The bestrophin mutation A243V, linked to adult-onset vitelliform macular dystrophy, impairs its chloride channel function.

Abstract

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 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.

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.

• Authors: Kuai Yu, Yuanyuan Cui, H Criss Hartzell
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 47 Issue 11 Pg. 4956-61 (Nov 2006) ISSN: 0146-0404 [Print] United States
• PMID: 17065513 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• BEST1 protein, human
• Bestrophins
• CLCA1 protein, human
• Chloride Channels
• Eye Proteins
• Membrane Proteins

Topics

• Bestrophins
• Biotinylation
• Cell Membrane
• Chloride Channels (metabolism)
• Eye Proteins (genetics)
• Gene Expression
• Humans
• Kidney (embryology)
• Macular Degeneration (genetics, metabolism)
• Membrane Proteins (metabolism)
• Mutagenesis, Site-Directed
• Patch-Clamp Techniques
• Point Mutation
• Transfection