Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1^−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1^−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.Tight regulation of cell volume is fundamental to proper cell function and survival. In general, rapid water influx across cell membranes leads to cell swelling, which in turn activates net efflux of K⁺ and Cl⁻, thereby triggering the release of osmotically obligated water from the cell. Essential to this process is the activation of a current primarily carried by chloride ions (I_swell). This current is gated by volume-regulated anion channels (VRACs) returning the cell to a controlled state of homeostatic integrity, a complex mechanism commonly referred to as regulatory volume decrease (RVD) (1, 2). Although VRACs share common features in almost all cell types, it is unclear whether there is one ubiquitous channel or a diversity of chloride channels with slightly differing functional properties. In this context, three families of proteins—the Ca²⁺- and/or volume-sensitive anoctamins, bestrophins, and the recently discovered LRRC8s—are presently at the center of interest (3–7).Bestrophin 1 (BEST1), a member of the human bestrophin family of four paralogous genes, encodes an integral membrane protein strongly expressed in the human retinal pigment epithelium (RPE) (8). Mutations in BEST1 have been associated with various macular dystrophies most prominently represented by Best disease (BD), a central retinopathy with autosomal dominant inheritance but variable penetrance and expressivity (9, 10). Key features of BD pathology include a striking lipofuscin accumulation in the macular RPE (11) and an abnormal light peak (LP)/dark trough ratio in the electro-oculogram (EOG) reflective of an impaired RPE (12). The abnormalities in the LP were suggested to be compatible with a function of BEST1 as a Ca²⁺-activated Cl⁻ channel (CaCC) (13, 14).Addressing BEST1 function, several studies have suggested a role of the protein in distinct basic cellular processes such as Ca²⁺ homeostasis, neurotransmitter release, and cell volume regulation. These studies mostly relied on BEST1 overexpression in HEK293 cells or conducted in vitro experiments with isolated cells from existing Best1-deficient mouse lines. In summarizing these data, BEST1 was shown to be (i) a calcium sensor localized to the endoplasmic reticulum (ER) of mouse RPE (15), (ii) an intracellular Cl⁻ channel activating anoctamin 1 (ANO1) located at the plasma membrane of mouse trachea (5), (iii) a modulator of voltage-gated Ca²⁺ channels in murine RPE (16), and (iv) a channel for tonic GABA or slow glutamate release in mouse glia cells and astrocytes (17, 18). To date, the functional role of Best1 has not been determined in the mouse testis, the site of highest endogenous Best1 expression in the mouse (19). In addition, using patient-derived hiRPE cells, the role of BEST1 in mediating ER calcium release and/or uptake was shown (20). In contrast, two independent studies in S2R+ cells from Drosophila melanogaster strongly suggested the invertebrate Drosophila Best1 (dBest1) to act as a volume-regulated chloride channel but with biophysical characteristics clearly distinct from a vertebrate VRAC (3, 21). By small interfering RNA (siRNA)-mediated knockdown of BEST1 in HEK293 cells (6) and mouse Best1 (mBest1) gene disruption in murine peritoneal macrophages (22), two studies could not show a functional effect of BEST1 on I_swell, thus questioning this protein as a candidate for mammalian VRAC in these cell types. Instead, two studies identified the LRRC8A gene as an essential component of a VRAC in various cultured cell lines (6, 7). In these latter studies, the authors propose a scenario where LRRC8A and the isoforms LRRC8B to LRRC8E form variable cell type-specific hexamers, explaining the variability of VRAC properties in different cell types.Together, the rather disparate reports on BEST 1 function underscore the need to further clarify its role in mammalian VRACs. To this end, we focused on two tissues with strong endogenous BEST1 protein expression—namely, human RPE (8) and mouse sperm (19). Major insight into BEST1 function was gained from (i) RPE cell culture models established via hiPSC technology from a healthy donor and two macular dystrophy patients with established pathologic mutations in BEST1 and (ii) a mouse strain deficient for Best1, the murine ortholog of the human BEST1 gene. When exposed to hypo-osmotic challenge, both the mutant hiPSC-RPE cells and Best1-deficient mouse spermatozoa exhibited severe phenotypes, suggesting BEST1 as a crucial component of VRAC function in these cell types. In addition, membrane rupture experiments and voltage-clamp recordings in oocytes from Xenopus laevis, coexpressing aquaporin-1 (AQP1) and BEST1 from mouse and human, respectively, demonstrated identical functional properties of the mammalian BEST1 orthologs.