Abstract
Bestrophin-1, mainly analyzed in overexpression experiments, functions as Ca2+-dependent Cl- channel. Analysis of endogenously expressed bestrophin-1 suggested an influence on intracellular Ca2+. The aim of the study is to analyze the influence of endogenously expressed bestrophin-1 on Ca2+ homeostasis. Primary cultures of retinal pigment epithelial (RPE) cells were established from wild-type and ...
Abstract
Bestrophin-1, mainly analyzed in overexpression experiments, functions as Ca2+-dependent Cl- channel. Analysis of endogenously expressed bestrophin-1 suggested an influence on intracellular Ca2+. The aim of the study is to analyze the influence of endogenously expressed bestrophin-1 on Ca2+ homeostasis. Primary cultures of retinal pigment epithelial (RPE) cells were established from wild-type and bestrophin-1-deficient mice. Intracellular free Ca2+ ([Ca2+](i)) was recorded by Ca2+ imaging; through immunocytochemistry and differential centrifugation, subcellular localization of bestrophin-1 was analyzed. RPE cells of bestrophin-1-deficient mice showed higher levels of resting [Ca2+](i) than cells from wild-type mice. In cells from knockout mice and wild-type mice, ATP led to increases in [Ca2+](i) subsequent to phospholipase C activation. ATP-induced Ca2+ in bestrophin-1-deficient mice rose faster and decayed slower. In cells from wild-type mice, ATP led to [Ca2+](i) increase via depletion of Ca2+ from thapsigargin-sensitive stores. In cells from bestrophin-1-deficient mice, ATP-dependent increase in [Ca2+](i) resulted in 40% of cells from depletion of bafilomycin-sensitive and in 60% from thapsigargin-sensitive Ca2+ stores. After differential centrifugation, bestrophin-1 was found in fractions enriched of ClC-3 Cl channel and myosin-7A. Co-localization analysis of bestrophin-1, with beta-catenin or pan-cadherin, in fresh sections of porcine retina, revealed bestrophin-1 in the basolateral membrane. A portion of endogenously expressed bestrophin-1,localized in the endoplasmic reticulum, influenced uptake of Ca2+ into Ca2+ stores. Therefore, bestrophin-1 possibly conducts Cl- as counter ion for Ca2+ uptake into cytosolic Ca2+ stores.
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