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S4 voltage-sensor mutations in CaV1.1 and NaV1.4 channels cause the human muscle disorder hypokalemic periodic paralysis (HypoPP). The mechanism whereby these mutations predispose affected sarcolemma to attacks of sustained depolarization and loss of excitability is poorly understood. Recently, three HypoPP mutations in the domain II S4 segment of NaV1.4 were shown to create accessory ionic permeation pathways, presumably extending through the aqueous gating pore in which the S4 segment resides. However, there are several disparities between reported gating pore currents from different investigators, including differences in ionic selectivity and estimates of current amplitude, which in turn have important implications for the pathological relevance of these aberrant currents. To clarify the features of gating pore currents arising from different DIIS4 mutants, we recorded gating pore currents created by HypoPP missense mutations at position R666 in the rat isoform of Nav1.4 (the second arginine from the outside, at R672 in human NaV1.4). Extensive measurements were made for the index mutation, R666G, which created a gating pore that was permeable to K(+) and Na(+). This current had a markedly shallow slope conductance at hyperpolarized voltages and robust inward rectification, even when the ionic gradient strongly favored outward ionic flow. These characteristics were accounted for by a barrier model incorporating a voltage-gated permeation pathway with a single cation binding site oriented near the external surface of the electrical field. The amplitude of the R666G gating pore current was similar to the amplitude of a previously described proton-selective current flowing through the gating pore in rNaV1.4-R663H mutant channels. Currents with similar amplitude and cation selectivity were also observed in R666S and R666C mutant channels, while a proton-selective current was observed in R666H mutant channels. These results add support to the notion that HypoPP mutations share a common biophysical profile comprised of a low-amplitude inward current at the resting potential that may contribute to the pathological depolarization during attacks of weakness.

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca(2+) channel) or NaV1.4 (the skeletal muscle voltage-gated Na(+) channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice ("gating-pore") in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K(+) channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K(+) and Cl(-) conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of V(REST) indirectly, for instance, by interfering with intracellular pH homeostasis.

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.

Hypokalemic periodic paralysis (HypoPP) is a familial skeletal muscle disorder that presents with recurrent episodes of severe weakness lasting hours to days associated with reduced serum potassium ([K.sup.+]). HypoPP is genetically heterogeneous, with missense mutations of a calcium channel ([Ca.sub.v]1.1) or a sodium channel ([Na.sub.v]1.4) accounting for 60% and 20% of cases, respectively. The mechanistic link between [Ca.sub.v]1.1 mutations and the ictal loss of muscle excitability during an attack of weakness in HypoPP is unknown. To address this question, we developed a mouse model for HypoPP with a targeted [Ca.sub.v]1.1 R528H mutation. The [Ca.sub.v]1.1 R528H mice had a HypoPP phenotype for which low [K.sup.+] challenge produced a paradoxical depolarization of the resting potential, loss of muscle excitability, and weakness. A vacuolar myopathy with dilated transverse tubules and disruption of the triad junctions impaired [Ca.sup.2+] release and likely contributed to the mild permanent weakness. Fibers from the [Ca.sub.v]1.1 R528H mouse had a small anomalous inward current at the resting potential, similar to our observations in the [Na.sub.v]1.4 R669H HypoPP mouse model. This 'gating pore current' may be a common mechanism for paradoxical depolarization and susceptibility to HypoPP arising from missense mutations in the S4 voltage sensor of either calcium or sodium channels., Introduction Hypokalemic periodic paralysis (HypoPP) is an inherited disorder of skeletal muscle in which recurrent attacks of weakness occur in association with low serum [K.sup.+] (> 2.8 mM) (1, 2). [...]

Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum [K. sup.+]. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel [(Ca.sub.V]1.1) and a sodium channel [(Na.sub.V]1.4) have been identified in HypoPP families. Mutations of [Na.sub.V]1.4 give rise to a heterogeneous group of muscle dis-orders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the [Na.sub.V]1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the [Na.sub.V]1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in [low-K.sup.+] challenge, insensitivity to [high-K. sup.+] challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the [Na. sup.+]/[K. sup.+]-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant [Na.sub.V]1.4 channels., Introduction Hypokalemic periodic paralysis (HypoPP) is the most prevalent form of familial periodic paralysis and is one of the many ion channelopathies affecting skeletal muscle (1, 2). These rare heritable [...]

The combination of sarcolemmal depolarization and hypokalemia exhibited by the different forms of hypokalemic paralysis has been attributed to abnormalities of the K+ conductance governing the resting membrane potential (VREST). Supportive data have been observed in muscle fibers biopsied from patients with familial hypokalemic periodic paralysis (HypoPP) that paradoxically depolarize at low K+. Although this observation is consistent with anomalous K+ conductance, rigorous experimental support is lacking. To establish a foundation for understanding the pathophysiology of hypokalemic paralysis, we studied Ba2+-treated muscle fibers under voltage clamp. As anticipated, Ba2+ blocked inward rectifying K+ (IRK) currents, and thereby promoted depolarization, supporting the notion that the IRK conductance governs VREST. The IRK conductance also declined when muscle was challenged with reduced external K+. When the external K+ declined below 1 mM, VREST became uncoupled from the K+ reversal potential and depolarized. Partial (∼50%) block of the IRK conductance with Ba2+ potentiated this uncoupling threshold, such that depolarization could be elicited by exposure to 2 mM external K+. A quantitative computer model of resting ionic conductances was constructed, and simulations demonstrated that small alterations to resting conductances, such as adding a low-amplitude aberrant inward current flowing through 'gating pores' in mutant Na+ channels causing HypoPP-2, can promote paradoxical depolarization in low K+. These findings offer a simple explanation for some of the heretofore poorly understood physiological abnormalities of HypoPP muscle and support the notion that pathological gating pore leakage currents may directly predispose to paralytic attacks. Muscle Nerve, 2007 [ABSTRACT FROM AUTHOR]

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca2+ channel) or NaV1.4 (the skeletal muscle voltage-gated Na+ channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice (‘gating-pore’) in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits u'ansmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K+ channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K+ and Cl- conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of VREST indirectly, for instance, by interfering with intracellular pH homeostasis. [ABSTRACT FROM AUTHOR]

Hypokalemic periodic paralysis (HypoPP) is a familial skeletal muscle disorder that presents with recurrent episodes of severe weakness lasting hours to days associated with reduced serum potassium (K+). HypoPP is genetically heterogeneous, with missense mutations of a calcium channel (CaV1.1) or a sodium channel (NaV1.4) accounting for 60% and 20% of cases, respectively. The mechanistic link between CaV1.1 mutations and the ictal loss of muscle excitability during an attack of weakness in HypoPP is unknown. To address this question, we developed a mouse model for HypoPP with a targeted CaV1.1 R528H mutation. The Cav1.1 R528H mice had a HypoPP phenotype for which low K+ challenge produced a paradoxical depolarization of the resting potential, loss of muscle excitability, and weakness. A vacuolar myopathy with dilated transverse tubules and disruption of the triad junctions impaired Ca2+ release and likely contributed to the mild permanent weakness. Fibers from the CaV1.1 R528H mouse had a small anomalous inward current at the resting potential, similar to our observations in the NaV1.4 R669H HypoPP mouse model. This "gating pore current" may be a common mechanism for paradoxical depolarization and susceptibility to HypoPP arising from missense mutations in the S4 voltage sensor of either calcium or sodium channels. [ABSTRACT FROM AUTHOR]