Fenestrations control resting-state block of a voltage-gated sodium channel

Potency of drug action is usually determined by binding to a specific receptor site on target proteins. In contrast to this conventional paradigm, we show here that potency of local anesthetics (LAs) and antiarrhythmic drugs (AADs) that block sodium channels is controlled by fenestrations that allow drug access to the receptor site directly from the membrane phase. Voltage-gated sodium channels initiate action potentials in nerve and cardiac muscle, where their hyperactivity causes pain and cardiac arrhythmia, respectively. LAs and AADs selectively block sodium channels in rapidly firing nerve and muscle cells to relieve these conditions. The structure of the ancestral bacterial sodium channel NaVAb, which is also blocked by LAs and AADs, revealed fenestrations connecting the lipid phase of the membrane to the central cavity of the pore. We cocrystallized lidocaine and flecainide with NavAb, which revealed strong drug-dependent electron density in the central cavity of the pore. Mutation of the contact residue T206 greatly reduced drug potency, confirming this site as the receptor for LAs and AADs. Strikingly, mutations of the fenestration cap residue F203 changed fenestration size and had graded effects on resting-state block by flecainide, lidocaine, and benzocaine, the potencies of which were altered from 51- to 2.6-fold in order of their molecular size. These results show that conserved fenestrations in the pores of sodium channels are crucial pharmacologically and determine the level of resting-state block by widely used drugs. Fine-tuning drug access through fenestrations provides an unexpected avenue for structure-based design of ion-channel–blocking drugs.

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Solution Structure and Alanine Scan of a Spider Toxin That Affects the Activation of Mammalian Voltage-gated Sodium Channels

Journal of Biological Chemistry ◽

10.1074/jbc.m605403200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4643-4652 ◽

Cited By ~ 27

Author(s):

Gerardo Corzo ◽

Jennifer K. Sabo ◽

Frank Bosmans ◽

Bert Billen ◽

Elba Villegas ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Solution Structure ◽

Receptor Site ◽

Amino Acid Sequences ◽

Structural Motif ◽

Dimensional Structure ◽

Voltage Gated Sodium Channel ◽

Voltage Gated ◽

Voltage Gated Sodium Channels

Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion β-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded β-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion β-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

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Indoxacarb, metaflumizone, and other sodium channel inhibitor insecticides: Mechanism and site of action on mammalian voltage-gated sodium channels

Pesticide Biochemistry and Physiology ◽

10.1016/j.pestbp.2013.03.004 ◽

2013 ◽

Vol 106 (3) ◽

pp. 101-112 ◽

Cited By ~ 25

Author(s):

Richard T. von Stein ◽

Kristopher S. Silver ◽

David M. Soderlund

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Site Of Action

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Sodium Channel Nav1.3 Is Expressed by Polymorphonuclear Neutrophils during Mouse Heart and Kidney Ischemia InVivo and Regulates Adhesion, Transmigration, and Chemotaxis of Human and Mouse Neutrophils In Vitro

Anesthesiology ◽

10.1097/aln.0000000000002135 ◽

2018 ◽

Vol 128 (6) ◽

pp. 1151-1166 ◽

Cited By ~ 6

Author(s):

Marit Poffers ◽

Nathalie Bühne ◽

Christine Herzog ◽

Anja Thorenz ◽

Rongjun Chen ◽

...

Keyword(s):

Endothelial Cells ◽

Sodium Channel ◽

Sodium Channels ◽

Polymorphonuclear Neutrophils ◽

Human Neutrophils ◽

Mouse Heart ◽

Voltage Gated ◽

Voltage Gated Sodium Channels

Abstract Background Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. Methods Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. Results Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. Conclusions Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

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Purification and Characterization of JZTx-14, a Potent Antagonist of Mammalian and Prokaryotic Voltage-Gated Sodium Channels

Toxins ◽

10.3390/toxins10100408 ◽

2018 ◽

Vol 10 (10) ◽

pp. 408 ◽

Cited By ~ 1

Author(s):

Jie Zhang ◽

Dongfang Tang ◽

Shuangyu Liu ◽

Haoliang Hu ◽

Songping Liang ◽

...

