Substitutions in the Domain III Voltage-sensing Module Enhance the Sensitivity of an Insect Sodium Channel to a Scorpion β-Toxin

Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT3, from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNav). Like other scorpion β-toxins, Lqh-dprIT3 shifts the voltage dependence of activation of BgNav channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNav splice variants tested for their sensitivity to the toxin, only BgNav1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT3. Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.

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Interpreting the functional role of a novel interaction motif in prokaryotic sodium channels

The Journal of General Physiology ◽

10.1085/jgp.201611740 ◽

2017 ◽

Vol 149 (6) ◽

pp. 613-622 ◽

Cited By ~ 11

Author(s):

Altin Sula ◽

B.A. Wallace

Keyword(s):

Sodium Channels ◽

Functional Role ◽

Structural Features ◽

Channel Activation ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Channel Opening ◽

Activated State ◽

Electrical Signaling

Voltage-gated sodium channels enable the translocation of sodium ions across cell membranes and play crucial roles in electrical signaling by initiating the action potential. In humans, mutations in sodium channels give rise to several neurological and cardiovascular diseases, and hence they are targets for pharmaceutical drug developments. Prokaryotic sodium channel crystal structures have provided detailed views of sodium channels, which by homology have suggested potentially important functionally related structural features in human sodium channels. A new crystal structure of a full-length prokaryotic channel, NavMs, in a conformation we proposed to represent the open, activated state, has revealed a novel interaction motif associated with channel opening. This motif is associated with disease when mutated in human sodium channels and plays an important and dynamic role in our new model for channel activation.

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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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Neutralization of Gating Charges in Domain II of the Sodium Channel α Subunit Enhances Voltage-Sensor Trapping by a β-Scorpion Toxin

The Journal of General Physiology ◽

10.1085/jgp.118.3.291 ◽

2001 ◽

Vol 118 (3) ◽

pp. 291-302 ◽

Cited By ~ 56

Author(s):

Sandrine Cestèle ◽

Todd Scheuer ◽

Massimo Mantegazza ◽

Hervé Rochat ◽

William A. Catterall

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Voltage Dependence ◽

Membrane Potentials ◽

Voltage Sensor ◽

Scorpion Toxin ◽

Channel Activation ◽

Α Subunit ◽

Gating Charges

β-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.

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Sodium channel activation underlies transfluthrin repellency in Aedes aegypti

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009546 ◽

2021 ◽

Vol 15 (7) ◽

pp. e0009546

Author(s):

Felipe Andreazza ◽

Wilson R. Valbon ◽

Qiang Wang ◽

Feng Liu ◽

Peng Xu ◽

...

Keyword(s):

Aedes Aegypti ◽

Sodium Channel ◽

Sodium Channels ◽

Human Disease ◽

Disease Vectors ◽

Channel Activation ◽

Principal Mechanism ◽

Pyrethroid Insecticides ◽

Voltage Gated Sodium Channels ◽

Sodium Channel Activation

Background Volatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial. Methodology/Principal findings Here, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency. Conclusions/Significance These results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.

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A general mechanism of KCNE1 modulation of KCNQ1 channels involving non-canonical VSD-PD coupling

Communications Biology ◽

10.1038/s42003-021-02418-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Xiaoan Wu ◽

Marta E. Perez ◽

Sergei Yu Noskov ◽

H. Peter Larsson

Keyword(s):

Intermediate State ◽

Voltage Dependence ◽

General Mechanism ◽

Beta Subunits ◽

Voltage Sensing ◽

Conserved Residues ◽

Voltage Gated ◽

Activated State ◽

And Shifts ◽

Pore Domain

AbstractVoltage-gated KCNQ1 channels contain four separate voltage-sensing domains (VSDs) and a pore domain (PD). KCNQ1 expressed alone opens when the VSDs are in an intermediate state. In cardiomyocytes, KCNQ1 co-expressed with KCNE1 opens mainly when the VSDs are in a fully activated state. KCNE1 also drastically slows the opening of KCNQ1 channels and shifts the voltage dependence of opening by >40 mV. We here show that mutations of conserved residues at the VSD–PD interface alter the VSD–PD coupling so that the mutant KCNQ1/KCNE1 channels open in the intermediate VSD state. Using recent structures of KCNQ1 and KCNE beta subunits in different states, we present a mechanism by which KCNE1 rotates the VSD relative to the PD and affects the VSD–PD coupling of KCNQ1 channels in a non-canonical way, forcing KCNQ1/KCNE1 channels to open in the fully-activated VSD state. This would explain many of the KCNE1-induced effects on KCNQ1 channels.

