Correspondence to: William A. Catterall, Department of Pharmacology, Mailstop 357280, University of Washington School of Medicine, Seattle, WA 98195-7280. Fax:(206) 685-3822 E-mail:wcatt{at}u.washington.edu.
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Abstract |
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ß-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 S3S4 loop at the extracellular end of the S4 voltage sensor in domain II of the subunit. Here, we probe the role of gating charges in the IIS4 segment in ß-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 ß-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the ß-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-sensortrapping model in which the ß-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.
Key Words: sodium channels, Centruroides suffusus suffusus toxin IV, ß-scorpion toxin, voltage sensor, voltage-dependent gating
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INTRODUCTION |
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Voltage-gated sodium channels are responsible for the voltage-dependent increase in sodium permeability and, therefore, play a critical role in the initiation and propagation of action potentials in excitable cells ( subunit of 260 kD associated with one or two smaller auxiliary subunits ß1, ß2, and ß3 (for review see
subunit consists of four homologous domains (IIV), each containing six transmembrane segments (S1S6) and one reentrant segment (SS1/SS2) connected by internal and external polypeptide loops (for review see
Voltage-gated sodium channels are the molecular target of several groups of neurotoxins, which bind to specific receptor sites and strongly alter sodium channel function (for review see -scorpion toxins, sea anemone toxins, and spider toxins bind to receptor site 3 and slow sodium channel inactivation (
-scorpion toxins and sea anemone toxins to receptor site 3 is proposed to slow inactivation by preventing the normal outward movement of the IVS4 transmembrane segment (
In this paper, we demonstrate that neutralization of the two outermost arginine residues of the IIS4 voltage sensor markedly enhances ß-scorpion toxin effects on sodium channels. This effect is proposed to result from an increase in mobility of the IIS4 segment within the membrane. Our results reveal crucial roles of the two first arginines of the IIS4 segment in voltage-dependent activation, ß-scorpion toxin action, and stabilization of the IIS4 segment within the membrane.
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MATERIALS AND METHODS |
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Materials
ß-Scorpion toxin Css IV was purified from the venom of Centruroides suffusus suffusus ( subunits (
Site-directed Mutagenesis
Mutations R850Q, R853Q, K859Q, and K862Q were produced using an M13 construct containing a XmaI-SphI fragment (nt 5411,897) of the Nav1.2a cDNA. Uracil-containing mutagenesis template was prepared from this construct and oligonucleotide-directed mutagenesis was performed using the dut- ung- selection procedure (
Transient Expression in tsA-201 Cells
The tsA-201 cells were maintained at 37°C in 10% CO2 in DMEM/Ham's F12 medium (GIBCO BRL and Life Technologies) supplemented with 10% FBS (Gemini Biological Products), 20 µg/ml penicillin, and 10 µg/ml streptomycin (Gemini Biological Products). TsA-201 cells were transiently cotransfected with cDNAs for the channel subunit and pEBFP-N1 vector encoding the enhanced green fluorescent protein (CLONTECH Laboratories, Inc.) using calcium phosphate precipitation (
Electrophysiological Recording and Analysis
Whole-cell sodium currents were recorded from tsA-201 cells expressing Nav1.2a wild-type or mutant sodium channel subunits. The external recording solution consisted of the following (in mM): 150 NaCl, 10 Cs-HEPES, 1 MgCl2, 2 KCl, and 1.5 CaCl2, pH 7.4. The internal recording solution consisted of the following (in mM): 190 N-methyl-D-glutamine, 10 HEPES, 4 MgCl2, 10 NaCl, and 5 EGTA, pH 7.4. Patch electrodes were pulled from 75-µl micropipette glass (VWR Scientific) and were fire-polished before use. Electrode resistances were typically 1.52.5 m
in the bath. Recordings were obtained using a patch-clamp amplifier (model Axopatch 200B; Axon Instruments, Inc.). Voltage pulses were applied and data were acquired using pClamp6 software (Axon Instruments Inc.). Linear leak and capacitance currents have been subtracted using an online P/-4 subtraction paradigm. Css IV, MTSEA, and MTSET were dissolved in the extracellular solution at the final concentration. Css IV was applied to cells using fast local perfusion of the cell with background perfusion of the chamber; MTSEA and MTSET were added to the extracellular solution in the recording bath. All experiments were performed at room temperature. Conductance-voltage (activation) curves were derived from peak sodium current versus voltage measurements according to: G = I/(V - VR) where I is the peak current, V is the test voltage, and VR is the apparent reversal potential. Normalized conductance-voltage and inactivation curves were fit with a Boltzmann relationship of the form 1/{1 + exp[(V1/2 - V)/k]} or with the sum of two such expressions, where V1/2 is the voltage for half-maximal activation or inactivation, and k is a slope factor with the dimensions of millivolts.
