Control of Strex BK-Channel Palmitoylation Via a Polybasic Domain

Abstract

Large conductance voltage and calcium sensitive potassium channels (BK) are widely expressed throughout the body and encoded by a single gene (KCNMA1). The splice insertion of the STREX exon at splice site C2, generates a channel phenotype with increased calcium sensitivity and differing regulation by phosphorylation. In mammals, splicing of the STREX exon is dynamically controlled by cellular excitability as well as circulating stress and sex hormones. With STREX insertion, a palmitoylation site and a polybasic region are introduced to the channel. The interaction of the polybasic region with the plasma membrane and palmitoylation of cysteine residues in the STREX-linker between RCK1 and RCK2 may serve as a membrane targeting motif that alters the phenotype of the BK-STREX channel.A GFP-tagged carboxyl terminal construct spanning from the S6 transmembrane domain to the COOH end region of the intracellular carboxyl terminus, localised at the plasma membrane. Membrane localisation was abolished when the STREX insert was excluded. To test whether the polybasic region is important for plasma membrane targeting, site directed mutagenesis was used to mutate positive residues in the polybasic domain into negative (E) or neutral (A) residues. These mutations abolished membrane targeting of S6-COOH-STX to the plasma membrane. Full-length channels with mutations in the polybasic region were studied in patch-clamp electrophysiology to determine calcium and voltage sensitivity, with disruption of the polybasic domain shifting apparent calcium sensitivity towards the zero (insertless) BK channel phenotype. The importance of the polybasic domain for palmitoylation of the BK-STREX channel was further confirmed by assaying 3H-palmitate incorporation into carboxyl terminal constructs.These data suggest that the polybasic region generated by inclusion of the STREX insert is an important determinant of BK-STREX channel palmitoylation and thus contributes to the altered channel properties upon STREX inclusion.