Screening the effects of cysteine mutations on BKCa channel function and trafficking
Large conductance voltage and calcium-activated potassium (BKCa) channels link electrical excitability with cellular signalling. A single gene (KCNMA1 or slo) encodes all four α-subunits that shape the native BKCa channel pore. Alternative splicing of the mRNA determines the channel phenotype, including its intrinsic voltage dependence and calcium sensitivity (Chen et al., 2005). The identification and characterization of BKCa channel splice variants and the functional impact of point mutations within regulatory motifs in the channel sequence normally depends on the use of low throughput patch clamp electrophysiology. Previously we have developed a robust, high throughput functional assay to screen BKCa splice variants using FLIPR (fluorescent-imaging plate reader) Membrane Potential (FMP) dyes (Saleem et al., 2009). We have now combined this technique with imaging of epitope-tagged channel constructs to determine cell surface expression in order to assess the impact of cysteine point mutations on channel function and trafficking.
Chen et al. (2005) J Biol Chem 280, 33599-33609.
Support from: The Wellcome Trust; MRC; and BBSRC.