Evaluation of four <i>KCNMA1</i> channelopathy variants on BK channel current under Ca _V 1.2 activation

Variants in <i>KCNMA1</i>, encoding the voltage- and calcium-activated K⁺ (BK) channel, are associated with human neurological disease. The effects of gain-of-function (GOF) and loss-of-function (LOF) variants have been predominantly studied on BK channel currents evoked under steady-state voltage and Ca²⁺ conditions. However, in their physiological context, BK channels exist in partnership with voltage-gated Ca²⁺ channels and respond to dynamic changes in intracellular Ca²⁺ (Ca²⁺_i). In this study, an L-type voltage-gated Ca²⁺ channel present in the brain, Ca_V1.2, was co-expressed with wild type and mutant BK channels containing GOF (D434G, N999S) and LOF (H444Q, D965V) patient-associated variants in HEK-293T cells. Whole-cell BK currents were recorded under Ca_V1.2 activation using buffering conditions that restrict Ca²⁺_i to nano- or micro-domains. Both conditions permitted wild type BK current activation in response to Ca_V1.2 Ca²⁺ influx, but differences in behavior between wild type and mutant BK channels were reduced compared to prior studies in clamped Ca²⁺_i. Only the N999S mutation produced an increase in BK current in both micro- and nano-domains using square voltage commands and was also detectable in BK current evoked by a neuronal action potential within a microdomain. These data corroborate the GOF effect of N999S on BK channel activity under dynamic voltage and Ca²⁺ stimuli, consistent with its pathogenicity in neurological disease. However, the patient-associated mutations D434G, H444Q, and D965V did not exhibit significant effects on BK current under Ca_V1.2-mediated Ca²⁺ influx, in contrast with prior steady-state protocols. These results demonstrate a differential potential for <i>KCNMA1</i> variant pathogenicity compared under diverse voltage and Ca²⁺ conditions.