title: Role of bestrophin-1 in store-operated calcium entry in retinal pigment epithelium.
abstract: The retinal pigment epithelium (RPE) expresses bestrophin-1 where mutant bestrophin cause retinal degenerations. Overexpression of bestrophin-1 demonstrated Ca(2+)-dependent Cl(-) channel function, whereas the RPE in bestrophin-1 knockout or mutant bestrophin-1 knock-in mice showed no change in Cl(-) conductance. To account for these apparently mutually exclusive findings, we investigated the function of endogenously expressed bestrophin-1 in a short-time RPE cell culture system by means of immunocytochemistry, Ca(2+) imaging, and siRNA knockdown. Immunocytochemical quantification of bestrophin-1 localization demonstrated 2.5 times higher co-localization with the endoplasmic reticulum (ER) Ca(2+)-sensor protein, Stim-1, than with the membrane protein β-catenin, implicating it in store-operated Ca(2+) entry (SOCE). Ca(2+) release from ER stores under extracellular Ca(2+)-free conditions using thapsigargin (1 μM) to inhibit endoplasmic Ca(2+) ATPase (SERCA) followed by re-adjustment of extracellular Ca(2+) to physiological levels activated SOCE, which was insensitive to the blocker of numerous transient receptor potential channels and voltage-dependent Ca(2+) channels SKF96563 (1 μM). SOCE was augmented at 5 μM and inhibited at 75 μM by 2-aminoethoxydiphenyl borate which indicates the involvement Orai-1 channels. In confirmation, SOCE was decreased by siRNA knockdown of Orai-1 expression. SOCE amplitude was strongly reduced by siRNA knockdown of bestrophin-1 expression, which was due to neither changes in Stim-1/Orai-1 expression nor Stim-1/bestrophin-1 interaction. The amount of Ca(2+) released by SERCA inhibition was reduced after siRNA knockdown of bestrophin-1, but not of Orai-1. In conclusion we found that a proportion of bestrophin-1 is functionally localized to ER Ca(2+) stores where it influences the amount of Ca(2+) and therefore Ca(2+) signals which result from activation of Orai-1 via Stim-1.
authors.affiliation: Experimental Ophthalmology, Eye Hospital, University Medical Center Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany.
authors.forename: Néstor Más
authors.initials: NM
authors.lastname: Gómez
authors.forename: Ernst R
authors.initials: ER
authors.lastname: Tamm
authors.forename: Olaf
authors.initials: O
authors.lastname: Strauβ
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D051176:beta Catenin / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D018118:Chloride Channels; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D051176:beta Catenin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium