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 Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) in Cav1.1 and Nav1.4. Vigorous structural and functional studies suggest that mutations of the arginine residues of VSDs destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of an aberrant ion permeation pathway, the gating pore. Presently, non-physiological leak currents through the gating pore, known as the gating pore currents, are thought to underlie HypoPP. In this study, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium ion channel (mKir2.1) and HypoPP2-associated Nav1.4 missense variants, p.R669H, p.R672H, p.R1135H or p.R669H/p.R672H (double mutations, DMT). Whole-cell patch clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane electrical potential of the cells to −90 mV or below (comparable to myofibers), and that p.R669H and DMT induced notable proton-based gating pore currents. Using a ratiometric pH indicator, SNARF-4F, we observed that the proton transports at higher rate in p.R669H and DMT compared to wild-type (WT) control. The expression of the HypoPP2-associated Nav1.4 variants was similar to the WT level based on our newly developed membrane protein targeting assay, indicating that the higher proton transport rates found in our fluorometric measurements for p.R669H and DMT are indeed ascribed to the gating pore currents. Our optical method has a potential to serve as an in vitro platform for rapidly characterizing variants associated with HypoPP and other channelopathies, and for subsequent high-throughput drug screen. Support: JP19ek0109230 and JP22bm0804005(AMED), JP18K07524 and JP22K07493(JSPS), Takeda Science Foundation, Mochida Memorial Foundation, and DC01782(NIH).