BK channels of five different subunit combinations underlie the de novo KCNMA1 G375R channelopathy

Abstract

De 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 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.

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

“…Limited data is available to consider the impact of these on heterozygous variants. Co-expression of WT and mutant (GOF) BK channel cDNAs supports the assumption that heterotetramers are the predominant channel type produced by 1:1 transcript ratios in Xenopus oocytes ( Geng et al, 2021 ). A few studies have shown that N999S and D434G confer similar ΔV 1/2 onto different splice variants ( Figure 1C–G ; Li et al, 2018 ; Moldenhauer et al, 2020a ; Wang et al, 2009 ) and maintain left-shifted V 1/2 values compared to WT in the presence of the β4 subunit ( Berkefeld and Fakler, 2013 ; Li et al, 2018 ; Wang et al, 2009 ).…”

“…From heterologous cells, we predicted that the variants would have a strong (N999S), intermediate (D434G), or weak (H444Q) potential to alter neuronal BK current levels in transgenic mice. However, heterozygous patient genotypes create the possibility for hetero-tetramer channel formation ( Geng et al, 2021 ), necessitating understanding the relative GOF and LOF effects in vivo from BK current levels in heterozygous transgenics compared to WT littermates. Recordings were made in the dentate gyrus of the hippocampus, where BK channels are highly expressed, regulate neuronal excitability, and where changes in BK channel properties are associated with seizure ( Kaufmann et al, 2010 ; Knaus et al, 1996 ; Misonou et al, 2006 ; Sailer et al, 2006 ; Sausbier et al, 2005 ; Sausbier et al, 2006 , Trimmer, 2015 ).…”

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

“…From heterologous cells, we predicted that the variants would have a strong (N999S), intermediate (D434G), or weak (H444Q) potential to alter neuronal BK current levels in transgenic mice. However, heterozygous patient genotypes create the possibility for hetero- tetramer channel formation (Geng et al ., 2021), necessitating understanding the relative GOF and LOF effects in vivo from BK current levels in heterozygous transgenics compared to WT littermates. Recordings were made in the dentate gyrus of the hippocampus, where BK channels are highly expressed, regulate neuronal excitability, and where changes in BK channel properties are associated with seizure (Kaufmann et al ., 2010; Knaus et al ., 1996; Misonou et al ., 2006; Sailer et al ., 2006; Sausbier et al ., 2005b; Trimmer, 2015).…”