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Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7

Biomedicines ◽

10.3390/biomedicines8020037 ◽

2020 ◽

Vol 8 (2) ◽

pp. 37

Author(s):

Kathleen Yin ◽

Jennifer R. Deuis ◽

Zoltan Dekan ◽

Ai-Hua Jin ◽

Paul F. Alewood ◽

...

Keyword(s):

Sodium Channels ◽

Rational Design ◽

Voltage Dependence ◽

Spider Venom ◽

Fast Inactivation ◽

Peak Current ◽

Native Peptide ◽

Voltage Gated Sodium Channels ◽

Nav Channels ◽

And Shifts

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at NaV channels in the future.

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β-Scorpion Toxin Modifies Gating Transitions in All Four Voltage Sensors of the Sodium Channel

The Journal of General Physiology ◽

10.1085/jgp.200609719 ◽

2007 ◽

Vol 130 (3) ◽

pp. 257-268 ◽

Cited By ~ 58

Author(s):

Fabiana V. Campos ◽

Baron Chanda ◽

Paulo S.L. Beirão ◽

Francisco Bezanilla

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Integrated Approach ◽

Voltage Sensor ◽

Scorpion Toxin ◽

Charge Voltage ◽

Voltage Sensors ◽

Fluorescence Measurements ◽

Voltage Gated Sodium Channels ◽

Activated State

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

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Dual role of Ca(V)1.1 voltage-sensing domain I in determining kinetics and voltage dependence of calcium channel activation

Intrinsic Activity ◽

10.25006/ia.7.s1-a3.11 ◽

2019 ◽

Vol 7 (Suppl. 1) ◽

pp. A3.11

Author(s):

Yousra El Ghaleb

Keyword(s):

Calcium Channel ◽

Voltage Dependence ◽

Dual Role ◽

Voltage Sensing ◽

Channel Activation ◽

Domain I ◽

Voltage Sensing Domain

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Pore- and voltage sensor–targeted KCNQ openers have distinct state-dependent actions

The Journal of General Physiology ◽

10.1085/jgp.201812070 ◽

2018 ◽

Vol 150 (12) ◽

pp. 1722-1734 ◽

Cited By ~ 6

Author(s):

Caroline K. Wang ◽

Shawn M. Lamothe ◽

Alice W. Wang ◽

Runying Y. Yang ◽

Harley T. Kurata

Keyword(s):

Voltage Dependence ◽

Drug Effects ◽

Drug Binding ◽

Voltage Sensor ◽

Voltage Sensing ◽

Channel Activation ◽

M Current ◽

State Dependent ◽

Mechanistic Basis ◽

Voltage Sensing Domain

Ion channels encoded by KCNQ2-5 generate a prominent K+ conductance in the central nervous system, referred to as the M current, which is controlled by membrane voltage and PIP2. The KCNQ2-5 voltage-gated potassium channels are targeted by a variety of activating compounds that cause negative shifts in the voltage dependence of activation. The underlying pharmacology of these effects is of growing interest because of possible clinical applications. Recent studies have revealed multiple binding sites and mechanisms of action of KCNQ activators. For example, retigabine targets the pore domain, but several compounds have been shown to influence the voltage-sensing domain. An important unexplored feature of these compounds is the influence of channel gating on drug binding or effects. In the present study, we compare the state-dependent actions of retigabine and ICA-069673 (ICA73, a voltage sensor–targeted activator). We assess drug binding to preopen states by applying drugs to homomeric KCNQ2 channels at different holding voltages, demonstrating little or no association of ICA73 with resting states. Using rapid solution switching, we also demonstrate that the rate of onset of ICA73 correlates with the voltage dependence of channel activation. Retigabine actions differ significantly, with prominent drug effects seen at very negative holding voltages and distinct voltage dependences of drug binding versus channel activation. Using similar approaches, we investigate the mechanistic basis for attenuation of ICA73 actions by the voltage-sensing domain mutation KCNQ2[A181P]. Our findings demonstrate different state-dependent actions of pore- versus voltage sensor–targeted KCNQ channel activators, which highlight that subtypes of this drug class operate with distinct mechanisms.

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