Role of Arginine Residues on the S4 Segment of the Bacillus halodurans Na+ Channel in Voltage-sensing

Chahine, Mohamed; Pilote, Sylvie; Pouliot, Valérie; Takami, Hideto; Sato, Chikara

doi:10.1007/s00232-004-0701-z

Cited by 38 publications

(38 citation statements)

References 30 publications

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“…4A). Evidently, mutation of charged amino acid residues results in channels that are more difficult to activate, similar to reports (29)(30)(31). The increases in energy required to activate these single-mutant channels compared with WT channels are similar (⌬⌬G°ϭ 1.97 to 2.67 kcal/mol) (Fig.…”

Section: Mutant Cycle Analysis Of the Voltage Dependence Of Activatiosupporting

confidence: 75%

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

DeCaen

Yarov‐Yarovoy

Zhao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

138

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The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus ''locking'' the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.voltage-sensing ͉ gating charge ͉ mutant cycle ͉ sliding helix V oltage-gated ion channels conduct electrical currents that are essential for a wide range of physiological processes including neuronal excitation, action potential conduction, synaptic neurotransmission, and muscle contraction. In prokaryotes, ion channels are necessary for pH homeostasis, chemotaxis, and motility. Voltage-gated ion channels respond to changes in membrane potential by opening and closing (''gating'') their ion-conducting pathway across the cell membrane. Gating is rapid, reversible, and steeply voltage-dependent, suggesting that charged gating particles associated with the channels move across the membrane in response to the electric field (1). Capacitative ''gating currents'' caused by these charge movements (2, 3) indicate that approximately 3-4 positive charges per voltage-sensing module move outward during gating of sodium or potassium channels (4-10). The primary structure of sodium channels (11) revealed four homologous domains containing six predicted alpha-helical segments (S1-S6) in each. Subsequently, the positively charged S4 segment was proposed to have a transmembrane position, serve as the voltage sensor that perceives changes in membrane potential, and move outward along a spiral path during channel activation to conduct the gating current and initiate a conformational change to open the pore (12, 13). A major thermodynamic obstacle to transmembrane movement of the S4 segment is stabilization of its positively charged amino acid residues in the membrane environment. The sliding helix model posits (12) that sequential formation of ion pairs with negatively charged amino acid residues in the S1, S2, and/or S3 segments serve to stabilize the S4 segments in the membrane and thereby catalyze their transmembrane movement. A detailed structural version of th...

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Section: Mutant Cycle Analysis Of the Voltage Dependence Of Activatiosupporting

confidence: 75%

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

DeCaen

Yarov‐Yarovoy

Zhao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

138

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“…Neutralization of the comparable residue in other mammalian sodium channel isoforms shifts the voltage dependence of activation in the positive direction, with a 12.6 mV shift and a decrease in the slope in Na v 1.2 (Cestèle et al, 2001) and a 5.7 mV shift with a decrease in slope in Na v 1.4 (Mitrovic et al, 1998). A similar effect was observed in the bacterial sodium channel, in which the homologous mutation shifted the V 1/2 of activation by 12.6 mV in the positive direction (Chahine et al, 2004). Therefore, it is not surprising that the R859C mutation altered the voltage dependence of activation in Na v 1.1.…”

Section: Discussionsupporting

confidence: 52%

An Epilepsy Mutation in the Sodium ChannelSCN1AThat Decreases Channel Excitability

