KCNQ1-R539W Mutation Substitutes Cholesterol for Phosphatidylinositol-4, 5-Bisphosphate in Channel Regulation

Abstract

Point mutation of nearby residues in ion channels can be associated with diametrically opposed clinical phenotypes despite the mutant channels exhibit similar biophysical characteristics. Hence a characterization of the channel structure/function at the amino-acid scale is required for better understanding of channel genotype/phenotype relationship. R555C and R539W KCNQ1 mutant channels are a good illustration of this idea: R555C mutation is associated with a fruste form of type 1 long QT syndrome, whereas R539W mutation is associated with sudden death. Puzzling enough, the genotype/phenotype relationship is difficult to understand because the mutated residues are in the same helix C module, they both concern arginine residues, both channels have the same biophysical properties, and the same sensitivity to short chain phosphatidylinositol-4,5-bisphosphate (PIP2).To better understand the genotype/phenotype relationship in the context of these mutations, we performed several tests in COS-7 cells expressing the WT or mutant channels and used tail-currents amplitudes as readout. We show that R539W is very peculiar: As opposed to WT and R555C channels, the R539W channel current is barely running down when available PIP2 is decreased, either by wortmannin application in whole-cell, or by magnesium application in inside-out configuration. Consistent with that, the R539W channel is also insensitive to extracellular osmolarity, known to modulate the channel activity via PIP2. These results suggest that KCNQ1-R539W mutation shortcuts PIP2 in the channel open pore stabilization. Both structural model prediction and functional analysis implicate membrane cholesterol in this effect. Indeed, structural model prediction suggests that the introduced tryptophan in R539W interacts with cholesterol. Both cyclodextrin application on R539W and substitution of R539 by residues other than tryptophan restore channel rundown, consistent with the supposed tryptophan-cholesterol interaction. We conclude that the R539W/cholesterol interaction substitutes for R539/PIP2 interaction in the channel open pore stabilization.