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MOLECULAR AND GENOMIC |
1 Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Abstract |
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(Received 9 June 2006;
accepted after revision 24 July 2006;
first published online 27 July 2006)
Corresponding author G. F. Tomaselli: Department of Medicine, Johns Hopkins University School of Medicine, 720 Rutland Ave/Ross 844, Baltimore, MD 21205, USA. Email: gtomase1{at}jhmi.edu
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Introduction |
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The clinical significance of slow inactivation in Na+ channels has been validated by abnormalities in slow inactivation that are linked to human neuromuscular diseases such as periodic paralysis (Cummins & Sigworth, 1996; Bendahhou et al. 2002) and epilepsy (Alekov et al. 2001; Spampanato et al. 2004), as well as several potentially lethal cardiac rhythm disorders including Brugada syndrome (Veldkamp et al. 2000), long-QT syndrome (Groenewegen et al. 2003) and cardiac atrioventricular conduction block (Wang et al. 2002). Unlike fast inactivation, however, little is known about the molecular mechanism of slow inactivation in Na+ channels. In Shaker K+ channels, C-type inactivation, named because the process is sensitive to mutations in the C-terminal portion of the channel (Hoshi et al. 1991), involves a conformational change in the outer vestibule (Yellen et al. 1994; Liu et al. 1996). Recent studies suggest that slow inactivation may consist of two structurally distinct processes in K+ channels: First, closure of the outer pore, known as P-type inactivation (De Biasi et al. 1993; Yang et al. 1997); then, stabilization of the closed conformation by C-type inactivation (Olcese et al. 1997; Loots & Isacoff, 1998; Pathak et al. 2005).
Located C-terminal to the putative DEKA selectivity filter, the outer ring of negatively charged residues Glu-Glu-Asp-Asp (EEDD) is highly conserved in mammalian voltage-gated Na+ channels (Table 1). The local electrostatic potential at the level of the EEDD ring is surprisingly large, more negative than –100 mV (Hui et al. 2003). Molecular motions of residues in this ring are coupled to slow inactivation gating based on crosslinking studies of introduced cysteine pairs (Xiong et al. 2003). However, the structural mechanism and specific role of EEDD in slow inactivation gating remain elusive. Here we show that the EEDD ring significantly facilitates the open state of the Na+ channel outer vestibule by, in part, destabilizing outer pore closure that mediates slow inactivation. Neutralization or charge reversal of any residue in this ring markedly favours slow inactivation. Double charge neutralization in the EEDD ring further enhances slow inactivation. The crucial modulation of slow inactivation by this highly conserved outer charged ring suggests that the EEDD ring is a molecular motif regulating the open/closed conformation of the outer vestibule of Na+ channels during slow inactivation.
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Methods |
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A 1.9 kb BamHI–SphI or 2.5 kb SphI–KpnI fragment of the rat µ1 skeletal muscle Na+ channel (rNav1.4) cDNA (Trimmer et al. 1989) was subcloned into pGEM-11Zf+ and pGEM-7Zf+ (Promega, Madison, WI, USA), respectively. Mutagenesis was conducted using the polymerase chain reaction with oligonucleotide primers. Mutations were confirmed by sequencing mutagenic cassettes, which were cloned backed into pSP64T. Complementary RNA was prepared via in vitro transcription using SP6 mMessage mMachine kit (Ambion, Austin, TX, USA). Stage V–VI oocytes were harvested from female Xenopus laevis that had been anaesthetized by immersion in tank water containing 0.15% tricaine methanesulphonate (pH 7.4; Sigma, St Louis, MO, USA). Adequate anaesthesia was accompanied by the absence of a withdrawal response, typically requiring immersion for 15 min. An additional 5–10 min was required in some animals. Frogs were generally not killed at the end of surgery. They were allowed to recover from anaesthesia and surgery. Agile swimming was required to be observed. After final collection of oocytes, frogs were humanely killed. Animal care and handling followed protocols approved by the Animal Care and Use Committee of the Johns Hopkins University.
