New approaches to understanding BK channelopathies at the molecular level of single channels

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ABSTRACT New approaches to understanding BK channelopathies at the molecular level of single channels. Mutations in KCNMA1 BK potassium channels produce a wide range of channelopathies which include epilepsy, dyskinesis, autism, multiple congenital abnormalities, intellectual disability, developmental delay, axial hypotonia, ataxia, cerebral and cerebellar atrophy, bone thickening, tortuosity of arteries, malformation syndrome, and others. We suggest that the majority of studies that seek to understand the biophysical basis of these BK channelopathies have not been studying the predominant channels; we plan to do so. The majority of these channelopathies arise in individuals who are heterozygous for the mutant gene. Since BK channels are tetrameric, composed of four like subunits, mutant subunits could assemble with wild-type (WT) subunits to form an ensemble of channels with different stoichiometries. The vast majority of the ensemble channels (88%) would be heterotetrameric hybrid channels containing a varied number of mutant subunits (from one to three). However, these hybrid channels are typically overlooked, and the mutations found in channelopathy patients have usually been studied in the laboratory by expressing only the mutant subunit in a laboratory expression system. Assuming random assembly of subunits, the purely mutant channels would not represent more than 6% of the channels found in the cells of a heterozygous patient. Based on these incomplete studies many mutations were categorized into gain-of-function (GOF) or loss-of-function (LOF) categories due to the findings of enhanced or reduced activation of BK channels, respectively based on homotetrameric mutant channels. We suggest that the omission of studying the full palette of channel types present in these patients has led to a chaotic and inaccurate categorization of phenotypes by not recognizing that most aberrant channels in these patients may be hybrids which constitute the majority of the aberrant channels in heterozygous channelopathies. In Aim 1 using a combination of electrophysiological techniques including single channel analysis, we propose to show that a cell carrying one mutant and one WT KCNMA1 allele expresses an ensemble of BK channels dominated by hybrid channels assembled from both mutant and WT subunits. In Aim 2 we will determine the functional properties and gating mechanisms to determine how the predominant channel forms associated with BK channelopathies (as determined in Aim 1) alter channel activation. In Aim 3 we will test the hypothesis that some genetic variants of BK channels result in a truncated subunit that leads to a reduced amount of BK current in cells when heterozygous with WT subunits. Although these variant genes circulate in the population they are not reported to cause neurological disease when heterozygous. Nevertheless, these mutations need to be studied because reduced BK currents could confer a furtive genetic pre-disposition to neurological disease. These aims will then characterize the actions of BK variants at the molecular level of single channels for all expressed channel types, providing information key towards understanding the channelopathies.