Marmorstein Alan D, Kinnick Tyson R, Stanton J Brett, Johnson Adiv A, Lynch Ronald M, Marmorstein Lihua Y
Department of Ophthalmology, Mayo Clinic, Rochester, MN.
Department of Physiology, University of Arizona, Tucson, AZ.
Mol Vis. 2015 Apr 1;21:347-59. eCollection 2015.
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 Ca2+ 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.
Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp.
Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. 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 Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling.
These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ 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.
编码Bestrophin-1（Best1）的BEST1基因突变会导致Best卵黄样黄斑营养不良（BVMD）及其他遗传性视网膜退行性疾病。Best1是一种整合膜蛋白，定位于视网膜色素上皮（RPE）的基底外侧质膜。众多体外和体内模型的数据表明，Best1可调节细胞内钙离子水平。尽管从体外和晶体结构数据已知Best1也是一种钙激活阴离子通道，但缺乏Best1在人RPE中作为通道发挥功能的证据。为评估RPE中与Best1相关的通道活性，我们检测了表达内源性Best1的人胎儿RPE（fhRPE）细胞的跨上皮电特性。
利用腺病毒介导的基因转移，我们在fhRPE细胞中过表达Best1和BVMD突变体Best1W93C，并评估静息跨上皮电位（TEP）、跨上皮电阻、短路电流（Isc）和细胞内钙离子水平。使用全细胞膜片钳直接测量转染的HEK293细胞中的氯离子电流。
Best1W93C表现出氯离子电流缺失，并且在共表达时会抑制HEK293细胞中Best1的通道活性。在fhRPE中，Best1的过表达增加了TEP和Isc，而Best1W93C则降低了TEP和Isc。在浴液培养基中替换氯离子会导致过表达Best1的单层细胞中Isc显著降低，但在表达Best1W93C的单层细胞中Isc没有显著变化。我们通过用离子霉素处理细胞来消除钙离子对跨上皮电特性的限制，发现表达Best1W93C的单层细胞中不存在表达Best1的单层细胞中Isc和TEP的变化。同样，用尼氟酸抑制钙激活阴离子通道会降低对照和Best1单层细胞的Isc和TEP，但对Best1W93C单层细胞没有显著影响。用细胞外ATP刺激会导致对照单层细胞中的TEP升高，且高于表达Best1（W93C）的单层细胞中观察到的升高。ATP刺激后对细胞内钙离子浓度（[Ca2+]i）的检测表明，Best1W93C的表达损害了细胞内钙离子信号传导。
这些数据表明，Best1活性强烈影响RPE细胞中的电生理学和钙离子信号传导，并且一种常见的BVMD突变会破坏这两个参数。我们的研究结果支持Best1在人RPE中作为阴离子通道发挥功能的假说。
