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Bikash R. Pattnaik, Patrick Halbach, Simran Brar, Sara Tokarz, David M. Gamm, De-Ann M. Pillers; A Bestrophin Mutation Associated with Best’s Disease Causes Altered Chloride Conductance. Invest. Ophthalmol. Vis. Sci. 2012;53(14):739.
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Macular degeneration in Best Vitelliform Macular Dystrophy (BVMD), is a serious pediatric problem. BVMD is caused by dominantly-inherited mutations of bestrophin, a protein localized to the basolateral membrane of retinal pigment epithelial (RPE) cells. Bestrophin proteins form Cl- channels, an important contributor to RPE cell function. We sought to determine the effect of a novel disease associated mutation, located within the Ca2+-binding domain of bestrophin on the bestrophinopathy.
Using site-directed mutagenesis, we introduced the novel N296H genetic mutation associated with BVMD into a vector containing PIRES, GFP, and human bestrophin 1 (hBest1). The vector was sequenced to verify that the mutation was present. The hBest1 wildtype (WT) and hBest1 N296H mutant were introduced into cultured Chinese Hamster Ovary (CHO-M1) cells and transiently expressed. Ionic flux due to the two clones was measured as current-amplitude using either the whole-cell or perforated patch configurations of patch-clamp electrophysiology. For the cells recorded in perforated patch mode, we used 100 μM carbachol to stimulate G-protein coupled receptor-induced physiological changes in intracellular Ca2+.
GFP-positive cells confirmed the ectopic expression of WT hBest1 and hBest1 N296H in CHO cells. Under conditions of high intracellular Ca2+, Cl- current due to hBest1 exhibited both inward and outward current depending on the membrane potential. The corresponding current-amplitude plot was linear with resting membrane potential close to 0 mV. Substitution of bath Cl- with HCO3- positively shifted the I-V plot by ~23 mV without much change in conductance, which matched the previously published characteristic of bestrophin Cl- channel current. In comparison, mutant hBest1 N296H transfected cells had significantly reduced current amplitudes and also preference for other anions. Physiological alteration of intracellular Ca2+ through the activation of muscarinic receptors resulted only in the activation of current due to the wildtype but not the mutant channel.
These results demonstrate a direct relationship between the Ca2+ binding site mutation and altered Cl- conductance. The absence of Cl- conductance in the N296H mutant therefore supports the hypothesis that hBest1 is a Ca2+-activated Cl- channel.
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