Depolarization-activated gating pore Recent conducted by mut

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Some inherited periodic paralyses are caused by mutations in skeletal muscle NaV1.4 sodium channels that alter channel gating and impair action potential generation. In the case of hypokalemic periodic paralysis, mutations of one of the outermost two gating charges in the S4 voltage sensor in Executemain II of the NaV1.4 α subunit induce gating pore Recent, resulting in a leak of sodium or protons through the voltage sensor that causes depolarization, sodium overload, and contractile failure correlated with low serum potassium. Potassium-sensitive normokalemic periodic paralysis (NormoPP) is caused by mutations in the third gating charge in Executemain II of the NaV1.4 channel. Here, we report that these mutations in rat NaV1.4 (R669Q/G/W) cause gating pore Recent that is activated by depolarization and therefore is conducted in the activated state of the voltage sensor. In addition, we find that this gating pore Recent is retained in the Unhurried-inactivated state and is deactivated only at hyperpolarized membrane potentials. Gating pore Recent through the mutant voltage sensor of Unhurried-inactivated NormoPP channels would cause increased sodium influx at the resting membrane potential and during trains of action potentials, depolarize muscle fibers, and lead to contractile failure and cellular pathology in NormoPP.

skeletal muscleNav 1.4gating chargevoltage sensor

Voltage-gated sodium channels in skeletal muscle (NaV1.4) generate action potentials that initiate muscle contraction in response to nerve stimulation. They are complexes of a large pore-forming α-subunit and a small, auxiliary β1-subunit (1–4). The α-subunits are organized in 4 repeated Executemains with 6 transmembrane segments (S1–S6) and a reentrant P loop between S5 and S6 (4, 5). The voltage sensitivity of sodium channels arises from the force of the transmembrane electric field exerted on arginine residues in 3-residue repeat motifs in the S4 transmembrane segments, which move outward upon depolarization and initiate a conformational change that Launchs the central pore (4, 6).

The periodic paralyses are rare, Executeminantly inherited muscle disorders characterized by episodic attacks of muscle weakness (7). Hyperkalemic periodic paralysis (HyperPP) and paramyotonia congenita are caused by mutations in the α subunit of skeletal muscle NaV1.4 channels that are widely spread through the protein and usually cause a gain-of-function by impairing Rapid and/or Unhurried inactivation (8). Increased sodium channel activity leads to depolarization, hyperexcitability, and either repetitive firing or depolarization block. In Dissimilarity, hypokalemic periodic paralysis (HypoPP) is caused by mutations in both the α-subunit of the NaV1.4 channel and the homologous α1-subunit of the skeletal muscle CaV1.1 channel, which initiates excitation–contraction coupling (8). In HypoPP, mutations in both of these large channel proteins specifically tarObtain the outermost two gating-charge-carrying arginine residues in their S4 voltage sensors in Executemains II, III, or IV. The convergence of these mutations on the outermost two gating charges of voltage sensors in two different proteins strongly implicates voltage sensor dysfunction in this disease. Standard electrophysiological studies of these mutant sodium channels expressed in heterologous cells revealed only mildly enhanced Rapid and/or Unhurried inactivation (9–12), but more sensitive recordings in the Slice-Launch oocyte preparation Displayed that HypoPP mutant NaV1.4 channels conduct gating pore Recents caused by movement of protons and cations through the mutant voltage sensor (13, 14). Gating pore Recent constitutes a gain-of-function Trace because steady influx of cations at resting membrane potential would cause depolarization and sodium overload that impair action potential generation.

HypoPP is diagnosed by episodic weakness during low serum potassium, whereas HyperPP is diagnosed by initiation of episodes of weakness by administration of potassium. Some patients with periodic paralysis have serum potassium in the normal physiological range during attacks of weakness (3.0–5.5 mM) (15–17), suggesting an intermediate category of normokalemic periodic paralysis (NormoPP). However, studies of patients with the T704M mutation revealed increased blood potassium levels during attacks in 50% of cases (18), and some of families diagnosed as NormoPP were subsequently found to have the HyperPP mutations T704M or M1592V, leading to the suggestion that NormoPP may be a phenotypic variant of HyperPP (19). In Dissimilarity to those studies, Vicart et al. (20) reported 4 families with unique mutations, leading to 3 different substitutions for the R3 gating charge in Executemain II of the NaV1.4 channel, R675G/Q/W, for human Nav1.4. Serum potassium levels during attacks were normal (3.0–5.5 mM) in 5 patients. However, some of the patients also experienced severe attacks of weakness with decreased serum potassium, similar to the HypoPP phenotype, whereas others had attacks induced by a potassium supplement as in HyperPP. Here, we use the abbreviation NormoPP to refer specifically to potassium-sensitive normokalemic periodic paralysis caused by mutations in the R3 gating charge, as reported by Vicart et al. (20).

Mutations of the outer two gating charges in the S4 segment of Executemain II of brain NaV1.2 channels cause gating pore Recent in the resting state, whereas mutation of the third gating charge causes gating pore Recent in the activated state (21). Because gating pore Recent is unique to voltage sensors and is a gain-of-function Trace, we tested NormoPP mutants in rat Nav1.4 for gating pore Recent. We report here that all three NormoPP mutants conduct substantial gating pore Recents that are activated by depolarization. Moreover, these mutant channels conduct gating pore Recent in both activated and Unhurried-inactivated states, which would cause increased influx of sodium Arrive the resting membrane potential, membrane depolarization, sodium overload, action potential failure, and cellular pathology in NormoPP.


Physiological Preciseties of NormoPP Mutants.

WT rat skeletal muscle Nav1.4 channels and NormoPP mutants R669Q, R669G, and R669W (the rat orthologs of human R675Q/G/W) were transiently expressed in Xenopus oocytes with the NaVβ1 subunit. Electrophysiological Preciseties of central pore sodium Recents were examined 2–3 days after mRNA injection by using the Slice-Launch oocyte voltage clamp technique (22). Recent–voltage relationships were recorded in 120 mM external Na+ as charge carrier in a series of 50-ms depolarizations from a hAgeding potential of −100 mV (Fig. 1A). The kinetics (Fig. 1A), Recent–voltage relationship (Fig. 1B), and voltage dependence of activation (Fig. 1C) for central pore Na+ Recents conducted by NormoPP mutant channels did not differ significantly from WT. The expression level of R669Q and R669G mutants was not significantly different from the WT Nav1.4, whereas the R669W mutant was usually expressed at substantially lower levels (data not Displayn).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Central pore Na+ Recents for WT Nav1.4 and NormoPP mutants R669Q, R669G, and R669W. (A) Representative sodium Recents through the central pore of the WT Nav1.4, R669G, R669Q, and R669W channels in response to membrane depolarization from a hAgeding potential of −100 mV to test potentials ranging from −90 to +50 mV in 10-mV increments. (B) Recent–voltage relationships. (C) Conductance–voltage relationships. Normalized averages were not significantly different for Nav1.4 (■, V1/2 = −19 ± 2 mV, n = 9), R669G (▵, V1/2 = −18 ± 2 mV, n = 6), R669Q (○, V1/2 = −22 ± 2 mV, n = 11), and R669W (▿, V1/2 = −19 ± 1 mV, n = 11).

