Comparative gain-of-function effects of the KCNMA1-N999S mutation on human BK channel properties

Abstract

KCNMA1, encoding the voltage- and calcium-activated potassium channel, has a pivotal role in brain physiology. Mutations in KCNMA1 are associated with epilepsy and/or dyskinesia (PNKD3). Two KCNMA1 mutations correlated with these phenotypes, D434G and N999S, were previously identified as producing gain-of-function (GOF) effects on BK channel activity. Three new patients have been reported harboring N999S, one carrying a second mutation, R1128W, but the effects of these mutations have not yet been reported under physiological K+ conditions or compared to D434G. In this study, we characterize N999S, the novel N999S/R1128W double mutation, and D434G in a brain BK channel splice variant, comparing the effects on BK current properties under a physiological K+ gradient with action potential voltage commands. N999S, N999S/R1128W, and D434G cDNAs were expressed in HEK293T cells and characterized by patch-clamp electrophysiology. N999S BK currents were shifted to negative potentials, with faster activation and slower deactivation compared with wild type (WT) and D434G. The double mutation N999S/R1128W did not show any additional changes in current properties compared with N999S alone. The antiepileptic drug acetazolamide was assessed for its ability to directly modulate WT and N999S channels. Neither the WT nor N999S channels were sensitive to the antiepileptic drug acetazolamide, but both were sensitive to the inhibitor paxilline. We conclude that N999S is a strong GOF mutation that surpasses the D434G phenotype, without mitigation by R1128W. Acetazolamide has no direct modulatory action on either WT or N999S channels, indicating that its use may not be contraindicated in patients harboring GOF KCNMA1 mutations. NEW & NOTEWORTHY KCNMA1-linked channelopathy is a new neurological disorder characterized by mutations in the BK voltage- and calcium-activated potassium channel. The epilepsy- and dyskinesia-associated gain-of-function mutations N999S and D434G comprise the largest number of patients in the cohort. This study provides the first direct comparison between D434G and N999S BK channel properties as well as a novel double mutation, N999S/R1128W, from another patient, defining the functional effects during an action potential stimulus.

A Gain‐of‐Function Mutation in KCNMA1 Causes Dystonia Spells Controlled With Stimulant Therapy

“…Another de novo GOF variant in KCNMA1 (c.2984 A > G (p.N995S)) was recently reported in the patients from two independent families who had absence epilepsy in early childhood, but did not develop paroxysmal dyskinesia 8 . Subsequent biophysical characterization showed that N995S, similar to N536H, also specifically increases voltage sensitivity without affecting Ca 2+ ‐dependent activation 8,19 . N995 is located at the C‐terminal end of the channel and is spatially distal from N536 and the voltage sensor (Fig.…”

Cataplexy in Patients Harboring the KCNMA1 p.N999S Mutation

“…[1][2][3] We describe 3 unrelated patients with cataplexy, all sharing the same KCNMA1 gene mutation identified by whole exome sequencing (KCNMA1 [NM_002247.3] c.2984 A > G [p.N999S]), previously described as conferring a gain of function and reported in patients with paroxysmal dyskinesia and developmental delay phenotype. [1][2][3][4] Parents do not harbor the mutation and are asymptomatic. Patient 1 has an additional variant in the KCNMA1 gene (c.3382 C > T [p.Arg1128Trp]) predicted to be benign.…”

“…Patient 1 has an additional variant in the KCNMA1 gene (c.3382 C > T [p.Arg1128Trp]) predicted to be benign. 4 Patient 1 is a 4-year-old female with cataplexy (Video S1), hypotonia, and global developmental delay, conceived via sperm donor (neither KCNMA1 variant is maternally inherited, father's genotype is unknown). MRI brain was normal.…”

“…Interestingly, acetazolamide has been suggested to potentiate BK channel activity, 5 while biophysical analysis of the N999S mutation shows a gain-of-function. 4 This paradox may be explained by non-BK effects of acetazolamide, 4 cell-typespecific effects, or other unknown factors.…”

Altered synaptic plasticity and central pattern generator dysfunction in aDrosophilamodel of PNKD3 paroxysmal dyskinesia

“…Dyskinetic attacks emerge in early infancy or childhood, are frequently co-morbid with absence or generalized tonic-clonic seizures 3 , and in some cases occur alongside developmental delay 4 . PNKD3 is caused by dominant gain-of-function (GOF) mutations in KCNMA1, encoding the pore forming αsubunit of the hSlo1 BK (big K + conductance) channel [3][4][5][6][7] . BK channels are voltage-and Ca 2+activated potassium channels, and modulate synaptic output from excitable cells by gating K + efflux during the repolarization and afterhyperpolarization (AHP) phases of the action potential (AP) [8][9][10] .…”