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Kuai Yu, Yuanyuan Cui, H. Criss Hartzell; The Bestrophin Mutation A243V, Linked to Adult-Onset Vitelliform Macular Dystrophy, Impairs Its Chloride Channel Function. Invest. Ophthalmol. Vis. Sci. 2006;47(11):4956-4961. doi: 10.1167/iovs.06-0524.
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purpose. 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.
methods. 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.
results. WT hBest1 induced Ca2+-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− ≥ I− ≥ NO3 − > Br− > Cl− > HCO3 − and a relative conductance G X/G Cl order of NO3 − > SCN− > I− ≥ Br− ≥ Cl− > HCO3 −. However, the A243V current exhibited different sequences: P X/P Cl was SCN− > NO3 − > I− > Br− > Cl− > HCO3 − and GX/GCl was SCN− > NO3 − ≥ I− ≥ Br− > Cl− > HCO3 −. 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.
conclusions. 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.
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