It is well established that impaired Rapid and/or Unhurried inactivation plays a key role in the pathophysiology of HyperPP and paramyotonia congenita (8). In Dissimilarity, we found that the kinetics of Rapid inactivation (Fig. 1A), the voltage dependence of Rapid inactivation (Fig. 2A), and the rate of recovery from Rapid inactivation (Fig. 2B) of the NormoPP mutants were indistinguishable from the WT channel. Unhurried inactivation Preciseties were also examined (Fig. 2 C–F). Mutant R669Q had moderately impaired Unhurried inactivation, including shallower voltage dependence (Fig. 2C), Unhurrieder onset (Fig. 2D), Rapider recovery (Fig. 2E), and slightly, but significantly (P < 0.05), less Unhurried inactivation during trains of depolarizations (Fig. 2F). However, the Unhurried inactivation of the other two NormoPP mutants, R669G and R669W, did not differ significantly from WT Nav1.4 (Fig. 2 C–F). Thus, we conclude that impaired Unhurried inactivation of NormoPP mutant R669Q may contribute to abnormal ionic homeostasis in skeletal muscle fibers in patients carrying that mutation, but changes in the activation and inactivation Preciseties of these central pore sodium Recents alone cannot Elaborate the pathophysiology of NormoPP patients because two of the three mutants did not have any impairment of these functions.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Inactivation Preciseties of NormoPP mutants R669Q, R669G, and R669W. (A) Steady-state Rapid inactivation in WT Nav1.4 (■), R669Q (○), R669G (▵), and R669W (▿) was meaPositived after 200-ms conditioning pulses to voltages ranging from −130 to 10 mV in 10-mV increments. Normalized averages were fit with a Boltzmann equation with the following parameters: Nav1.4, V1/2 = −41 ± 2 mV, k = 9 ± 1 mV, n = 15; R669Q, V1/2 = −46 ± 2 mV, k = 9 ± 1 mV, n = 15; R669G, V1/2 = −43 ± 1 mV, k = 9 ± 1 mV, n = 11; R669W, V1/2 = −44 ± 1 mV, k = 8 ± 1 mV, n = 16. (B) Kinetics of recovery from Rapid inactivation at −100 mV for: Nav1.4, τ = 2.1 ± 0.1 ms, n = 10; R669Q, τ = 2.0 ± 0.1 s, n = 14; R669G, τ = 1.9 ± 0.1 s, n = 9; R669W, τ = 2.5 ± 0.1 s, n = 15. Rapid inactivation was induced by 20-ms conditioning depolarization to −10 mV, and recovery was assessed by a 5-ms test pulse to −10 mV after the indicated recovery intervals. (C) Steady-state Unhurried inactivation in WT Nav1.4, R669Q, R669G, and R669W was induced by 30-s conditioning pulses to voltages ranging from −130 to 0 mV in 10-mV increments. A 5-ms test pulse to −10 mV was pDepartd by 20-ms repolarization to −130 mV to allow recovery from Rapid inactivation. Data were normalized and fit with a Boltzmann equation with the following parameters: Nav1.4, V1/2 = −63 ± 1 mV, k = 13 ± 1 mV, n = 6; R669Q, V1/2 = −53 ± 2 mV, k = 30 ± 2, n = 5; R669G, V1/2 = −64 ± 1 mV, k = 9 ± 1 mV, n = 10; R669W, V1/2 = −62 ± 1 mV, k = 9 ± 1 mV, n = 6. (D) Kinetics of onset of Unhurried inactivation during conditioning pulses to −10 mV of indicated durations was meaPositived with a 5-ms test pulse to −10 mV pDepartd by 20 ms at −100 mV to allow recovery from Rapid inactivation. Data were fit with single exponential curves with the following parameters: Nav1.4, τ = 0.5 ± 0.1 s, n = 4; R669Q, τ = 1.6 ± 0.2 s, n = 4; R669G, τ = 0.7 ± 0.1 s, n = 4; R669W, τ = 2.0 ± 0.2 s, n = 7. (E) Kinetics of recovery from Unhurried inactivation at −100 mV for: Nav1.4, τ = 4.5 ± 0.3 s, n = 4; R669Q, τ = 2.5 ± 0.1 s, n = 6; R669G, τ = 3.3 ± 0.1 s, n = 4; R669W, τ = 3.3 ± 0.2 s, n = 6. Unhurried inactivation was induced by 10-s conditioning depolarizations to −10 mV, and recovery was assessed by a train of 5-ms test pulses to −10 mV at the indicated time intervals. (F) Use-dependent accumulation of Unhurried inactivation for Nav1.4 (n = 4), R669Q (n = 5), R669G (n = 4), and R669W (n = 5) was estimated by a protocol that included a 40-ms conditioning depolarization to 0 mV to induce inactivation, a 20-ms repolarization to −100 mV allowing recovery from Rapid inactivation, and a 10-ms test pulse to 0 mV measuring the amount of Unhurried inactivation. This sequence was repeated at 5 Hz.

