BK channel properties correlate with neurobehavioral severity in three KCNMA1-linked channelopathy mouse models.

KCNMA1 forms the pore of BK K+ channels, which regulate neuronal and muscle excitability. Recently, genetic screening identified heterozygous KCNMA1 variants in a subset of patients with debilitating paroxysmal non-kinesigenic dyskinesia, presenting with or without epilepsy (PNKD3). However, the relevance of KCNMA1 mutations and the basis for clinical heterogeneity in PNKD3 has not been established. Here, we evaluate the relative severity of three KCNMA1 patient variants in BK channels, neurons, and mice. In heterologous cells, BKN999S and BKD434G channels displayed gain-of-function (GOF) properties, whereas BKH444Q channels showed loss-of-function (LOF) properties. The relative degree of channel activity was BKN999S > BKD434G>WT > BKH444Q. BK currents and action potential firing were increased, and seizure thresholds decreased, in Kcnma1N999S/WT and Kcnma1D434G/WT transgenic mice but not Kcnma1H444Q/WT mice. In a novel behavioral test for paroxysmal dyskinesia, the more severely affected Kcnma1N999S/WT mice became immobile after stress. This was abrogated by acute dextroamphetamine treatment, consistent with PNKD3-affected individuals. Homozygous Kcnma1D434G/D434G mice showed similar immobility, but in contrast, homozygous Kcnma1H444Q/H444Q mice displayed hyperkinetic behavior. These data establish the relative pathogenic potential of patient alleles as N999S>D434G>H444Q and validate Kcnma1N999S/WT mice as a model for PNKD3 with increased seizure propensity.

So far, only 70 patients around the world have been diagnosed with a newly identified rare syndrome known as KCNMA1-linked channelopathy. The condition is characterised by seizures and abnormal movements which include frequent 'drop attacks', a sudden and debilitating loss of muscle control that causes patients to fall without warning. The disease is associated with mutations in the gene for KCNMA1, a member of a class of proteins important for controlling nerve cell activity and brain function. However, due to the limited number of people affected by the condition, it is difficult to link a particular mutation to the observed symptoms; the basis for the drop attacks therefore remains unknown. Park et al. set out to 'model' KCNMA1-linked channelopathy in the laboratory, in order to determine which mutations in the KCNMA1 gene caused these symptoms. Three groups of mice were each genetically engineered to carry either one of the two most common mutations in the gene for KCNMA1, or a very rare mutation associated with the movement symptoms. Behavioural experiments and studies of nerve cell activity revealed that the mice carrying mutations that made the KCNMA1 protein more active developed seizures more easily and became immobilized, showing the mouse version of drop attacks. Giving these mice the drug dextroamphetamine, which works in some human patients, stopped the immobilizing attacks altogether. These results show for the first time which specific genetic changes cause the main symptoms of KCNMA1-linked channelopathy. Park et al. hope that this knowledge will deepen our understanding of this disease and help develop better treatments.

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

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 & NOTEWORTHYKCNMA1-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.