et al. 2006

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Mutations in three voltage-gated sodium channel genes, SCN1A, SCN2A, and SCN1B, and two GABA A receptor subunit genes, GABRG2 and GABRD, have been identified in families with generalized epilepsy with febrile seizures plus (GEFSϩ). A novel mutation, R859C, in the Na v 1.1 sodium channel was identified in a four-generation, 33-member Caucasian family with a clinical presentation consistent with GEFSϩ. The mutation neutralizes a positively charged arginine in the domain 2 S4 voltage sensor of the Na v 1.1 channel ␣ subunit. This residue is conserved in mammalian sodium channels as well as in sodium channels from lower organisms. When the mutation was placed in the rat Na v 1.1 channel and expressed in Xenopus oocytes, the mutant channel displayed a positive shift in the voltage dependence of sodium channel activation, slower recovery from slow inactivation, and lower levels of current compared with the wild-type channel. Computational analysis suggests that neurons expressing the mutant channel have higher thresholds for firing a single action potential and for firing multiple action potentials, along with decreased repetitive firing. Therefore, this mutation should lead to decreased neuronal excitability, in contrast to most previous GEFSϩ sodium channel mutations, which have changes predicted to increase neuronal firing.

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“…A neutralizing mutation in S4 (R114C) produced similar effects on NaChBac as isoflurane: a positive shift in the voltage dependence of activation and a negative shift in voltage dependence of inactivation (Chahine et al, 2004). The positive shift in the voltage dependence of NaChBac activation and the net decrease in channel conductance by isoflurane suggest a reduction in voltage sensitivity via direct or indirect interaction with the voltage sensor.…”

Section: Isoflurane Inhibits Nachbac 1081mentioning

confidence: 97%

“…In current models of Na v gating, the S4 helix, which contains highly conserved positively charged amino acids, plays a critical role as voltage sensor for activation (Chahine et al, 2004). Similar to Na v , NaChBac undergoes several gating transitions involving gating charge movement (Kuzmenkin et al, 2004), the kinetics of which closely follow those of , p Ͻ 0.001 for Ϫ100 mV holding vs. Ϫ80 mV holding by unpaired t test.…”

Section: Figmentioning

confidence: 99%

Isoflurane Inhibits NaChBac, a Prokaryotic Voltage-Gated Sodium Channel

Ouyang¹,

Jih²,

Zhang³

et al. 2007

J Pharmacol Exp Ther

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Volatile anesthetics inhibit mammalian voltage-gated Na ϩ channels, an action that contributes to their presynaptic inhibition of neurotransmitter release. We measured the effects of isoflurane, a prototypical halogenated ether volatile anesthetic, on the prokaryotic voltage-gated Na ϩ channel from Bacillus halodurans (NaChBac). Using whole-cell patch-clamp recording, human embryonic kidney 293 cells transfected with NaChBac displayed large inward currents (I Na ) that activated at potentials of Ϫ60 mV or higher with a peak voltage of activation of 0 mV (from a holding potential of Ϫ80 mV) or Ϫ10 mV (from a holding potential of Ϫ100 mV). Isoflurane inhibited I Na in a concentration-dependent manner over a clinically relevant concentration range; inhibition was significantly more potent from a holding potential of Ϫ80 mV (IC 50 ϭ 0.35 mM) than from Ϫ100 mV (IC 50 ϭ 0.48 mM). Isoflurane positively shifted the voltage dependence of peak activation, and it negatively shifted the voltage dependence of end steady-state activation. The voltage dependence of inactivation was negatively shifted with no change in slope factor. Enhanced inactivation of I Na was 8-fold more sensitive to isoflurane than reduction of channel opening. In addition to tonic block of closed and/or open channels, isoflurane enhanced use-dependent block by delaying recovery from inactivation. These results indicate that a prokaryotic voltage-gated Na ϩ channel, like mammalian voltage-gated Na ϩ channels, is inhibited by clinical concentrations of isoflurane involving multiple state-dependent mechanisms. NaChBac should provide a useful model for structure-function studies of volatile anesthetic actions on voltage-gated ion channels.

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Role of Arginine Residues on the S4 Segment of the Bacillus halodurans Na+ Channel in Voltage-sensing

Cited by 38 publications

References 30 publications

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

An Epilepsy Mutation in the Sodium ChannelSCN1AThat Decreases Channel Excitability

Isoflurane Inhibits NaChBac, a Prokaryotic Voltage-Gated Sodium Channel

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