Structural mapping of patient-associated KCNMA1 gene variants

KCNMA1-linked channelopathy is a neurological disorder characterized by seizures, motor abnormalities, and neurodevelopmental disabilities. The disease mechanisms are predicted to result from alterations in KCNMA1-encoded BK ^K+ channel activity; however, only a subset of the patient-associated variants have been functionally studied. The localization of these variants within the tertiary structure or evaluation by pathogenicity algorithms has not been systematically assessed. In this study, 82 nonsynonymous patient-associated KCNMA1 variants were mapped within the BK channel protein. Fifty-three variants localized within cryo-EM resolved structures, including 21 classified as either gain-of-function (GOF) or loss-of-function (LOF) in BK channel activity. Clusters of LOF variants were identified in the pore, the AC region (RCK1), and near the Ca ²⁺ bowl (RCK2), overlapping with sites of pharmacological or endogenous modulation. However, no clustering was found for GOF variants. To further understand variants of uncertain significance (VUS), assessments by multiple standard pathogenicity algorithms were compared, and new thresholds for sensitivity and specificity were established from confirmed GOF and LOF variants. An ensemble algorithm was constructed (KCNMA1 Meta Score), consisting of a weighted summation of this trained dataset combined with a structural component derived from the Ca ²⁺ bound and unbound BK channels. KMS assessment differed from the highest performing individual algorithm (REVEL) at 10 VUS residues, and a subset were studied further by electrophysiology in HEK293 cells. M578T, E656A, and D965V (KMS+;REVEL-) were confirmed to alter BK channel properties in voltage-clamp recordings, and D800Y (KMS-;REVEL+) was assessed as benign under the test conditions. However, KMS failed to accurately assess K457E. These combined results reveal the distribution of potentially disease-causing KCNMA1 variants within BK channel functional domains and pathogenicity evaluation for VUS, suggesting strategies for improving channel-level predictions in future studies by building on ensemble algorithms such as KMS.