PubMed:17065513 JSONTXT

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bestrophin mutation A243V, linked to adult-onset vitelliform macular dystrophy, impairs its chloride channel function.\nPURPOSE: 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.\nMETHODS: 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.\nRESULTS: WT hBest1 induced Ca(2+)-activated Cl(-) currents in HEK cells that were \u003e1 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(-) \u003e or = I(-) \u003e or = NO(3)(-) \u003e Br(-) \u003e Cl(-) \u003e HCO(3)(-) and a relative conductance G(X)/G(Cl) order of NO(3)(-) \u003e SCN(-) \u003e I(-) \u003e or = Br(-) \u003e or = Cl(-) \u003e HCO(3)(-). However, the A243V current exhibited different sequences: P(X)/P(Cl) was SCN(-) \u003e NO(3)(-) \u003e I(-) \u003e Br(-) \u003e Cl(-) \u003e HCO(3)(-) and G(X)/G(Cl) was SCN(-) \u003e NO(3)(-) \u003e or = I(-) \u003e or = Br(-) \u003e Cl(-) \u003e 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.\nCONCLUSIONS: 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."}

{"project":"PubmedHPO","denotations":[{"id":"T1","span":{"begin":175,"end":192},"obj":"HP_0007754"}],"text":"The bestrophin mutation A243V, linked to adult-onset vitelliform macular dystrophy, impairs its chloride channel function.\nPURPOSE: 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.\nMETHODS: 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.\nRESULTS: WT hBest1 induced Ca(2+)-activated Cl(-) currents in HEK cells that were \u003e1 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(-) \u003e or = I(-) \u003e or = NO(3)(-) \u003e Br(-) \u003e Cl(-) \u003e HCO(3)(-) and a relative conductance G(X)/G(Cl) order of NO(3)(-) \u003e SCN(-) \u003e I(-) \u003e or = Br(-) \u003e or = Cl(-) \u003e HCO(3)(-). However, the A243V current exhibited different sequences: P(X)/P(Cl) was SCN(-) \u003e NO(3)(-) \u003e I(-) \u003e Br(-) \u003e Cl(-) \u003e HCO(3)(-) and G(X)/G(Cl) was SCN(-) \u003e NO(3)(-) \u003e or = I(-) \u003e or = Br(-) \u003e Cl(-) \u003e 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.\nCONCLUSIONS: 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."}