX and STX.
Collapse
|
|
45
|
Salgado VL, Yeh JZ, Narahashi T. Use- and voltage-dependent block of the sodium channel by saxitoxin. Ann N Y Acad Sci 1986; 479:84-95. [PMID: 2434010 DOI: 10.1111/j.1749-6632.1986.tb15563.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
|
|
46
|
|
|
47
|
Strichartz G, Rando T, Hall S, Gitschier J, Hall L, Magnani B, Bay CH. On the mechanism by which saxitoxin binds to and blocks sodium channels. Ann N Y Acad Sci 1986; 479:96-112. [PMID: 2434011 DOI: 10.1111/j.1749-6632.1986.tb15564.x] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
|
|
48
|
Bougis PE, Marchot P, Rochat H. Characterization of elapidae snake venom components using optimized reverse-phase high-performance liquid chromatographic conditions and screening assays for alpha-neurotoxin and phospholipase A2 activities. Biochemistry 1986; 25:7235-43. [PMID: 3801413 DOI: 10.1021/bi00370a070] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The vast majority of Elapidae snake venoms, genus Naja, includes three classes of toxic polypeptides: alpha-neurotoxins, phospholipases A2, and cardiotoxins. A new experimental approach using reverse-phase high-performance liquid chromatography in particular has been developed, allowing their respective resolution, identification, and quantitation from milligram quantities of venom. First, definition of optimal chromatographic conditions for Naja mossambica mossambica toxins has been ascertained. Different column packing and solvent systems were compared for their efficiency, with particular attention to the ionic strength of the aqueous solvent. A medium-chain alkyl support (octyl) in conjunction with a volatile ammonium formate (0.15 M, pH 2.70)/acetonitrile solvent system was found to be particularly effective. All the components known until now from this venom could be resolved in a single step, and the elution order was alpha-neurotoxins, phospholipases A2, and cardiotoxins with a total recovery of absorbance and toxicity. Then, with these suitable conditions, we describe a new major cardiotoxin molecule in this venom by hydrophobic and not ionic-charge discrimination. Second, specific assays were designed to detect alpha-neurotoxin and phospholipase A2 activities in chromatographic fractions: alpha-neurotoxin activity was determined by competition for the binding of a radiolabeled alpha-neurotoxin to the acetylcholine receptor of the ray electric organ, and phospholipase A2 activity was defined by the enzymatic activity of these toxins with a fluorescent phospholipid as substrate. Finally, the applicability of these new methods to study other Naja snake venoms was demonstrated.
Collapse
|
|
49
|
Worley JF, French RJ, Krueger BK. Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium. J Gen Physiol 1986; 87:327-49. [PMID: 2419487 PMCID: PMC2217599 DOI: 10.1085/jgp.87.2.327] [Citation(s) in RCA: 60] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.
Collapse
|
|
50
|
Uehara A, Moczydlowski E. Blocking mechanisms of batrachotoxin-activated Na channels in artificial bilayers. MEMBRANE BIOCHEMISTRY 1986; 6:111-47. [PMID: 2427915 DOI: 10.3109/09687688609065446] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The effects of various pharmacological agents that block single batrachotoxin-activated Na channels from rat muscle can be described in terms of three modes of action that correspond to at least three different binding sites. Guanidinium toxins such as tetrodotoxin, saxitoxin, and a novel polypeptide, mu-conotoxin GIIIA, act only from the extra-cellular side and induce discrete blocked states that correspond to residence times of individual toxin molecules. Such toxins apparently do not deeply penetrate the channel pore since the voltage dependence of block is insensitive to toxin charge and block is not relieved by internal Na+. Many nonspecific organic cations, including charged anesthetics, exhibit a voltage-dependent block that is enhanced by depolarization when present on the inside of the channel. This site is probably within the pore, but binding to this site is weak, as indicated by fast blockade that often appears as lowered channel conductance. A separate class of neutral and tertiary amine anesthetics such as benzocaine and procaine induce discrete closed states when added to either side of the membrane. This blocking effect can be explained by preferential binding to closed states of the channel and appears to be due to a modulation of channel gating.
Collapse
|