Endogenously Expressed Bestrophin-1 Influences Store-operated Ca2+ Entry Mediated By Orai-1 In The RPE

Abstract

Purpose: : : Recent publications suggest that endogenously expressed bestrophin-1 might function as an intracellular Cl channel localized to cytosolic Ca²⁺ stores. With this localization bestrophin-1 was found to interfere with intracellular Ca²⁺ signalling . In order to clarify the intracellular function of bestrophin-1 we investigated store-operated Ca²⁺ entry (SOCE) in primary porcine retinal pigment epithelial (RPE) cells.

Methods: : Ca²⁺ signalling was investigated by means Ca²⁺ imaging using fura-2 as Ca²⁺sensitive fluorescence dye. Expression of Orai-1 and bestrophin-1 was down-regulated by siRNA transfection. Expression of Orai-1 Stim-1 and bestrophin-1 was investigated by means of western-blot analysis and immunohistochemistry.

Results: : Short-time cultured primary porcine RPE cells robustly expressed bestrophin-1 and the proteins of SOCE: Orai-1 and stromal-interacting molecule-1 (Stim-1). SOCE was elicited by application of sarcoplasmic Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (1 µM) under extracellular Ca²⁺-free conditions followed by re-addition of extracellular Ca²⁺. Amplitude of SOCE was increased by 5 µM 2-APB, reduced by 75 µM 2-APB and not influenced by 50 µM SKF96563. RNAi knock-down of Orai-1 strongly reduced SOCE amplitude by 70%. RNAi knock-down of bestrophin-1 also reduced the SOCE amplitude by 80 %. Quantification of the amount of Ca²⁺ released from Ca²⁺ stores by thapsigargin application showed that less Ca²⁺ is stored in cells which have treated with RNAi against bestrophin-1 but not in cells treated with RNAi against Orai-1. Co-localization analysis by immunohistochemistry showed stronger co-localization of bestrophin-1 and Stim-1 than bestrophin-1 with beta-catenin. Immunoprecipitation experiments revealed that Stim-1 does not interact with bestrophin-1.

Conclusions: : Porcine RPE cells exhibit SOCE mediated by activation of Orai-1 Ca²⁺ channels in response to release of Ca²⁺ from cytosolic stores. Bestrophin-1 function as Cl channel which conducts the counter-ion for Ca²⁺ uptake into cytosolic stores by SERCA activity. A loss of bestrophin-1 function would change intracellular Ca²⁺ signalling which involves release of Ca²⁺ from cytosolic stores.

Keywords: signal transduction: pharmacology/physiology • ion channels • retinal pigment epithelium