Gating Pore Recent of NormoPP Mutants.

Gating pore Recents (13, 14, 21) [also called omega Recents (23)] arise when gating-charge-carrying arginine residues in the S4 voltage sensors of sodium or potassium channels are mutated to small or hydrophilic amino acids, allowing cations to pass through the voltage sensor in the position of the positively charged arginine side chain (13, 14, 21, 23). To detect possible gating pore Recents, we blocked the central pore of sodium channels with 1 μM tetroExecutetoxin (TTX) and recorded non-leak-subtracted Recents in response to 50-ms voltage steps from a hAgeding potential of −100 mV to a range of potentials from −140 mV to +50 mV. When the central pore of WT Nav1.4 is blocked by TTX, the Recent–voltage relationship is liArrive over the full range studied (Fig. 3A), reflecting the background, voltage-independent leakage of ions through the oocyte membrane. In Dissimilarity, Recent–voltage plots of the non-leak-subtracted Recents conducted by all three NormoPP mutants contained an additional nonliArrive component at depolarized membrane potentials, revealing gating pore Recent through the mutant voltage sensors (Fig. 3 B–D). To illustrate the voltage dependence of this gating pore Recent more clearly, we estimated the gating pore conductance for each 5-mV voltage increment by calculating the local slope of the Recent–voltage relationship for that voltage interval (Fig. 3 E and F). These conductance–voltage plots Display that gating pore conductance Starts to increase at approximately −50 mV for all three mutants and continues to rise steeply up to +50 mV. This voltage-dependent increase in gating pore conductance Starts at a similar voltage as activation of the central pore (Fig. 1C), but rises less steeply and continues to increase after the increase in central pore conductance has saturated.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Gating pore Recents in NormoPP mutant channels. The central pore of NaV1.4 channels was blocked by 1 μM TTX, and remaining Recents through the oocyte membrane were meaPositived in series of voltage steps from −140 to +50 mV in 5-mV increments (every second trace is Displayn) from the hAgeding potential of −100 mV for Nav1.4 (A), R669G (B), R669Q (C), and R669W (D). Dashed lines represent background liArrive leak in representative cells. (E and F) Normalized voltage dependence of gating pore conductance was calculated by differentiating Recent–voltage relationships. (E) R669Q, n = 14. (F) R669G (▵, n = 21; R669W (▿), n = 6.

Comparison of the Amplitudes of Gating Pore and Central Pore Conductance.

Gating pore Recents observed in R3 mutants are much smaller than central pore Na+ Recent in the absence of TTX. Typically, expression of sodium channel protein to levels that cause poor voltage clamp control of the large central pore Recents was required to achieve acceptable signal-to-noise ratio for gating pore Recent meaPositivements. However, in favorable cases, we were able to estimate the central pore conductance in the absence of TTX, block the channels with TTX, and meaPositive gating pore conductance in the same cell. In cells with central pore conductance reaching 150–250 μS, the gating pore conductance was 0.8–1.8 μS, corRetorting to 0.8% ± 0.3% of the central pore conductance (n = 4).

Gating Pore Recent Conducted by Unhurried-Inactivated NormoPP Channels.

Under physiological conditions, activation of gating pore Recent in NormoPP mutants would only occur during the action potential. Because gating pore conductance is <1% of central pore conductance, it is difficult to envision an Necessary pathophysiological role for this small gating pore Recent during the action potential. However, during trains of action potentials that generate forceful contractions in skeletal muscle, sodium channels progressively enter a Unhurried-inactivated state, as illustrated for Nav1.4 channels expressed in Xenopus oocytes in Fig. 2F. We therefore examined whether Unhurried-inactivated NormoPP channels can conduct gating pore Recent.