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RESULTS |
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Effects of Mutations of the Gating Charges in Domain II on Activation and Inactivation
Previous studies have demonstrated the important role of the positively charged amino acid residues of the S4 segments in the voltage-dependent gating of sodium channels (
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Measurements of steady-state inactivation for Nav1.2a, R850Q, and R853Q using 150-ms prepulses under control conditions indicate that these mutations do not significantly modify the voltage of half-maximal inactivation when compared with wild-type sodium channels (Fig 1 B and Table 1), despite the difference in voltage dependence of activation. On the other hand, mutations R856Q, K859Q, and K862Q cause small but significant positive shifts in the voltage dependence of steady-state inactivation (Fig 1 B and Table 1). These data agree with previous results implicating the IIS4 segment in activation, rather than inactivation (
Effects of Css IV on Mutant Sodium Channels with Neutralized Gating Charges in Transmembrane Segment IIS4
As previously described for the wild-type Nav1.2a channels (
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Effects of Cysteine Substitution and Reaction with Methanethiosulfonate (MTS) Reagents at Positions 850 and 853
To further examine the role of the two outermost arginines of the IIS4 segment in the activation process of sodium channels as well as in the mechanism of action of ß-scorpion toxins, we replaced the two outermost arginine residues by cysteine. Those mutants allowed us to use cysteine-modifying MTS reagents to chemically modify the channel at these positions. This method has been extensively used to monitor the movement of the S4 segments during activation and the inactivation gate during inactivation of voltage-gated sodium channels (
Neutralizing R850 or R853 by substitution with cysteine reduced the slope of the activation curve and shifted the voltage of half activation by 512 mV in the depolarizing direction (Fig 3 and Table 1). Compared with glutamine substitution, neutralization of R850 and R853 by cysteine substitution induces a 23-mV additional positive shift in the voltage dependence of activation (Table 1). We studied the modification of the mutants R850C and R853C by MTSEA and MTSET. These two reagents covalently modify accessible cysteinyl sulfhydryl groups by the transfer of a positively charged, substituted amino group, but MTSEA is smaller and can reach less accessible sites of reaction. Our experiments were performed by adding MTSET or MTSEA to the extracellular recording solution, allowing modification of the cysteine residues by MTS reagents at the resting membrane potential. Modification of cysteine residues in S4 segments by the MTS reagents is fast and complete under similar conditions (
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Effects of Css IV on R850C-MTSET and R853C-MTSEA
Because R850C-MTSET and R853C-MTSEA have activation curves that are strongly shifted relative to wild-type Nav1.2a or the unmodified cysteine-containing channels, they represent powerful tools for elucidating the relationship between the voltage dependence of channel gating and the ability of ß-scorpion toxins to trap the IIS4 segment in its activated position. Therefore, we analyzed the effects of 200 nM Css IV on R850C-MTSET and R853C-MTSEA (Fig 4, AD).
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For R850C without MTSET modification, 200 nM Css IV causes a negative shift in the conductance-voltage relationship of a small fraction of sodium channels in the absence of a depolarizing prepulse (Fig 4 A). Although R850C-MTSET channels have a negative voltage dependence of activation (Fig 3), treatment with 200 nM Css IV causes an additional negative shift of activation of a similar small fraction of the sodium channels (Fig 4 A). Comparison of the effects of Css IV on unmodified R850C channels and R850C-MTSET channels shows that they are similar in magnitude, but shifted in absolute voltage dependence by the -17-mV difference in voltage-dependent activation between the two channels. Thus, the negative shifts of activation caused by modification by MTSET and modification by Css IV are additive and independent. Consistent with this, depolarizing prepulses in the presence of Css IV cause a larger negative shift in the activation curves for R850C and R850C-MTSET, and the voltage dependence of a large fraction of the modified channels is shifted (Fig 4 B). Thus, modification by MTSET causes a negative voltage shift in sodium channel activation, but once channels are fully modified after a depolarizing prepulse, the effects of Css IV are similar before and after treatment with the MTS reagent.
