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

Abstract

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.bestrophin 1 | volume-regulated anion channel | induced pluripotent stem cell | retinal pigment epithelium | mouse sperm

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

“…[7][8][9] Monomers of Best1 oligomerize [10][11][12][13][14] to form a highly conserved homopentameric Ca 2þ -activated ion channel. 8,9 Data from heterologous systems, 15,16 primary human RPE cell culture, 17,18 mouse models, 19,20 the structurally similar paralog bestrophin-2, 21,22 as well as recently published crystal structures 8,9 all unambiguously demonstrate that mammalian Best1 is a calcium-activated anion channel. In addition to functioning as an anion channel, Best1 regulates intracellular Ca 2þ signaling and influences Ca 2þ homeostasis.…”

Cl− channels in apoptosis

“…However, while LRRC8A is a crucial element of volume regulation in some cell lines, it is less important for VRAC and RVD in others. In fact, it might even be irrelevant for VRAC and volume regulation in retinal pigment epithelial cells Milenkovic et al 2015). At any rate, shortly after LRRC8A had been reported as VRAC, a study reported the role of LRRC8A in cisplatin resistance (Sorensen et al 2014).…”

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

“…Nearly 200 mutations in BEST1 have been associated with retinal degenerative disorders, and the vast majority of those that have been characterized cause Cl − channel dysfunction (7)(8)(9)(10)(11). Studies have also indicated that certain bestrophin channels are important for cell volume regulation (12)(13)(14).…”