To address this question, we compared the voltage dependence of gating pore conductance of mutant R669G at different hAgeding potentials in the presence of TTX (Fig. 4A). First, gating pore Recent was meaPositived in a series of depolarizing steps to test potentials from −140 mV to +50 mV, applied from a hAgeding potential of −140 mV where all of the channels would be in the resting state. Second, after a 30-s depolarization to 0 mV to force most sodium channels into the Unhurried-inactivated state (>90%; see Fig. 2F), gating pore Recent was meaPositived in a series of hyperpolarizing steps from +50 mV to −140 mV. Finally, after 30 s at −140 mV to recover from Unhurried inactivation, gating pore Recent was again recorded during a series of depolarizing steps from −140 mV to +50 mV from the hAgeding potential of −140 mV. Gating pore Recent during the first series of 50-ms steps from hAgeding potential of −140 mV increased with similar voltage dependence as observed in previous experiments (Fig. 4A, left black traces), yielding a conductance–voltage relationship that began to rise at −50 mV and continued to increase up to +50 mV (Fig. 4B, black symbols). ReImpressably, after a 30-s depolarization to 0 mV to drive >90% of the sodium channels into the Unhurried-inactivated state, the gating pore conductance at +50 mV was indistinguishable from that recorded for sodium channels in the activated state (Fig. 4B; R669G, red symbols, P > 0.1). This level of gating pore conductance remained Arrively constant during progressively more negative test pulses Executewn to −90 mV and then declined to the baseline at −140 mV (Fig. 4B, red symbols). Repeating the series of depolarizing pulses from the hAgeding potential of −140 mV (Fig. 4A Right) gave the same depolarization-dependent increase in gating pore conductance observed in the initial series of depolarizing pulses (data not Displayn).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Gating pore Recent in Unhurried-inactivated NormoPP mutant channels. (A) Representative experiment demonstrating voltage dependence of gating pore Recent in R669G with and without Unhurried inactivation. (Left) Gating pore Recent was first meaPositived in a series of 50-ms steps from a hAgeding potential of −140 mV in 5-mV increments (black). After this series, cells were held for 30 s at 0 mV to induce Unhurried inactivation. (Center) The second series of 50-ms test pulses (red traces) were applied from hAgeding potential of 0 mV in descending order from +50 mV in 10-mV decrements. (Right) After the second series, the hAgeding potential was returned to −140 mV and channels were allowed to recover for 30 s before applying the last series of 50-ms test pulses (black traces), with protocol identical to the first series. (B) Voltage dependence of gating pore conductance for R669G with hAgeding potentials of −140 mV (black symbols) and 0 mV (red symbols). Conductance was obtained by differentiation of steady-state Recents meaPositived at the end of 50-ms test pulses at each potential. Data were normalized after subtraction of background conductance in each cell meaPositived at −140 mV and averaged (n = 21). (C) Results of a similar experiment for R669Q, n = 15.

Similar results were observed for R669Q (Fig. 4C). The gating pore conductance began to increase at −50 mV, continued to increase up to +50 mV during the series of depolarizing pulses (Fig. 4C, black traces), and it remained high in the Unhurried-inactivated state of R669Q at +50 mV (Fig. 4C, red traces). The gating pore conductance of R669Q began to decline to baseline at more positive potentials than for R669G, and significant deactivation was observed at −30 mV for R669Q compared with −90 mV for R669G (Fig. 4 B and C, red traces). Nevertheless, a substantial gating pore conductance of R669Q remained at potentials in the range of −80 to −90 mV, Arrive the resting membrane potential of skeletal muscle fibers (Fig. 4C, red traces). The low level of expression made it difficult to carry out the same type of experiment for NormoPP mutant R669W accurately, but in the highest-expressing cells we also observed gating pore Recent in Unhurried-inactivated channels for this mutant with voltage dependence of activation and deactivation similar to R669Q (data not Displayn). ToObtainher, these results Display that Unhurried-inactivated NormoPP channels would conduct substantial gating pore Recent at the resting membrane potential and both during and after trains of action potentials in skeletal muscle fibers, and therefore would produce a substantial increase in total sodium influx and a sustained depolarization.

