BPS2026 – Single BK channel activity from skin biopsies of heterozygous individuals with neurological disorder

In ultra-rare disorders such as KCNMA1 channelopathy, assessing variant pathogenicity through changes in BK channel function is a primary source of evidence for disease causality; however, in vitro assays are not typically conducted in heterozygous KCNMA1 genotypes found in affected individuals. In this study, fibroblasts were obtained from two unrelated individuals presenting with epilepsy and paroxysmal dyskinesia that harbor distinct heterozygous KCNMA1 variants. KCNMA1 -N999S was previously shown to produce strong gain-of-function (GOF) properties in homomeric BK channels expressed in vitro and heterozygous animal models , while the changes produced by KCNMA1 -E656A in homomeric channels were inconclusive with both GOF and benign effects reported. Neither variant has been validated in heterozygous patient-derived tissue. Comparing KCNMA1 levels in primary human skin fibroblasts of heterozygous patients and unaffected controls, we found WT and N999S-containing transcripts in equal ratios in fibroblasts from case 1. Conversely, E656A-containing transcripts and the number of functional BK channels were reduced compared to WT from case 2. In patch-clamp recordings from single BK channels (-40 mV to +160 mV), the open probability (P o ) in both heterozygous KCNMA1 -E656A and KCNMA1 -N999S fibroblasts was increased compared to controls by 1.7- and 3-fold, respectively. Dwell time histograms showed decreased closed time and increased open time constants. Slope conductance for BK channels containing either variant was unchanged. These data demonstrate that both E656A and N999S variants produce GOF gating changes in single BK channel currents from heterozygous patient cells. However, the increased gating was offset by decreased BK channel expression in KCNMA1 -E656A fibroblasts, mitigating its effect compared to N999A. This study shows that single BK channels can be reliably detected from human fibroblasts and suggests the utility for using skin biopsies to establish variant pathogenicity for heterozygous KCNMA1 patient genotypes.