Correspondence to: Nikolaus G. Greeff, Physiologisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland., greeff{at}physiol.unizh.ch (E-mail), Fax: 41-1-635-6814; (fax)
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Abstract |
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The highly charged transmembrane segments in each of the four homologous domains (S4D1S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.
Key Words: mutagenesis, voltage sensor, gating current, two-electrode voltage clamp
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Introduction |
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Voltage-gated sodium channels are highly specialized membrane proteins that react rapidly to small changes of the membrane potential (
Previously, the special role of S4D4 in sodium channel gating was analyzed mainly by measuring ionic currents at single channel or whole cell level. An additional and more direct insight into the gating machinery of these channels is obtained from gating current measurements (
By performing both gating current and ionic current studies of rat brain (rB)IIA1 sodium channel mutants expressed in Xenopus oocytes we were able to give closer insights into the tight structural and functional coupling of S4D4 to the inactivation machinery of the channel. The data show that the mutation of the central arginine residues (R1635H, R1635/1638H, and R1638H) have profound and specific effects on both the inactivation and immobilization properties of the channel. These findings strongly support the hypothesis that S4D4 is the outstanding voltage sensor involved in sodium channel fast inactivation.
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Materials and Methods |
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Mutagenesis and Expression of Channels
The cDNA of wild-type rBIIA sodium channel subunit used in this study was derived from plasmid pVA2580 and transferred into high expression vector pBSTA (both plasmids kindly provided from Dr. A. Goldin, Department of Microbiology and Molecular Genetics, University of California, Irvine, CA). The resulting plasmid pBSTA(
) contains a T7 RNA polymerase promoter and Xenopus-ß-globin 5' and 3' untranslated sequences that greatly increase the expression of exogenous proteins in oocytes (
) subcloned into vector pBSTA. The supercoiled double-stranded DNA template was annealed with two synthetic oligonucleotide primers that contained the desired mutation and were complementary to opposite strands of the vector. Primer extension was performed during temperature cycling using high fidelity Pfu DNA polymerase. Subsequently, the parental dammethylated DNA template was destroyed by DpnI digestion and the mutation-containing synthesized DNA was transformed into Escherichia coli XL1-Blue supercompetent cells (Stratagene Corp.). Mutagenic oligonucleotides were designed such that restriction endonuclease recognition sites were created or deleted. Thus, the desired mutations could be identified by restriction endonuclease analysis of the recombinant plasmid clones. In addition, every mutation was verified by DNA sequencing. Finally, the mutated BglII-SacII subfragment was transferred back into pBSTA(
). At least two independent clones of each mutant were tested to exclude effects of inadvertent mutations in other regions of the channel. Capped, full-length transcripts were generated from SacII linearized plasmid DNA using T7 RNA polymerase (mMessage mMachine In vitro Transcription Kit; Ambion Inc.). Oocytes (stage VVI) from Xenopus laevis (NASCO) were used. 1 d before injection of complementary RNA (cRNA), the oocytes were defolliculated in a Ca2+-free solution containing 2 mg/ml collagenase (Boehringer Mannheim) for ~1 h at room temperature. Oocytes were microinjected with 2040 ng of cRNA (50 nl) and maintained at 18 ± 1°C in Modified Barth's Solution (88 mM NaCl, 2.4 mM NaHCO3, 1 mM KCl, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 10 mM Hepes-CsOH, pH 7.5, supplemented with 25 U penicillin, 25 µg/ml streptomycin-sulfate, and 50 µg/ml gentamycin-sulfate. For the recording of gating currents, 2 µM tetrodotoxin (TTX; RBI-Research Biochemicals International) was added.