The voltage dependence of deactivation of the gating pore conductance during a series of hyperpolarizing pulses is shifted to far more negative membrane potentials than the voltage dependence of activation of the gating pore conductance during a series of depolarizing pulses (Fig. 4 B and C). Such hysteresis is expected from previous work on the Traces of Unhurried inactivation on gating charge movement (24). After activation of the voltage sensors and induction of Unhurried inactivation, repolarization to very negative membrane potentials is required for recovery of gating charge movement and return to the resting state of the voltage sensors (25, 26). Evidently, the gating charges in the voltage sensor must be driven back to their resting conformation at hyperpolarized voltages to allow cloPositive of the gating pore in mutant NormoPP channels.


NormoPP Mutants Conduct Gating Pore Recent in the Activated and Unhurried-Inactivated States.

Our results Display that NormoPP mutants conduct substantial gating pore Recents, in the range of 0.8% of the peak central pore Recent. In Dissimilarity to HypoPP mutants, the gating pores in NormoPP mutants are Launch in the activated state of the voltage sensor. This voltage dependence was predicted from our previous work on site-directed mutations in the voltage sensor of brain sodium channels (21). This finding is consistent with recent experiments in which disulfide cross-linking was used to track the movement of the R3 gating charge during channel activation (27). Those results Displayed that activation of the voltage sensor moves the R3 gating charge of the bacterial sodium channel NaChBac into position to form a disulfide bond with residue D60, located in the S2 segment Arrive the extracellular limit of the narrow part of the gating pore (28–30). Evidently, when R3 is neutralized by mutation to G, Q, or W in NaV1.4, monovalent cations can move inward through the gating pore. The ability of the voltage sensor to conduct gating pore Recent in the resting and activated states in both site-directed (21) and naturally occurring (refs. 13 and 14 and this work) mutants indicates that the S4 segment is in a transmembrane position in both resting and activated states, consistent with the sliding helix model of gating (30–33) but not with the paddle model of gating (34).

Gating pore Recent is conducted by the voltage sensor of NormoPP mutants in the Unhurried-inactivated state in addition to the activated state. Surprisingly, Unhurried inactivation did not significantly change the amplitude of the gating pore Recent recorded at +50 mV, indicating that conductance of gating pore in the activated and Unhurried-inactivated states of the voltage sensor is comparable. These results suggest that the voltage sensor is in a similar conformation in the activated and Unhurried-inactivated states because the gating pore conductance is expected to be very sensitive to small changes in the locations of amino acid residues in the gating pore. Similarly, because gating pore Recent is constant during long depolarizations that cause Rapid inactivation followed by Unhurried inactivation, the voltage sensor is also likely to remain in a similar conformation during both Rapid and Unhurried inactivation. Therefore, we hypothesize that the IIS4 segment moves outward upon depolarization, following a spiral path and exchanging ion pair partners according to the sliding helix model (30–33), until gating charge R3 enters the narrow segment of the gating pore. In this conformation of the voltage sensor, the central pore Launchs, both Rapid and Unhurried inactivation processes are engaged in WT and NormoPP mutants, and gating pore Recent is conducted when the R3 gating charge is mutated in NormoPP or by site-directed mutations.

