Molecular Determinants for Alpha-Scorpion Toxin Binding to the Resting State of a Voltage Sensor of Brain Sodium Channels

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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Binding of scorpion toxin to receptor sites associated with sodium channels in frog muscle. Correlation of voltage-dependent binding with activation.

The Journal of General Physiology ◽

10.1085/jgp.74.3.375 ◽

1979 ◽

Vol 74 (3) ◽

pp. 375-391 ◽

Cited By ~ 114

Author(s):

W A Catterall

Keyword(s):

Sodium Channels ◽

Voltage Dependence ◽

Binding Capacity ◽

Specific Binding ◽

Frog Muscle ◽

Scorpion Toxin ◽

Sartorius Muscle ◽

Single Class ◽

Bathing Medium ◽

Toxin Binding

Purified scorpion toxin (Leiurus quinquestriatus) slows inactivation of sodium channels in frog muscle at concentrations in the range of 17-170 nM. Mono[125I]iodo scorpion toxin binds to a single class of sites in frog sartorius muscle with a dissociation constant of 14 nM and a binding capacity of 13 fmol/mg wet weight. Specific binding is inhibited more than 90% by 3 microM sea anemone toxin II and by depolarization with 165 mM K+. Half-maximal inhibition of binding is observed on depolarization to -41 mV. The voltage dependence of scorpion toxin binding is correlated with the voltage dependence of activation of sodium channels. Removal of calcium from the bathing medium shifts both activation and inhibition of scorpion toxin binding to more negative membrane potentials. The results are considered in terms of the hypothesis that activation of sodium channels causes a conformational change in the scorpion toxin receptor site resulting in reduced affinity for scorpion toxin.

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Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin

Journal of Biological Chemistry ◽

10.1074/jbc.m603814200 ◽

2006 ◽

Vol 281 (30) ◽

pp. 21332-21344 ◽

Cited By ~ 107

Author(s):

Sandrine Cestèle ◽

Vladimir Yarov-Yarovoy ◽

Yusheng Qu ◽

François Sampieri ◽

Todd Scheuer ◽

...

Keyword(s):

Sodium Channels ◽

Structure And Function ◽

Voltage Sensor ◽

Scorpion Toxin ◽

And Function

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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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Localization of voltage-sensitive sodium channels on the extrasynaptic membrane surface of mouse skeletal muscle by autoradiography of scorpion toxin binding sites

Journal of Neurocytology ◽

10.1007/bf01188407 ◽

1990 ◽

Vol 19 (3) ◽

pp. 408-420 ◽

Cited By ~ 15

Author(s):

T. Le Treut ◽

J. -L. Boudier ◽

E. Jover ◽

P. Cau

Keyword(s):

Skeletal Muscle ◽

Sodium Channels ◽

Binding Sites ◽

Membrane Surface ◽

Scorpion Toxin ◽

Mouse Skeletal Muscle ◽

Toxin Binding ◽

Extrasynaptic Membrane

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Scorpion toxin-potassium channel interaction law and its applications.

Venoms and Toxins ◽

10.2174/2666121701999200531143349 ◽

2020 ◽

Vol 01 ◽

Author(s):

Zheng Zuo ◽

Zongyun Chen ◽

Zhijian Cao ◽

Wenxin Li ◽

Yingliang Wu

Keyword(s):

Potassium Channel ◽

Scorpion Toxin ◽

Scorpion Toxins ◽

Toxin Binding ◽

Channel Interaction ◽

Binding Interface ◽

Α Helix ◽

Interaction Law ◽

Binding Interfaces ◽

Β Sheet

: The scorpion toxins are the largest potassium channel-blocking peptide family. The understanding of toxin binding interfaces is usually restricted by two classical binding interfaces: one is the toxin α-helix motif, the other is the antiparallel β-sheet motif. In this review, such traditional knowledge was updated by another two different binding interfaces: one is BmKTX toxin using the turn motif between the α-helix and antiparallel β-sheet domains as the binding interface, the other is Ts toxin using turn motif between the β-sheet in the N-terminal and α-helix domains as the binding interface. Their interaction analysis indicated that the scarce negatively charged residues in the scorpion toxins played a critical role in orientating the toxin binding interface. In view of the toxin negatively charged amino acids as “binding interface regulator”, the law of scorpion toxin-potassium channel interaction was proposed, that is, the polymorphism of negatively charged residue distribution determines the diversity of toxin binding interfaces. Such law was used to develop scorpion toxin-potassium channel recognition control technique. According to this technique, three Kv1.3 channel-targeted peptides, using BmKTX as the template, were designed with the distinct binding interfaces from that of BmKTX through modulating the distribution of toxin negatively charged residues. In view of the potassium channel as the common targets of different animal toxins, the proposed law was also shown to helpfully orientate the binding interfaces of other animal toxins. Clearly, the toxin-potassium channel interaction law would strongly accelerate the research and development of different potassium channelblocking animal toxins in the future.

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