Electrophysiology and Data Acquisition
Two-electrode voltage clamp (TEVC) recordings were performed 18 d after cRNA injection with a TEC-05 (npi-electronic) that had been modified for optimized compensation of the series resistance (Rs) and for fast charging of the membrane capacitance (. Macroscopic ionic and gating current signals were recorded using a PDP-11/73 computer (Digital Equipment Corp.) controlling a 16-bit A/D and 12-bit D/A interface (CED). The oocytes were clamped at a holding potential of -100 mV for at least 5 min to ensure recovery from slow inactivation before recording started. The experiments were done at a temperature of +15 ± 1°C controlled by a Peltier element, unless otherwise stated. Rs compensation was adjusted to accelerate the settling time of capacitance transients within 200 µs (without low pass filtering, see below). No analogue subtraction was used, since the 16-bit ADC had a sufficiently fine resolution for digital subtraction of the linear transient and leak currents by scaled averages from pulses between -120 and -150 mV. Reduction of the remaining asymmetry was achieved by finding a compromise between clamping speed and asymmetry, i.e., low-pass filtering the command signal at 5 kHz (eight-pole Bessel). Signals were low-pass filtered at 5 kHz (-3 dB) and sampled at 10 or 20 kHz. The actual clamp speed at the oocyte membrane was determined from the integrated capacitance transient to have a time constant between 150 and 200 µs. A small nonlinearity in leak subtraction appearing occasionally was compensated by baseline correction. Data analysis was performed on the PDP-11 and additionally with the Windows-compatible programs UN-SCAN-ITTM (Silk Scientific Corp.) and PRISMTM (GraphPad Software, Inc.).
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Results |
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Ionic Current Properties of Wild-Type and Mutant Sodium Channels
Na+ currents obtained from Xenopus oocytes injected with either wild-type (WT) or mutant (R1635H; R1635/1638H; R1638H; subsequently named by the position in the S4 segment R4H; R4/5H; R5H) rBIIA sodium channel cRNA display characteristic patterns of voltage-dependent activation and inactivation (Figure 1 A). For well-resolved gating current recordings in Xenopus oocytes, a very high expression of rBIIA sodium channels was necessary. For this purpose the genes of both WT and mutant sodium channels were expressed by use of a high expression vector (see Materials and Methods). Sodium peak currents of 1040 µA, elicited between -10 and -20 mV in 88 mM external sodium, were obtained 24 d after injection of the corresponding cRNA. During this period, only ionic current measurements were performed, because the corresponding gating currents were still too small (<0.5 µA). Rs errors were <5 mV unless the currents exceeded ~2030 µA, because an optimized TEVC was used (see Materials and Methods). Between days 5 and 8, gating currents increased to peaks of 310 µA, whereas ionic currents started to decline after reaching maximum levels of up to -100 µA (
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Compared with WT channels, the time course of inactivation is markedly slowed in R5H and R4/5H but moderately in R4H channels. In contrast, the activation kinetics appear rather similar. The biphasic current decay most clearly visible in WT channels (Figure 1 A) indicates a mixture of fast and slow gating channels in the oocyte membrane. This phenomenon is typical for rBIIA expression in Xenopus oocytes if coexpression of the ß1 subunit is omitted ( subunit because of two reasons: first, at the desired high expression levels, a negative effect of ß1 coexpression on the durability of the oocytes was observed; second, we could not exclude different effects of the ß1 subunit on the inactivation properties of WT and S4D4 mutant channels.
The normalized currentvoltage plots of WT and mutant channels superimpose rather well (Figure 1 B). Nevertheless, the interpretation is difficult as the inactivation kinetics of the analyzed channels are different. Besides of possible Rs effects, wild-type and mutant channels presumably have different inactivation time constant (h) to activation time constant (
m) ratios. This might result in different peak open probabilities and therefore would distort the currentvoltage curve unless
h and
m change in parallel.
The kinetics of macroscopic sodium currents were analyzed by performing single or double exponential fits from normalized current traces at -20, -5, and 20 mV in order to determine the corresponding h and
m values (Figure 2). WT sodium current inactivation was well fit only by a double exponential because of the coexistence of slow and fast gating channels, as already mentioned. In contrast, the mutant sodium currents were well fit by a single exponential. The speed of our TEVC was fast enough to detect an acceleration of the activation kinetics for more depolarizing potentials (Figure 2 B). The
m values of WT and mutant channels do not differ significantly, but the
h values are profoundly increased in the mutant channels according to the sequential order: WT (
h (fast)) < R4H < R4/5H < R5H
WT (
h (slow)). Thus, the S4D4 mutants display a strong effect on inactivation rather than on activation kinetics. The unequal effects of S4D4 mutants on the gating properties of sodium channels were also observed by other groups (
WT and mutant channels display different effects on steady-state inactivation (Figure 1 C). Compared with WT (-61.1 ± 1.2 mV), the midpoint of steady-state inactivation is shifted to more hyperpolarizing potentials in R4H (-74.7 ± 1.2 mV) and shifted to more depolarizing potentials in R5H (-54.2 ± 0.9 mV). No significant shift occurs in the double mutant R4/5H (-61.8 ± 1.4 mV). The slopes of the steady-state inactivation curves, and thus the voltage dependencies, do not differ significantly: WT, 9.55 ± 1.0 mV; R4H, 10.4 ± 1.6 mV; R4/5H, 10.8 ± 1.1 mV; R5H, 10.8 ± 0.7 mV. The steady-state inactivation of R4H indicates that at more depolarizing potentials a substantial portion of the channels is maintained in the inactivated state. Therefore, R4H and R5H show opposite preferences (for the inactivated state and the open state, respectively).