The 
subunit was coexpressed with the rat brain ß1 subunit (1: 1 weight ratio) (Isom et al. 1992) in Xenopus laevis oocytes as previously described (Tomaselli et al. 1995; Benitah et al. 1997). Injected oocytes were stored in the following solution (mM): NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, Hepes 5, Pyruvate 5, theophylline 0.5, supplemented with penicillin 100 U ml–1 and streptomycin 100 µg ml–1 (pH 7.6 with NaOH) for 1–2 days.
Molecular model and electrostatic computations
The molecular structure model of the NaV1.4 channel (Lipkind & Fozzard, 2000) and electrostatic isopotential surface were generated with Insight II (MSI, Inc., San Diego, CA, USA). The calculations of electrostatic potentials were performed with DelPhi using a finite difference algorithm and a non-linear Poisson–Boltzmann equation (Gilson & Honig, 1987; Sharp & Honig, 1990). The deepest descents and conjugate gradients were employed for the energy minimization procedures. The dielectric constants were set to 80 for the solvent and 10 for the protein interior. The electrostatic potentials are given in units of kT/e, where k is the Boltzmann constant, T is absolute temperature and e is elementary charge. Online data supplementary figures were generated with DeepView.
Electrophysiology and data analysis
Sodium currents were recorded 24–48 h after injection of cRNA using a two-microelectrode voltage-clamp (OC-725B; Warner Instrument Corp.) from oocytes perfused with frog Ringer solution containing (mM): NaCl 96, KCl 2, MgCl2 1, Hepes 5 (pH 7.6 with NaOH). All experiments were performed at room temperature. The methanethiosulphonate (MTS) reagents sodium (2-sulphonatoethyl)-methanethiosulphonate (MTSES) and [2-(trimethylammonium)ethyl] methanethiosulphonate bromide (MTSET) were purchased from Toronto Research Chemicals (Ontario, Canada). Acquisition and analysis of whole-cell currents was performed with custom-written software. The voltage-clamp protocols are provided as insets in Figs 2 and 6. The steady-state fast- and slow-inactivation curves were fitted with the Boltzmann function:
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is the midpoint of inactivation and k is the slope factor. The recovery from fast inactivation and development of slow inactivation were fitted with a first-order exponential function of the form:
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| (2) |
is the time constant. Recovery from slow inactivation was best fitted with double-exponential function:
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Results |
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The outer charged ring E403, E758, D1241 and D1532 (Fig. 1) in the external vestibule of rNav1.4 channel is three to four residues C-terminal to the putative selectivity filter DEKA. However, it is highly conserved in mammalian voltage-gated Na+ channels (Table 1). The EEDD ring, serving to concentrate cations in the channel entryway, has recently been shown to be associated with slow-inactivation gating (Xiong et al. 2003).
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Figure 2 shows the effects of neutralization of single acidic residues in the outer charged ring on the fast and slow inactivation in skeletal muscle Na+ channels (rNav1.4). Neutralization of these charged residues individually (E403C, E758C, D1241C or D1532C) produced no significant changes in the steady state channel availability (Fig. 2A). Recovery from fast inactivation was not different in these single-cysteine-mutant channels from that in the wild type (Fig. 2B). Charge neutralization, however, showed marked enhancement of slow inactivation. The development of slow inactivation was significantly facilitated in E403C, E758C, D1241C and D1532C mutant channels (Fig. 2C), whereas the recovery from slow inactivation was delayed (Fig. 2D). We also observed that slow inactivation, particularly its development, by cysteine substitution at position D1241 was less pronounced when compared with that in other cysteine-mutant channels.
