It has been proposed that Best vitelliform macular dystrophy (BVMD) is caused by dysfunction in the Cl(-) channel function of human bestrophin-1 (hBest1), but some patients with BVMD who have the hBest1 A243V mutation have normal electro-oculograms, suggesting that this mutation may not affect Cl(-) channel function. The purpose of this study was to determine whether the A243V mutation affects the Cl(-) channel function of hBest1.Wild-type alanine at position 243 was changed to valine by PCR-based mutagenesis. Wild-type (WT) and A243V hBest1 were transfected into HEK-293 cells, and Cl(-) currents were measured with the whole-cell patch-clamp technique. The trafficking of proteins to the plasma membrane was tested by cell-surface biotinylation.WT hBest1 induced Ca(2+)-activated Cl(-) currents in HEK cells that were >1 nA in amplitude. The currents produced by the A243V mutant, however, were only approximately 10% as large as WT. This was not due to the inability of the A243V mutant to reach the plasma membrane, as shown by cell-surface biotinylation. The A243V mutation changed channel anion selectivity. The WT current exhibited a relative permeability P(X)/P(Cl) order of SCN(-) > or = I(-) > or = NO(3)(-) > Br(-) > Cl(-) > HCO(3)(-) and a relative conductance G(X)/G(Cl) order of NO(3)(-) > SCN(-) > I(-) > or = Br(-) > or = Cl(-) > HCO(3)(-). However, the A243V current exhibited different sequences: P(X)/P(Cl) was SCN(-) > NO(3)(-) > I(-) > Br(-) > Cl(-) > HCO(3)(-) and G(X)/G(Cl) was SCN(-) > NO(3)(-) > or = I(-) > or = Br(-) > Cl(-) > HCO(3)(-). Unlike several other hBest1 mutations that have dominant-negative effects on wild-type channels, the A243V-mutation did not influence the wild-type current when A243V and WT hBest1 were transfected together.The disease-causing A243V mutation is associated with altered hBest1 Cl(-) channel activity. The absence of a dominant negative effect of A243V is consistent with the more mild symptoms associated with this mutation. These results are interpreted in terms of the hypotheses that bestrophins are Cl(-) channels and regulators of Ca signaling.