The impact of the mutations on the rate of recovery from fast inactivation was tested at potentials from -80 to -140 mV. Recovery is drastically slowed in R4H, whereas R5H recovers at a speed similar to that of the WT channel (Figure 1 D). These findings agree well with the data of
The results obtained from Figure 1 and Figure 2 clearly demonstrate an antagonism between R4H, which predominantly stabilizes the inactivated state by increasing the recovery time constant (R), and R5H, which mainly impedes the entry into the inactivated state by increasing the inactivation time constant (
h). The inactivation properties of the double mutant are in between the extreme positions of the single mutants with
h values quite similar to R5H and
R values also increased as in R4H, albeit to a much lesser extent.
Gating Current Properties of Wild-type and Mutant Channels
To gain further insights into the coupling of the S4D4 voltage sensor to the inactivation structure of the channel, we analyzed gating currents at the whole oocyte membrane either simultaneously with ionic currents in the same cell or in separate experiments. Compared with the cut-open oocyte method (
In sodium channels the inactivation process is closely coupled to the partial gating charge immobilization, which was demonstrated by the fact that both recover with the same time course (
A summary of chargevoltage plots of WT and mutant sodium channels, generated in the presence or absence of an inactivating pulse and fitted to a standard Boltzmann distribution with slopes, half-activation potentials, and degrees of gating charge immobilization, is given in Figure 4 and Table 1. R5H shows similar gating charge immobilization (48%) compared with WT (56%), whereas both R4H (34%) and R4/5H (34%) display strongly reduced gating charge immobilization. For R4H this observation reflects the fact that at more depolarizing potentials a substantial portion of the channels persists in the inactivated state (see DISCUSSION), as also indicated by the left-shift of the steady-state inactivation curve (Figure 1 C). The slopes and half-activation potentials of WT and R5H only differ slightly from the corresponding values of R4H and R4/5H (Table 1), which indicates the similar activation behavior of WT and mutant channels. The larger values of the immobilized gating charges at potentials more negative than -30 mV compared with the nonimmobilized total ON charges (Figure 4) are most probably due to one of two possibilities. The first possibility is a contamination of the small total ON charges below -30 mV with residual ionic current in absence of an inactivating prepulse. This contamination could not be avoided at the very high expression levels and bath temperatures of 15°C despite of the presence of 2 µM TTX in the bath solution. The second possibility is an integration artifact resulting from a common baseline adjustment for the integration of gating current traces with different kinetics. However, our results were not distorted for that reason because the voltage ranges of main interest were not significantly affected.
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The observed strong effects of R4H and R4/5H on gating charge immobilization clearly support the findings of
Recovery from Fast Inactivation: Correlation of Ionic and Gating Current Studies
The comparison of the recovery time courses of ionic and gating currents yields additional information about the kinetics and voltage dependence of fast inactivation in WT and mutant channels. Figure 5 A illustrates recordings of WT ionic and gating current recoveries obtained from separate oocytes at different stages of expression. We used an alternating pulse protocol with and without prepulse for each recovery time in order to be able to normalize for the slow peakcurrent decay of the reference traces (without prepulse). This current decay results from the presence of a subpopulation of slow gating channels that predominantly appear in the absence of ß1 coexpression (as discussed in the context of Figure 1). During recovery, the fast gating channels recover first, followed by the slow gating channels. The gating currents that were recorded in the presence of 2 µM TTX show a more stable reference current compared with the ionic current, thus suggesting little decay. In view of the large differences of the recovery rates of WT and R4H obtained from ionic current data (see Figure 1 D), we decided that it was more important to analyze the close correlation of ionic and gating current recovery concerning the time course and its voltage dependence, rather than attempting to discriminate the overlapping fast and slow gating channels. The gating current recovery shows a characteristic pattern: (a) the basic level where recovery starts is determined by the degree of immobilization that occurs when the duration and potential of the prepulse fully inactivate ionic current (