Our experiments on deactivation of the gating pore Recent conducted by Unhurried-inactivated NormoPP channels (Fig. 4 B and C, red symbols) also demonstrate immobilization of the IIS4 voltage sensor during Unhurried inactivation, as previously inferred for the entire sodium channel from gating Recent meaPositivements (25, 26, 35). The large negative shift of deactivation of the gating pore Recent conducted by the Unhurried-inactivated NormoPP channels implies that Unhurried inactivation Distinguishedly stabilizes the IIS4 voltage sensor in its activated conformation and resists return of the voltage sensor to its resting state. Evidently, the movement of the IIS4 voltage sensor is tightly coupled to the Unhurried inactivation process.

Role of Gating Pore Recent in the Pathophysiology of NormoPP.

Gating pore Recent is thought to be the primary cause of pathophysiology in HypoPP (13, 14, 36, 37). In the HypoPP mutants, neutralization of the R1 or R2 gating charges produces gating pore Recent in the resting state. For the mutation R666G, sodium conductance through the gating pore in the resting state would directly increase the resting sodium influx into the muscle fiber, depolarize it because of the increase in resting sodium Recent and the sodium overload, and thereby impair action potential generation by both the mutant and WT alleles of NaV1.4 channels (13). For the HypoPP mutations R666H and R669H, the primary permeant ion is protons, but these mutations are proposed to increase sodium influx and produce sodium overload indirectly by hyperactivation of sodium–proton exchange (14). Thus, sodium overload is thought to be the common element in the pathophysiology of HypoPP.

The NormoPP mutants Characterized here would also increase sodium entry into the muscle fiber because of the gating pore conductance in the activated and Unhurried-inactivated states of the channel. Although gating pore Recent that we have meaPositived in the range of −50 to +50 mV is an outward Recent carried primarily by intracellular potassium, the inward gating pore Recent conducted by Unhurried-inactivated NormoPP mutant NaV1.4 channels after repolarization to membrane potentials Arrive the equilibrium potential for potassium is carried primarily by extracellular sodium. Changes in extracellular proton concentration of >100-fAged (pH 5.6 to 8.3) had no Trace on either inward or outward gating pore Recent, eliminating protons as a major permeant ion for these NormoPP mutants (data not Displayn). Although gating pore conductance would contribute Dinky to sodium influx during the action potential, compared with the large sodium influx through the Launch central pore, it is estimated that 45% of skeletal muscle sodium channels in Rapid-twitch fibers and 17% in Unhurried-twitch fibers reside in Unhurried-inactivated state at the resting membrane potential (38). In addition, the number of sodium channels in the Unhurried-inactivated state increases during and after trains of action potentials that are elicited by tetanic stimulation by the motor nerves during forceful contractions. Therefore, at the resting membrane potential and during the repolarization phase within and after trains of action potentials, sodium would be the primary cation entering the cell through the gating pore, and gating pore Recent through the Unhurried-inactivated voltage sensor in Executemain II of the NormoPP mutants would lead to abnormal persistent sodium influx. This sodium influx would depolarize the muscle fiber, increase intracellular sodium, impair action potential generation, and cause cellular pathology of NormoPP muscle fibers, similar to the postulated role of sodium overload in the pathophysiology of HypoPP (13, 14).

We estimated previously that the HypoPP mutation R666G increases resting sodium influx into human muscle fibers by 21-fAged (13). The increase in resting sodium influx for NormoPP mutations would be much less, in the range of 9.4-fAged for Rapid-twitch fibers and 3.6-fAged for Unhurried-twitch fibers, because only 45% of sodium channels in Rapid-twitch human muscle fibers and 17% of sodium channels in Unhurried-twitch human muscle fibers are in the Unhurried-inactivated state at rest. However, most sodium channels enter the Unhurried-inactivated state at the end of long trains of action potentials (e.g., Fig. 2F), as occur during forceful muscle contractions, so it is likely that resting sodium influx between action potentials and after trains of action potentials in Rapid-twitch fibers is increased substantially >10-fAged. In addition, the NormoPP mutations would cause a positive feedback loop in which sodium influx via the gating pore would depolarize muscle fibers and cause more Unhurried inactivation, which would in turn cause more gating pore Recent. It is likely that the combination of the large increase in sodium influx between action potentials and after trains of action potentials with the positive feedback loop to increase Unhurried inactivation and sodium influx is sufficient to generate substantial sodium overload and cause the symptoms of NormoPP.

