BK channel activity in skin fibroblasts from patients with neurological disorder.

Seventy-five unique variants in the KCNMA1 gene have been identified from individuals with neurological disorders. However, variant pathogenicity and evidence for disease causality are lacking in most cases. In this study, the KCNMA1 variants N999S and E656A (rs886039469 and rs149000684, respectively) were investigated from two individuals presenting with neurological disorders. N999S was previously shown to produce strong gain-of-function (GOF) changes in homomeric BK channel properties in vitro and is found as a heterozygous allele associated with epilepsy and paroxysmal dyskinesia in humans. Although its pathogenicity has been demonstrated in heterozygous animal models, the GOF classification for N999S has not been validated in a heterozygous patient-derived tissue. Conversely, the GOF pathogenicity for E656A is based solely on homomeric channels expressed in vitro and is inconclusive. For either variant, the properties of single heterozygous channels and allele expression is unknown. In this study, we profiled the wild-type and mutant KCNMA1 transcripts from primary human skin fibroblasts of heterozygous patients and unaffected controls and performed patch-clamp electrophysiology to characterize endogenous BK channel current properties. GOF gating was observed in single BK channel recordings from both channel types. Fibroblasts from the individual harboring the E656A variant showed decreases in the number of BK channels detected and E656A-containing transcripts compared to controls. These results show that single BK channels can be reliably detected in primary fibroblasts obtained from human skin biopsies, suggesting their utility for establishing variant pathogenicity, and reveal the BK channel expression and functional changes associated with two heterozygous patient genotypes.