Representative gating current recoveries of WT and mutant channels are given in Figure 5 B. The reduced degree of immobilization in R4H and R4/5H compared with WT as already shown in Figure 4 and Table 1 is reflected here by the increased level of the nonimmobilized gating current fraction (Ig,n) at the onset of recovery. On the other hand, the similar levels of Ig,n in R5H and WT indicate that the degree of immobilization is not considerably altered in the mutant. A comparison of the recovery of ionic and gating current at different recovery potentials is illustrated in Figure 6. Three different recovery potentials (-80, -100, and -120 mV) were tested in one cell, with and without an inactivating pulse, and normalized to account for current decay as described in Figure 5. The ionic currents of both WT and mutant channels show a clear voltage dependence of recovery. At -120 mV the recovery potential is strong enough to elicit most of the slow gating channels (Figure 6 A). The normalized gating charge recovery starts at a degree of immobilization of ~0.4 in WT and R5H at all recovery potentials, whereas R4H and R4/5H start at ~0.6 and thus show smaller fractions that recover (Figure 6 B). This reflects the different immobilization properties of the channels, which is consistent with the data of Figure 4 and Figure 5. In view of the preferred occupancy of the inactivated state by R4H during more depolarizing potentials (see Figure 1 C), one should expect that the starting point of gating current recovery in R4H depends strictly on the effective recovery potential. Indeed, the fraction of immobilized channels that recover increases for more hyperpolarizing potentials (Figure 6 B). As observed for the ionic current, a recovery potential of -120 mV is necessary to activate the majority of the channels, and therefore the degree of immobilization in R4H gets closer to the WT level.
One of our main findings is that the time constants of gating current recovery in R4H are drastically slowed down compared with WT and parallel the recovery of the corresponding ionic current (Figure 7 A). This observation is also true if we fit double exponential curves (data not shown). In particular, the effects of the mutations are equally pronounced in both time constants. Correspondingly, the mutations cause no shift in the relative proportions of different kinetic components. Both ionic and gating current recoveries in WT and R4H channels display a similar voltage dependence for most of the voltage range analyzed. However, at -80 mV there is obviously no correlation between the voltage dependence of ionic and gating current recovery in R4H. This apparent mismatch is due to the fact that at more depolarizing potentials a majority of the channels stay immobilized. As can be clearly derived from Figure 6 B, the starting point of the gating charge recovery in R4H strongly depends on the effective holding potential. Consequently, at more depolarizing potentials only a minor portion of the channels participates in recovery from immobilization, yielding recovery curves with low amplitude that are difficult to fit. The strong correlation of ionic and gating current recovery concerning time course and voltage dependence was also observed in R5H and in the double mutant (data not shown).
With respect to the observation that the Ig recovery is considerably slower than the INa recovery in both WT and mutant channels we theorized that this could be due to the fact that the corresponding recoveries were accomplished in separate oocytes at different stages of expression. Consequently, we performed some recovery experiments immediately one after the other in the same cell at 8°C in order to enhance the durability of the oocytes and measured gating current recovery at the sodium reversal potential (ENa). Subsequently, we recorded the ionic current recovery at a potential slightly below ENa, yielding relatively small sodium currents minimally distorted by Rs errors. The partial reduction of sodium currents with submaximal concentrations of TTX was avoided due to the phenomenon of use-dependent block (
The most important conclusions from these experiments are that a point mutation in the central part of S4D4 (R4H) is able to slow down both the release of the inactivation loop and the return of the immobilized voltage sensors similarly in a drastic and voltage-dependent manner, suggesting that these two processes are structurally interconnected; and that the mutation R4H considerably reduces the degree of immobilization in both the single and double mutant, most probably by stabilizing the inactivated state.