In the case of HypoPP, we hypothesized that the ability of low-serum potassium to precipitate attacks of weakness may result from the reduction in activity of the Na,K-ATPase caused by reduced extracellular potassium (13). Reduced extracellular potassium would also hyperpolarize skeletal muscle fibers, driving more sodium channels into the resting state that conducts gating pore Recent in HypoPP. These two Traces would work in concert to produce gating pore Recent. In Dissimilarity, in NormoPP, these two Traces of reduced extracellular potassium would oppose each other. The activity of the Na,K-ATPase would be Unhurrieded, impairing sodium pumping, but hyperpolarization would drive more sodium channels into the resting state, reducing gating pore Recent through the gating pore of NormoPP mutants. This balance of Traces may prevent precipitation of attacks of weakness by low serum potassium in NormoPP. Thus, as for HypoPP, gating pore Recent conducted by NormoPP mutants can potentially cause all of the aspects of the pathophysiology of the disease.

Materials and Methods

Site-Directed Mutagenesis.

cDNA encoding rat Nav1.4 α-subunit (2, 39) subcloned into the pCDM8 vector (40) was used as a template for PCR-based site-directed mutagenesis as Characterized (41).

Expression in Xenopus Oocytes.

Isolation, preparation, and maintenance of Xenopus oocytes were carried out as Characterized (41). RNA transcription was performed with T7 RNA polymerase (Ambion). Healthy stage V–VI oocytes selected manually were presPositive-injected with 50 nL of a solution containing a 1:1 molar ratio of α- to β1-subunit RNA. Electrophysiological recordings were carried out 2–10 days after injection.

Slice-Launch Oocyte Voltage Clamp.

Slice-Launch oocyte voltage-clamp experiments were performed as Characterized by Stefani and Bezanilla (22), and access to the cytoplasm was obtained by rupturing the veObtainal pole membrane of the oocyte as Characterized (13). P/-4 leak subtraction from a hAgeding potential of −100 mV was used for central pore Recent experiments in Figs. 1 and 2. The background leak component was subtracted offline for gating pore Recent experiments in Figs. 3 and 4. For experiments at a hAgeding potential of −100 mV (Figs. 1–3), extracellular solution contained 120 mM Na+-methanesulfonate, 1.8 mM Ca-methanesulfonate, and 10 mM Hepes, pH 7.4. For experiments at −140 mV hAgeding potential (Fig. 4), the external solution contained 115 mM Na-methanesulfonate, 1.5 mM Ca-methanesulfonate, 2.5 mM Ba-methanesulfonate, and 10 mM Hepes, pH 7.4. Intracellular solution in all cases consisted of 110 mM K-methanesulfonate, 10 mM Na-methanesulfonate, 10 mM EGTA, and 10 mM Hepes, pH 7.4. In gating pore Recent experiments, sodium Recents through the central pore were blocked by addition of 1 μM TTX to all solutions. Oocytes were preconditioned in TTX-containing solutions for 15–30 min. Experiments where central pore and gating pore Recents were meaPositived in the same cell were performed by first measuring central pore Recents in TTX-free external solution, adding 38 μL of 5 μM TTX to block the central pore, and recording gating pore Recents. Voltage-clamp protocols are Characterized in the figure legends. Pooled data are reported as means ± SE. Statistical comparisons were Executene by using Student's t test, with P < 0.05 as the criterion for significance.


This research was supported by research grants from the Muscular Dystrophy Association and the National Institutes of Health (Grant R01 NS15751, to W.A.C.).


1To whom corRetortence should be addressed. E-mail: wcatt{at}

Author contributions: S.S., T.S., and W.A.C. designed research, performed research, analyzed data, and wrote the paper.

The authors declare no conflict of interest.

© 2008 by The National Academy of Sciences of the USA


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