The best disease-linked Cl- channel hBest1 regulates Ca V1 (L-type) Ca2+ channels via src-homology-binding domains

Abstract

Mutations in the bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although bestrophins have been shown clearly to be Cl- ion channels, it is controversial whether Cl- channel dysfunction can explain the diseases. It has been suggested that bestrophins are multifunctional proteins: they may regulate voltage-gated Ca2+ channels in addition to functioning as Cl - channels. Here, we show that human Best1 gene (hBest1) differentially modulates CaV1.3 (L-type) voltage-gated Ca 2+ channels through association with the CaVβ subunit. In transfected human embryonic kidney 293 cells, hBest1 inhibited CaV1.3. Inhibition of CaV1.3 was not observed in the absence of the β subunit. Also, the hBest1 C terminus binds to Ca Vβ subunits, suggesting that the effect of hBest1 was mediated by the CaVβ subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330-370) in the cytoplasmic C terminus that contains a predicted src-homology-binding domain that is not present in other bestrophin subtypes. Mutation of Pro330 and Pro 334 abolished the effects of hBest1 on CaV1.3. The effect was specific to hBest1; it was not observed with mouse Best1 (mBest1), mBest2, or mBest3. Wild-type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited CaV currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Ca V channels by interacting with the CaVβ subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of CaV channels and suggest a possible mechanism for the role of hBest1 in macular degeneration. Copyright © 2008 Society for Neuroscience.