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Discussion |
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The currently available data suggest that only the outermost arginines (R1R3) represent the voltage-sensing part of S4D4 (
According to the present understanding (Figure 8 A), the S4S5 linker in domain 4 represents part of the putative receptor that binds the docking region of the intracellular loop connecting domains 3 and 4 (
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We found that the mutation of neighbored arginines in the central part of S4D4 either markedly increases the inactivation time constant (in R5H; Figure 2) or drastically increases the recovery time constant (in R4H; Figure 1 D and 7 A). Therefore, the two mutants display opposite preferences for the open state and the inactivated state, respectively (Figure 1 C). On the other hand, the voltage dependencies are hardly changed in either the single or the double mutant (Figure 1 D, 2, and 7 A). This supports the results of
The actual inactivation process is commonly regarded as a binding of L34 to a receptor site that occurs without voltage dependence in the cytoplasm. With respect to our data we propose that the binding of the inactivation loop to a receptor site at the intracellular mouth of the channel depends on the movement of S4D4; the receptor must first be accessible and then immediately a strong binding of L34 occurs. On one hand, the presentation of the receptor is delayed in R5H, which results in a slowed inactivation, and on the other, the release of L34 from the receptor is delayed in R4H, which extends recovery time from inactivation. In the state diagram (Figure 8 B), this is interpreted as a voltage-dependent conformational change to reach OR, the open state that presents the receptor instantly followed by the voltage-independent binding of L34 leading to the closure of the pore. For recovery from fast inactivation, hyperpolarization should cause the reverse movement of S4D4 and thereby disrupts the binding of the loop to its receptor. Therefore, any mutation that impedes the mobility of S4D4 should have a strong impact on either the inactivation time constant or the recovery time constant.
The macroscopic detectable degree of charge immobilization also reflects the distribution of the channels between the level of the inactivated states (Ig,n level in Figure 8 B) and the level of the C/O states (Ig,t level in Figure 8 B). This means that the actual ratio of channels producing the total gating current fraction (Ig,t) and channels that produce the nonimmobilized gating current fraction (Ig,n) determines the apparent immobilization properties due to an inactivating prepulse. Accordingly, the maximum degree of charge immobilization is obtained when all channels move from the leftmost closed state (C0) to the rightmost inactivated state (I0) during the test pulse and will switch between the I states during the short recovery period after an inactivating prepulse (ratio of Ig,n/Ig,t is minimal). However, if the inactivated states are already occupied at the holding potential by a fraction of channels, this fraction will always produce Ig,n even without inactivating prepulse. Hence, the fraction of channels producing Ig,t is smaller, leading to a reduced degree of apparent charge immobilization. As can be clearly deduced from Figure 6, the degree of immobilization in R4H depends on the effective membrane potential. This means that for more hyperpolarizing potentials (-120 mV) the number of channels passing along the Ig,t level is markedly increased whereas, for more depolarizing potentials (-80 mV) the majority of the channels stay on the Ig,n level. This is consistent with our observation that at a holding potential of -80 mV the ionic current of R4H is profoundly decreased (not recognizable in Figure 6 A because data were normalized). The slightly reduced degree of immobilization in R5H channels compared with WT (Table 1) is due to the fact that fast inactivation and with it the immobilization process is slowed and incomplete (indicated by the distinct plateau current of R5H in Figure 2 A). Consequently, there are less channels at the Ig,n level and more channels moving in both directions at the Ig,t level even after an inactivating prepulse. Finally, the double mutant represents a combination of the R4H and R5H phenotypes showing both slowed and incomplete immobilization and likewise a moderately delayed recovery from immobilization that lead to a degree of immobilization similar to R4H. An additional explanation for the reduction of charge immobilization in R4H and R4/5H may be that the (partial) neutralization of the positively charged arginine R4 leads to a small reduction of Ig,t. In contrast, for R5H this would be less the case, because according to
Regarding the theoretical capacity of S4D4 to contribute to the total gating charge, the degree of immobilization in WT channels implies that not only S4D4 is immobilized during inactivation but that S4 segments of other domains are at least partially involved. Our data do not permit a conclusion as to whether S4D4 and one additional S4 segment of another domain are completely blocked, whereas the two remaining S4 segments are free to move, or whether the return of several S4 segments is partially limited during inactivation. Meanwhile,
According to our molecular model (Figure 8), the open state that presents the receptor (OR) is reached under control of S4D4. The nature of the preceding states (Figure 8 B, shown in brackets) will be discussed now: if OR were preceded by a closed state, S4D4 would simultaneously participate in activation and inactivation. Hence, the S4D4 mutations should slow the inactivation and activation kinetics in parallel, which is not the case as far we can judge (see Figure 2 B), even taking into account some limitations of the clamp speed. Therefore, an activation step from a closed into an open state (C O) that is not affected by the S4D4 mutations appears necessary. Accordingly, the channel stays open during a voltage-dependent phase (O
OR), which is terminated by the voltage-independent attachment of the inactivation gate L34 to the receptor. Moreover, this concept implicates that the mutation R5H impedes the entry into the OR state and not the transition into the I state. This means that the mean open time of R5H should be prolonged and voltage dependent both in WT and mutant channels, and that the R5H channels were not absorbed into the I state since the OR
I rates should be undisturbed, which is indicated by the macroscopic plateau currents of this mutant (see Figure 2 A).