If neutralization of the negative charge in part accounts for the destabilization of the open conformation of the outer pore and enhancement of slow inactivation, then modification of the cysteine substitutions by negatively charged MTS reagents such as MTSES should restore the stabilization of the open state of the channels and retard slow inactivation. Thus, MTSES was applied to these cysteine-mutant channels. Modification by MTSES had no effect on recovery from fast inactivation (Fig. 3A). However, MTSES restored the recovery from slow inactivation in E403C, E758C, D1241C and D1532C mutant channels when compared to wild type (Fig. 3B). Interestingly, entry into slow inactivation in the presence of MTSES was only partially yet significantly restored in E403C (Fig. 3C), but not the other single-cysteine-mutant channels (E758C, D1241C or D1532C, Fig. 3D–F). To further test the effect of charge on slow inactivation, the mutant E403C was modified by the positively charged MTSET. MTSET application had no effect on recovery from fast inactivation in E403C mutant channels (Fig. 4A). In contrast, despite little alteration in the rate of entry into slow inactivation, recovery from slow inactivation was further delayed in E403C mutant channels (Fig. 4B–C). Thus, these results were consistent with the notion that the charge in the ring is an important modulator of slow inactivation, particularly the recovery from slow inactivation.
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If neutralization of one negative charge in the EEDD ring reduced the electrostatic repulsion, it is possible that charge reversal of a single residue could induce additional constriction of the outer charged ring via electrostatic attraction. Additionally, a different amino acid mutant might reveal changes in gating that result from steric effects. Thus, we substituted each of the negative charges in the EEDD ring with an arginine. No significant changes in steady-state fast inactivation and recovery from fast inactivation were found in E403R, E758R, D1241R and D1532R mutant channels (Fig. 5A and B). Entry into slow inactivation was hastened in these arginine-mutant channels compared to the wild type (Fig. 5C); however, enhancement of the development of slow inactivation was no greater than that produced by charge neutralization with single-cysteine-mutant substitutions (E403C versus E403R, E758C versus E758R, D1241C versus D1241R and D1532C versus D1532R) (Figs 5C and 3C) (n.s.). In contrast, recovery from slow inactivation was generally further delayed in arginine substitution mutants, compared with the single-cysteine-mutant channels (Fig. 5D–F). The time constants of recovery from slow inactivation of E758R (2627 ± 152 ms) and D1532R (5077 ± 942 ms) were significantly longer than those for E758C (1345 ± 407 ms) and D1532C (1707 ± 388 ms), respectively (P < 0.05). The time constants of recovery from slow inactivation of E403R (3276 ± 1173) and D1241R (2640 ± 521 ms) tended be longer than those for E403C (2204 ± 698 ms) and D1241C (1741 ± 497 ms), but the difference did not reach statistical significance.
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Mutations changing the charge of the EEDD ring significantly altered entry into slow inactivation. However, we found that in comparison with recovery from slow inactivation, rates of entry into slow inactivation were relatively less sensitive to charge alterations. Both charge neutralization and reversal similarly slowed entry into slow inactivated states. Modification of single-cysteine mutants by negatively charged MTSES nearly completely restored recovery from slow inactivation, but had little effect on the development of slow inactivation in the majority of these cysteine-mutant channels, with the exception of partial restoration in E403C (Fig. 3B–F). In addition, charge reversal had potent effects on the kinetics of recovery, but not the kinetics of the development of slow inactivation when compared with charge neutralization mutants (Figs 2C and 5C). Taken together, these results suggest that entry into slow inactivation, and recovery from slow inactivation involve different structural rearrangements and are not simply forward and reverse processes. Recovery from slow inactivation may contain an additional component that is very sensitive to charge.
Double charge neutralization and enhanced slow inactivation
If neutralization of a single amino acid in the EEDD ring could destabilize the open conformation of the slow inactivation gate, double cysteine mutations might produce further destabilization. The voltage dependence and kinetics of recovery from fast inactivation were not altered in E403C + E758C, E403C + D1241C, E403C + D1532C, E758C + D1532C and D1241C + D1532C mutant channels compared with wild-type or single-cysteine-mutant channels (Fig. 6). The steady-state slow inactivation curve shows that in wild-type channels approximately 20% of channels did not slow inactivate (Fig. 7), a finding consistent with other reports (Featherstone et al. 1996; Richmond et al. 1998). With the exception of D1532R (V= –62.0 ± 3.1 mV versus –53.7 ± 1.58 mV in wild type, P < 0.01), the single-mutant channels did not produce significant changes in the steady-state slow inactivation curves (Fig. 7A and B, Table 2). In contrast, most of the double-cysteine-mutant channels exhibited significant changes in V, slope factor and residual currents (Fig. 7C, Table 2). The E403C + E758C, E758C + D1532C and D1241C + D1532C channels exhibited an approximately 10–20 mV shift of the V in the hyperpolarized direction, a reduction in slope factor and more complete slow inactivation (92–100% versus 80% for the wild type).
