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Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

ABSTRACT: Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A encoding the voltage-gated Ca2+ channel Cav1.1 or the voltage-gated Na+ channel Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal that separates external fluid and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, based on HEK293T cells and by using the Sleeping Beauty transposon system, we generated HypoPP-model cell lines that co-express the mouse inward-rectifier K+ channel (mKir2.1) and HypoPP2-associated Nav1.4 channel. Whole-cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarizes the membrane potential to levels comparable to those of myofibers, and that some Nav1.4 variants induce notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants by using a ratiometric pH indicator. Our optical method provides a potential in vitro platform for high-throughput drug screening, not only for HypoPP but also for other channelopathies caused by VSD mutations.

SUBMITTER: Kubota T

PROVIDER: S-EPMC10320719 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

Kubota Tomoya T Takahashi Satoe S Yamamoto Risa R Sato Ruka R Miyanooto Aya A Yamamoto Reina R Yamauchi Kosuke K Homma Kazuaki K Takahashi Masanori P MP

Disease models & mechanisms 20230627 6

Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A encoding the voltage-gated Ca2+ channel Cav1.1 or the voltage-gated Na+ channel Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal that separates external fluid and the internal cytosolic crevices, resulting in the gener ...[more]

PMID: 37139703

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Project description: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.

Project description:Missense mutations at arginine residues in the S4 voltage-sensor domains of NaV1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K(+). Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K(+) during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K(+), yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na(+) current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type NaV1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na(+) was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼ 10 µS/cm(2) or 1% of the total resting conductance. Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na(+) current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na(+) current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of NaV, CaV, and KV channels associated with human disease.

Dataset Information

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

Publications

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

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