An ensemble of toxic channel types underlies the severity of the de novo variant G375R of the human BK channel

Abstract

AbstractDe novo mutations play a prominent role in neurodevelopmental diseases including autism, schizophrenia, and intellectual disability. Many de novo mutations are dominant and so severe that the afflicted individuals do not reproduce, so the mutations are not passed into the general population. For multimeric proteins, such severity may result from a dominant-negative effect where mutant subunits assemble with WT to produce channels with adverse properties. Here we study the de novo variant G375R heterozygous with the WT allele for the large conductance voltage- and Ca2+-activated potassium (BK channel, Slo1. This variant has been reported to produce devastating neurodevelopmental disorders in three unrelated children. If mutant and WT subunits assemble randomly to form tetrameric BK channels, then ~6% of the assembled channels would be wild type (WT, ~88% would be heteromeric incorporating from 1-3 mutant subunits per channel, and ~6% would be homomeric mutant channels consisting of four mutant subunits. To test this hypothesis, we analyzed the biophysical properties of the single BK channels in the ensemble of channels expressed following a 1:1 injection of mutant and WT cRNA into oocytes. We found ~3% were WT channels, ~85% were heteromeric channels, and ~12% were homomeric mutant channels. All of the heteromeric channels as well as the homomeric mutant channels displayed toxic properties, indicating a dominant negative effect of the mutant subunits. The toxic channels were open at inappropriate negative voltages, even in the absence of Ca2+, which would lead to altered cellular function and decreased neuronal excitability.SignificanceWe examined the molecular basis for a severe channelopathy associated with developmental and neurological disorders for a de novo variant of the Ca2+-activated potassium (BK) channel, Slo1. To identify the types of BK channels underlying the pathogenicity of the variant, we recorded from BK channels expressed for the G375R mutation heterozygous with the WT allele. We found five different functional types of BK channels, four of which displayed a gain of function toxicity by shifting channel opening to more negative voltages in proportion to the number of mutant subunits per channel and WT. The aberrant channels would disrupt cellular function and the normal electrical activity of neurons, providing a molecular basis for the severity of the phenotype.