Effects of alternative splicing on function of Bestrophin-1 calcium-activated chloride channels

The proposed Ca2+-activated Cl- channel protein Bestrophin 1 (Best1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK293 cells, and compared its properties to Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl- channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2 expressing cells had no detectable Ca2+-activated Cl- currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Dex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing...
Source: BJ Cell - Category: Biochemistry Authors: Tags: BJ Cell Source Type: research