The Best Disease-Linked Cl−ChannelhBest1Regulates CaV1 (L-type) Ca2+Channels via src-Homology-Binding Domains

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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 humanBest1gene (hBest1) differentially modulates CaV1.3 (L-type) voltage-gated Ca2+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 CaVβ 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 Pro330and Pro334abolished 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 CaVcurrents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates CaVchannels by interacting with the CaVβ subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of CaVchannels and suggest a possible mechanism for the role of hBest1 in macular degeneration.

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