Purpose: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca<sup>2+</sup> levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. Methods: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1<sup>W93C</sup> in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (I<inf>sc</inf>), and intracellular Ca<sup>2+</sup> levels. Cl<sup>-</sup> currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. Results: Best1<sup>W93C</sup> showed ablated Cl<sup>-</sup> currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and I<inf>sc</inf>, while Best1<sup>W93C</sup> diminished TEP and I<inf>sc</inf>. Substitution of Cl<sup>-</sup> in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1<sup>W93C</sup>. We removed Ca<sup>2+</sup> as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in I<inf>sc</inf> and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1<sup>W93C</sup>. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1<sup>W93C</sup> monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1<sup>W93C</sup>. Examination of [Ca<sup>2+</sup>]i following ATP stimulation demonstrated that the expression of Best1<sup>W93C</sup> impaired intracellular Ca<sup>2+</sup> signaling. Conclusions: These data indicate that Best1 activity strongly influences electrophysiology and Ca<sup>2+</sup> signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.
|Original language||English (US)|
|Number of pages||13|
|State||Published - Apr 1 2015|
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