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Discussion |
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Voltage-dependent ion channels exhibit multiple kinetically and mechanistically distinct forms of inactivation. In Shaker K+ channels, N-type inactivation is mediated by an intracellular region at the amino-terminus acting as a tethered ‘ball’ occluding the pore (Hoshi et al. 1990). Shaker K+ channels also exhibit C-type inactivation that is affected by mutations in the S6 segments (Hoshi et al. 1991; Boland et al. 1994). C-type inactivation is impaired by application of TEA to the extracellular side of the channels, whereas intracellular application affects N-type inactivation (Choi et al. 1991). In Shaker K+ channels, a consensus has been reached that C-type inactivation is associated with constriction of the outer pore related to structural rearrangements in the P-segments (Yellen et al. 1994; Liu et al. 1996; Harris et al. 1998; Kiss & Korn, 1998), although the precise molecular nature of this inactivation gate is not clear. However, similar to our data, a recent study revealed the interesting observation that P-loop mutants drastically affect C-type inactivation gating in KcsA channels (Cordero-Morales et al. 2006). In KcsA channels, Glu71 forms a strong interaction with Asp80 located at the end of Gly-Tyr-Gly-Asp signature sequence (Zhou et al. 2001). The E71A mutation significantly alters the C-type inactivation by eliminating its interaction with Asp80 and thus initiating further conformational changes in the P-loop that stabilize the open state (Cordero-Morales et al. 2006).
In Na+ channels, fast inactivation is mediated by the cytoplasmic III–IV linker that functions as a tethered pore blocker binding to an inactivation gate receptor in the intracellular mouth of the channel (Vassilev et al. 1989; Patton et al. 1992; West et al. 1992). The structural basis of slow inactivation in Na+ channels is much less clear. Mutations located in the regions other than P-loops, including S6 segments, have been described to influence slow inactivation in Na+ channels (Hayward et al. 1997; Wang & Wang, 1997; Bendahhou et al. 1999; O'Reilly et al. 2001; Wang et al. 2005). On the other hand, a growing body of evidence suggests that the outer pore of Na+ channels is linked to slow inactivation (Tomaselli et al. 1995; Balser et al. 1996; Townsend & Horn, 1997; Kambouris et al. 1998; Todt et al. 1999; Ong et al. 2000; Hilber et al. 2001; Vilin et al. 2001; Hilber et al. 2002; Xiong et al. 2003; Zhang et al. 2003; Fukuda et al. 2005; Pavlov et al. 2005; Tsang et al. 2005). A previous report did not detect a difference in the aqueous accessibility of residues in the outer vestibule between open and inactivated states (Struyk & Cannon, 2002), however, our recent data suggest the accessibility to cysteine-modifying reagents of G1530C and Y401C, located deep in the outer vestibule, is significantly impaired during slow inactivation compared with other channel states (Xiong & Tomaselli, 2005). Taken together, it is most likely that slow inactivation in Na+ channels resembles C-type inactivation in Shaker K+ channels (Kass, 2004).
Does slow inactivation in Na+ channels take place in two steps?