However, the analysis of our mutants on the single channel level has to be studied further. Interestingly, h and furthermore has a prolonged single channel mean open time. In contrast, the mutations in the proposed receptor region, L1660A and N1662A show a burst of short openings as would be expected when the attachment of L34 to the receptor in S4S5D4 is changed. Finally, the putative receptor region identified by
The C O transition either could be caused exclusively by the movement of the S4 voltage sensors of domain 13, or by S4D4 participating in activation during a first step and initiating inactivation during a second step (illustrated in Figure 8). There could also be another two-step process, where the first step of S4D4 just produces some delay before the second step starts inactivation by presentation of the receptor. Although these alternative pathways remain to be cleared in further studies, the main conclusion of this study is that sodium channel fast inactivation is strongly coupled to the mobility of the S4D4 voltage sensor. Then a gating current component should exist that parallels the movement of the inactivation gate. This component is expected to be slow and small in amplitude, as discussed in a previous study, where evidence for such a component had been obtained in high resolution recordings at the squid giant axon (
The observed strong but antagonistic effects of R4H and R5H on the inactivation properties of the sodium channel as well as the phenotype of the double mutant R4/5H support the idea that the central section of S4D4 plays an important role in controlling the movement of the voltage sensor in either direction. Therefore, we propose that the residues R4 and R5 are localized at a critical position concerning the interaction of S4D4 with surrounding channel structures.
Accordingly, it is conceivable that R4 and R5 could be critical determinants for the voltage-driven shift of R2 and R3 involved in the structural interactions that are necessary for this movement. With respect to the observed antagonism, we propose that negative counter charges affect the movement of the positively charged S4D4 residues inside the hydrophobic protein core. For Shaker potassium channels it was demonstrated that there are electrostatic interactions between the positively charged residues of the central to innermost section in S4 and the negatively charged residues in S2 (
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Thus, if either R4 or R5 is replaced by a histidine this leads to a local electrostatic asymmetry and a putative negative countercharge stabilizes either R4(+)-H5 in the open state or H4-R5(+) in the inactivated state (Figure 9). In contrast, in WT the electrostatics are more symmetrical, which allows S4 to move readily in both directions. The electrostatic asymmetry is less pronounced in R4/5H than in the single mutants, leading only to a moderate increase of the inactivation and recovery time constants when compared with WT. The results of
In general, it is rather difficult to compare different mutations without having precise information about the local secondary and tertiary structure of the protein. However, it is possible that the exchange of arginine by the bulky histidine in our study generally slows the mobility of S4D4 in the mutant channels in both directions (O OR and I2
C2; see Figure 8 B), which is particularly apparent in the double mutant. This effect could cover the clear antagonism of the charge neutralizing mutations R4Q and R5Q observed by
Consistent with this idea is the hypothesis of
Finally, another obvious explanation for the different results could be the coexpression of the ß1 subunit by
In our view, the ball and chain hypothesis would be well compatible with voltage-dependent inactivation if one assumes an interaction of the inactivation loop with the cytoplasmic extension of the S4D4 voltage sensor, i.e., the S4S5 linker. Considering the presently available data, it seems very likely that the actual receptor for the inactivation gate resides in the vicinity of S4D4.
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Acknowledgements |
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We thank Dr. Alan L. Goldin for providing the cDNA of the WT rBIIA sodium channel (pVA2580) and the high expression vector (pBSTA), as well as for valuable advice and discussions; we also thank Christian Gasser for his graphical assistance as well as Wolfgang Kathe and Dr. Kevin Martin for helpful discussion and comments on the manuscript.
The work was supported by the Swiss National Science Foundation (grant 31-37987.93) and the Hartmann-Müller-Stiftung.
Submitted: March 18, 1999; Revised: May 17, 1999; Accepted: May 27, 1999.
1used in this paper: cRNA, complementary RNA; ENa, sodium reversal potential; Ig,n, non-immobilized gating current fraction; Ig,t, total gating current fraction; rBIIA, rat brain IIA; Rs, series resistance; TEVC, two-electrode voltage clamp; TTX, tetrodotoxin; WT, wild-type
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References |
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