Effects of MTSEA modification of R853C were quite different. For unmodified R853C channels, a prepulse was not required to observe a substantial shift in the activation curve for nearly all of the sodium channels using 200 nM Css IV (Fig 4 C). However, after modification with MTSEA, a prepulse was required to observe a strongly shifted activation curve (Fig 4C and Fig D). This result contrasts with those for unmodified R853Q and R853C sodium channels where the prepulse was not required to observe the complete shift in the activation curve (Fig 2 C and 4 C). Thus, MTSEA modification of R853C markedly inhibited the effects of Css IV on R853C sodium channels and restored the requirement for a prepulse for Css IV to cause a negative shift in the voltage dependence of activation. Restoration of the positive charge by reaction with MTSEA may stabilize the IIS4 segment in an inward position by restoring ionic interactions.
Voltage Dependence of Wild-Type and Mutant Sodium Channels with IIS4 Segments Trapped in Their Activated Position by Css IV
Although the wild-type and mutant sodium channels studied here had different voltage dependence of activation (V1/2 from -16 to -39 mV; Table 1 and Fig 3), successful modification by Css IV shifted the voltage dependence of these sodium channels to approximately the same position on the voltage axis (Fig 5). This is most clearly illustrated for the channels that are strongly shifted to a more negative activation curve after a depolarizing prepulse in the presence of Css IV (i.e., R850Q, R853Q, R850C, and R850C-MTSET; Fig 5). The activation curves for these toxin-modified channels are all similar, within the experimental error for measurements of the voltage dependence of activation and fits to the sum of two Boltzmann functions (Fig 5). When only a fraction of the channels are shifted negatively by a prepulse in the presence of toxin (e.g., WT), the activation curves are well-fit with two Boltzmann components: a negative one having approximately the same voltage dependence as the fully shifted activation curves and a positive one resembling unmodified channels (Fig 5). Thus, approximately the same negative voltage dependence is attained by these toxin-modified channels, whether their activation curves are relatively positive or negative under control conditions and whether their activation curves are fully (R850Q, R853Q, R850C, and R850C-MTSET) or partially (WT) shifted by the prepulse in the absence of toxin (Fig 5). Exceptions to this rule are presented by toxin-modified R853C and R853C-MTSEA, whose voltage dependence of activation is not as negatively shifted by Css IV. Nevertheless, our results overall indicate that, once wild-type and most mutant sodium channels are successfully modified by toxin binding alone or in response to a depolarization, the channels adopt the same or similar toxin-modified conformation for wild-type, mutants, and MTS-modified mutants, perhaps having the IIS4 segment in its outward, activated position bound to Css IV.
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Accessibility of the IIS4 Segment in R853C Sodium Channels
The R853C mutant is modified by binding of Css IV at the holding potential of -100 mV, since a strong shift in the voltage dependence of activation is observed immediately on the application of Css IV. To test whether it might be possible to reverse effects of this mutation on Css IV action by using a strongly hyperpolarizing holding potential, we analyzed the effects of Css IV on R853C by measuring the threshold currents elicited by a test pulse at -65 mV when the holding potential was maintained at -100, -120, and -140 mV. In the absence of Css IV, no sodium current is activated at -65 mV in this mutant (Fig 3 B and 4 C). Transfected cells were exposed to 200 nM Css IV for 2 min at the holding potential. A subsequent test pulse to -65 mV resulted in an inward sodium current that was as large as that normally observed at this potential in the current-voltage curve (Fig 6, AC). Thus, R853C renders IIS4 directly accessible for voltage-sensor trapping by the toxin, and hyperpolarizing the membrane potential to -120 or -140 mV is not sufficient to prevent toxin action. This result indicates that neutralization of R853C alters the accessibility of IIS4 to the toxin at all membrane potentials positive to -140 mV. Modification of R853C with MTSEA prevents this change in accessibility of IIS4. When R853C-MTSEA is exposed to 200 nM Css IV using the same holding potential protocol, the test pulse to -65 mV does not result in sodium current, even when the membrane potential is maintained at -100 mV (Fig 6 D). These data reinforce the hypothesis that modification of R853C by MTSEA stabilizes the IIS4 segment in an inward position, thus, altering dramatically its accessibility to the toxin and preventing voltage-sensor trapping.