Recent evidence has shown that slow inactivation has two distinct components in Shaker K+ channels. The closure of the outer pore, known as P-type inactivation (De Biasi et al. 1993; Yang et al. 1997), precedes the second step, referred to as ‘C-type inactivation’, which stabilizes the closed conformation via shifting the voltage dependence of recovery from slow inactivation (Olcese et al. 1997; Loots & Isacoff, 1998; Pathak et al. 2005). Upon prolonged depolarization, the gate closes and enters into a less stable P-type inactivation first. Further sustained depolarization leads to a slower entry into more stable C-type inactivation associated with additional rearrangement of or around S4, and a shift in the voltage dependence of the gating charge in the hyperpolarized direction. Therefore, the onset of slow inactivation is time dependent, whereas recovery from slow inactivation becomes intrinsically voltage dependent and involves additional S4 movements (Loots & Isacoff, 1998).
Our data demonstrate that a number of modifications of the outer charged ring in the external channel vestibule (charge neutralization or reversal, charge restoration by MTSES modification) produce significant changes in recovery from slow inactivation. In contrast, charge neutralization altered entry into slow inactivation, but charge restoration or reversal was not associated with additional alterations in the kinetics of the development of slow inactivation. These results suggest that the charged outer ring has a stronger impact on the recovery from slow inactivation (‘C-type inactivation’) than entry into slow inactivation (‘P-type inactivation’), analogous to a two-step slow inactivation in Shaker K+ channels. However, it is unclear how the charges in S4 interact with the charges in the EEDD ring in Na+ channels, although in Shaker K+ channels, the outermost charge in the S4 (R362) has been shown to interact with the outer pore domain, generating an allosteric effect on slow inactivation (Loots & Isacoff, 2000; Elinder et al. 2001).
The role of electrical charge in modulation of slow inactivation
The outer negatively charged ring EEDD located at three to four amino acid residues external to the selectivity filter DEKA is highly conserved in the mammalian voltage-gated Na+ channels (Table 1). The local electrostatic potential at the level of the outer charged ring EEDD has been estimated to be –92.8 to –117 mV (Khan et al. 2002; Hui et al. 2003), with a lower-limit estimate of –58 to –65 mV (Green et al. 1987; Khan et al. 2002). Neutralization of one of these residues enhanced slow inactivation.
A previous report found that only E758C significantly disrupted the kinetics of slow inactivation, but three other cysteine substitutions in this ring, namely E403C, D1241C and D1532C, did not (Struyk & Cannon, 2002). However, the relatively brief depolarization period of 3 s at –10 mV used in this study (Struyk & Cannon, 2002) may have been insufficient to induce slow inactivation (Vilin et al. 1999; Ong et al. 2000; Tsang et al. 2005). In contrast, E403C has been shown to hasten recovery from slow inactivation and enhance entry into slow inactivation (Zhang et al. 2003), consistent with our findings. In our study, restitution of negative charge with MTSES reagents in E403C, E758C, D1241C and D1532C mutant channels restored the recovery from slow inactivation to that of wild-type channels. Reversal of the negative charge of the side chain further facilitated slow inactivation. Some of the double-cysteine mutants such as E403C + E758C drastically enhanced slow inactivation. The time constant of recovery from slow inactivation was increased by 2500%. The strong electrical repulsion present between E403 and E758 at close proximity in the wild-type channel vanishes in the double-mutant channel (Fig. 9). Thus, it is likely that these double mutations would permit a change in the spatial relationship between these two residues and others within the outer pore, facilitating constriction of the external vestibule and channel closure.
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Asymmetrical geometry and electric field
Unlike K+ channels, the Na+ channel is structurally and electrically asymmetrical. Although the electrostatic interactions play an important role in modulation of slow inactivation, the electrical field could be crucially influenced by the asymmetrical geometry of the outer pore. The distance between domain I and domain III is the largest compared with others (Benitah et al. 1997). D1241C in domain III appears isolated from the other three residues in the EEDD ring (Fig. 1B), consistent with a study by Khan et al. (2002). Therefore, the slowest rate of entry into slow inactivation of D1241C compared with the other single-cysteine mutants (Fig. 2C), the absence of additional effects on slow inactivation by either charge reversal (Fig. 5F) or the double-cysteine substitution E403C + D1241C, can be rationalized by the location of D1241 in the outer pore. We further employed computational simulations to examine the feasibility of an electrostatic contribution by the outer charged ring to slow inactivation gating in a contemporary model of the Na+ channel pore (online Supplementary Figs 2 and 3). The effect of the D1241R mutation on the local electrostatic potential is the least among the four EEDD residues, consistent with the insignificant difference in recovery from slow inactivation in D1241R mutant channels compared with D1241C (Fig. 5F). In contrast, the impact of the change from D1532C to D1532R on the local electrostatic potential is the most profound, consistent with the potent enhancement of slow inactivation by D1532R (Figs 5E and 7B). On the other hand, the C to R substitutions at E403 and E758 appear to confer an additional effect on electrostatic potentials in the absence of a significant difference in slow inactivation recovery kinetics between E403R and E403C (Fig. 5F). However, E403 appears to have a unique role in slow inactivation (Xiong et al. 2003). Moreover, modification of E403C by positively charged MTSET further delayed recovery from slow inactivation (Fig. 4C). Thus, we cannot rule out that the structure at the position E403 might be particularly important for slow inactivation.
Unequal enhancement of slow inactivation by different double-cysteine mutants may also be related to asymmetrical geometries and electrostatic fields. The residues in the P-segment of domain IV are mobile, and the distances between residues in domain IV and other domains may be quite large (particularly between domain I and IV) or close, depending on the equilibrium between different states (Benitah et al. 1997). The order of distances between P-loops appears to be: I + III > I + IV (or II + IV) > III + IV > I + II. P-loops in domains I and II are functionally close. Domains I and II are thought to be particularly important for slow inactivation (Kontis & Goldin, 1997; O'Reilly et al. 1999; also see Mitrovic et al. 2000). It has been suggested that if slow inactivation depends on S4 segment mobility, immobilization of the S4 segment by fast inactivation may inhibit slow inactivation (Featherstone et al. 1996; Richmond et al. 1998). Furthermore, in Na+ channels, the S4 voltage sensors in domains III and IV, but not I and II, are immobilized by fast inactivation (Cha et al. 1999). Thus, S4 segments in domains I and II may make a more significant contribution to slow inactivation, although it remains unclear that how S4 segments couple to slow inactivation in Na+ channels.
A molecular motif regulating the outer pore conformation during slow inactivation
We have shown that the EEDD ring plays a crucial role in modulation of slow inactivation. The natural question is whether this EEDD ring is the slow-inactivation gate or part of the gate. It does not appear to us that the EEDD ring alone constitutes the slow-inactivation gate, although we cannot exclude that it could be part of a slow-inactivation gate if slow inactivation involves a more global rearrangement of the outer pore (Cha & Bezanilla, 1997; Loots & Isacoff, 1998). Instead, it is a molecular motif regulating the open versus closed state of the outer pore during slow inactivation of Na+ channels. In Shaker K+ channels, the gating motion of S4 is linked to slow inactivation (Pathak et al. 2005). The interaction coupling S4 motion and pore closure is likely to be an allosteric effect, possibly involving a strain on the S4–S5 linker and P–S6 loop that subsequently rotate and cause the outer pore to close. In Na+ channels, the S4s of all domains are involved in the slow inactivation, particularly domain I and II (Kontis & Goldin, 1997). Interestingly, the µ-conotoxin with its the strongest interaction sites at E403 and E758 (Dudley et al. 1995; Chang et al. 1998) can inhibit slow inactivation by serving as a ‘splint’ to stabilize the open conformation of the outer vestibule (Todt et al. 1999).
In summary, E403, E758, D1241 and D1532 in each of the four domains work in concert to regulate the open/closed conformation of the Na+ channel outer pore. In contrast to previous reports of disparate residues in cytoplasmic S4–S5 linkers (Bendahhou et al. 2002), S5 (Bendahhou et al. 1999) and S6 segments (Hayward et al. 1997; Wang & Wang, 1997), the EEDD ring serves as a molecular motif critically modulating slow inactivation in mammalian voltage-gated Na+ channels.
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References |
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