Keyword(s):

Sodium Channels ◽

Resting State ◽

Purification And Characterization ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Steady State Inactivation ◽

Ic50 Values ◽

Gating Modifier ◽

Potent Antagonist

Exploring the interaction of ligands with voltage-gated sodium channels (NaVs) has advanced our understanding of their pharmacology. Herein, we report the purification and characterization of a novel non-selective mammalian and bacterial NaVs toxin, JZTx-14, from the venom of the spider Chilobrachys jingzhao. This toxin potently inhibited the peak currents of mammalian NaV1.2–1.8 channels and the bacterial NaChBac channel with low IC50 values (<1 µM), and it mainly inhibited the fast inactivation of the NaV1.9 channel. Analysis of NaV1.5/NaV1.9 chimeric channel showed that the NaV1.5 domain II S3–4 loop is involved in toxin association. Kinetics data obtained from studying toxin–NaV1.2 channel interaction showed that JZTx-14 was a gating modifier that possibly trapped the channel in resting state; however, it differed from site 4 toxin HNTx-III by irreversibly blocking NaV currents and showing state-independent binding with the channel. JZTx-14 might stably bind to a conserved toxin pocket deep within the NaV1.2–1.8 domain II voltage sensor regardless of channel conformation change, and its effect on NaVs requires the toxin to trap the S3–4 loop in its resting state. For the NaChBac channel, JZTx-14 positively shifted its conductance-voltage (G–V) and steady-state inactivation relationships. An alanine scan analysis of the NaChBac S3–4 loop revealed that the 108th phenylalanine (F108) was the key residue determining the JZTx-14–NaChBac interaction. In summary, this study provided JZTx-14 with potent but promiscuous inhibitory activity on both the ancestor bacterial NaVs and the highly evolved descendant mammalian NaVs, and it is a useful probe to understand the pharmacology of NaVs.

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Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1992.262.4.r712 ◽

1992 ◽

Vol 262 (4) ◽

pp. R712-R715 ◽

Cited By ~ 11

Author(s):

M. A. Perez-Pinzon ◽

M. Rosenthal ◽

T. J. Sick ◽

P. L. Lutz ◽

J. Pablo ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Tissue Oxygenation ◽

Mammalian Brain ◽

Survival Strategy ◽

Voltage Gated ◽

Channel Density ◽

Spike Threshold ◽

Channel Arrest ◽

Key Factor

In contrast to mammalian brain, which exhibits rapid degeneration during anoxia, the brains of certain species of turtles show an extraordinary capacity to survive prolonged anoxia. The decrease in energy expenditure shown by the anoxic turtle brain is likely to be a key factor for anoxic survival. The "channel arrest" hypothesis proposes that ion channels, which regulate brain electrical activity in normoxia, may be altered during anoxia in the turtle brain as a mechanism to spare energy. Goals of present research were to test this hypothesis and to determine whether down-regulation of sodium channels is a possible explanation for spike threshold shifts seen during anoxia in isolated turtle cerebellum. We report here that anoxia induced a significant (42%) decline in voltage-gated sodium channel density as determined by studies of the binding of a sodium channel ligand, [3H]brevetoxin. This study demonstrates that sodium channel densities in brain may be regulated by tissue oxygenation or by physiological events associated with anoxia. Moreover, it also suggests that downregulation of sodium channels may be a basis for changes in action potential thresholds, the electrical depression and energy conservation that provide the unique anoxic tolerance of turtle brain.

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Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1514943113 ◽

2016 ◽

Vol 113 (7) ◽

pp. 1823-1828 ◽

Cited By ~ 23

Author(s):

Carolina González ◽

José Cánovas ◽

Javiera Fresno ◽

Eduardo Couve ◽

Felipe A. Court ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Sodium Channel ◽

Sodium Channels ◽

Neural Function ◽

Axonal Membrane ◽

Local Synthesis ◽

Voltage Gated ◽

Voltage Gated Sodium Channels

The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons.

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Allosteric interactions between receptor site 3 and 4 of voltage-gated sodium channels: a novel perspective for the underlying mechanism of scorpion sting-induced pain

Journal of Venomous Animals and Toxins including Tropical Diseases ◽

10.1186/s40409-015-0043-6 ◽

2015 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Yi-Jun Feng ◽

Qi Feng ◽

Jie Tao ◽

Rong Zhao ◽

Yong-Hua Ji

Keyword(s):

Sodium Channels ◽

Receptor Site ◽

Underlying Mechanism ◽

Scorpion Sting ◽

Voltage Gated ◽

Allosteric Interactions ◽

Voltage Gated Sodium Channels

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Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

The Journal of General Physiology ◽

10.1085/jgp.201411210 ◽

2014 ◽

Vol 144 (2) ◽

pp. 147-157 ◽

Cited By ~ 19

Author(s):

Tamer M. Gamal El-Din ◽

Todd Scheuer ◽

William A. Catterall

Keyword(s):

Membrane Potential ◽

Sodium Channel ◽

Sodium Channels ◽

Voltage Dependence ◽

Periodic Paralysis ◽

Membrane Potentials ◽

Structural Basis ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Gating Charges

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.

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