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Toxin-induced Tail Current Reflects Voltage-Sensor Trapping
If the IIS4 segment is trapped in its outward, activated position by binding of Css IV, the toxin would be expected to induce long-lasting tail currents due to slowed deactivation and inward movement of the trapped voltage sensor. In fact, Css venom induces long-lasting tail currents after depolarizing pulses (
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DISCUSSION |
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Gating Charges in the IIS4 Segment of Sodium Channels
The activation process of sodium channels corresponds to a voltage-driven transition from a resting, closed conformation to an open conformational state, which is accompanied by the translocation of several positive charges outward across the membrane (
Comparison of the voltage dependence of gating of sodium channels having mutations in each of the five positively charged amino acid residues in segment IIS4 indicates that all five mutations affect activation gating. Neutralization of each of the five positive charges shifts the voltage dependence of activation in the positive direction. Moreover, the slope of the activation curve was reduced by neutralization of R850, R853, R856, and K859. Neutralization of K862 did not have an effect on the steepness of the activation curve. These results are consistent with the idea that R850, R853, R856, and K859 all move outward through the membrane electric field as the sodium channel activates. However, these charges are not equally important for activation gating. Neutralization of K859 produced the largest shift in the voltage dependence of activation, whereas neutralization of R856 has the smallest effect (Fig 1 and Table 1;
Neutralization of the Two Outermost Arginines of the IIS4 Segment Favors Voltage-Sensor Trapping by Css IV
Bound ß-scorpion toxins induce only a partial shift of the activation curve of wild-type sodium channels, even after a strong depolarizing prepulse, as indicated by the biphasic activation curves for wild-type channels (
Neutralization of R850 and R853 Increases the Mobility of IIS4 Segment within the Membrane
We interpret the enhanced effects of Css IV on activation of R850C/Q and R853C/Q in terms of a role for these two amino acid residues in stabilizing the position of the IIS4 segment with respect to interactions with Css IV. The ability of Css IV to shift the voltage dependence of activation of R853C/Q in the negative direction without a depolarizing prepulse supports the idea that this mutation increases the mobility of the IIS4 segment, allowing the toxin to induce IIS4 movement without depolarization. We consider two mechanisms that may contribute to this stabilizing influence of R850 and R853.
First, it is possible that these neutralizing mutations increase the mobility of the IIS4 segment in the presence of Css IV because the normal positive charges at these positions make unfavorable electrostatic interactions with the amino acid residues in the strongly positively charged toxin. Neutralization of these charges may enhance ß-toxin action by removing this unfavorable electrostatic interaction, allowing easier outward movement of the IIS3S4 loop and the IIS4 segment. This would promote more effective interaction with the bound Css IV toxin and more complete shift of the voltage dependence of activation.
Alternatively, it has been shown for K+ channels that basic amino acid residues in the S4 segment interact with acidic residues in the S2 and S3 segments. These electrostatic interactions are structural constraints that can stabilize the S4 segments and impede their motion (
Our results obtained with R853Q/C suggest a dominant role of R853 in stabilizing the position of the IIS4 segment within the membrane, because voltage-sensor trapping and the resulting negative shift of activation occur in the absence of a depolarizing prepulse with these mutations. The voltage-sensor trapping mechanism can also account for the marked effect of MTSEA on the activation of R853C. We suggest that reaction of MTSEA with this residue stabilizes the IIS4 segment in its inward position through steric hindrance and/or ion pair formation, and thereby impedes movement of the IIS4 segment. In contrast to the unmodified-R853C channel, R853C-MTSEA requires a depolarizing prepulse to observe a shift in the activation curve due to Css IV application, and the voltage dependence of only a fraction of the sodium channels is shifted. These results indicate that the modification of R853C by MTSEA stabilizes the IIS4 segment within the membrane, opposes depolarization-induced movement of the IIS4 segment toward the extracellular side of the membrane, and inhibits voltage-sensor trapping by Css IV.
Toxin-induced Tail Currents Caused by Slow Movement of the IIS4 Segment
The toxin-induced tail currents observed upon repolarization give direct information about the behavior of the toxin-modified channels. The appearance of this tail current upon repolarization is a direct consequence of the IIS4 voltage-sensor trapping and its slow reversal with time after repolarization. The tail currents of toxin-modified channels are different in waveform than tail currents of unmodified channels. For unmodified sodium channels, voltage pulses long enough to allow complete inactivation are not followed by tail currents because one or more of the S4 voltage sensors deactivate before the inactivation gate opens to allow current flow (
Alternative Interpretations of the Results
We have interpreted our results in terms of the voltage-sensor trapping hypothesis presented previously to explain the actions of - and ß-scorpion toxins (
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Footnotes |
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* Abbreviations used in this paper: MTS, methanethiosulfonate; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide.
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Acknowledgements |
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We thank Dr. Marie-France Martin-Eauclaire (CNRS, UMR 6560, Marseille, France) for purifying the ß-scorpion toxin, Css IV.
Submitted: 27 February 2001
Revised: 2 August 2001
Accepted: 3 August 2001
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References |
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