Purpose: 

Endogenously expressed bestrophin-1 was found to play a role in intracellular Ca²⁺ stores. Overexpressed bestrophin-1 shows Cl channel function. Thus, it is likely that Cl channel function is of importance for calcium signaling involving Ca²⁺-stores. 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: 

Western blot, immunocytochemistry, immunoprecipitation, siRNA transfection and Ca²⁺ imaging (Fura- 2AM).

Results: 

Short-time primary porcine RPE cells showed a robust expression of bestrophin-1 and SOCE proteins: Stim-1 and Orai-1. SOCE was elicited by thapsigargin (1mM), a SERCA ATPase blocker, followed by Ca²⁺ free conditions and later by re-addition of extracellular Ca²⁺. SOCE was found to be remarkably blocked by 2-APB (75mM), enhanced by 2-APB (5mM) and no affected by SKF96563 (50mM). Porcine RPE cells transfected with Orai-1siRNA showed a SOCE amplitude reduction by 70%. Bestrophin-1siRNA was found to reduce SOCE amplitude by 80%. Quantification of the amount of Ca²⁺ released from stores in response to thapsigargin application showed a remarkably reduction in those cells transfected with bestrophin-1siRNA compared to Orai-1siRNA transfected cells. Co-localization analysis by immunocytochemistry showed stronger co-localization of bestrophin-1 and Stim-1 than bestrophin-1 with b-catenin. Immunoprecipitation experiments revealed that Stim-1 does not interact with bestrophin-1.

Conclusions: 

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