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Title of Journal: Pflugers Arch Eur J Physiol

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Abbravation: Pflügers Archiv - European Journal of Physiology

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Springer-Verlag

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10.1016/0013-4694(77)90250-4

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1432-2013

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Role of bestrophin1 in storeoperated calcium ent

Authors: Néstor Más Gómez Ernst R Tamm Olaf Strauβ
Publish Date: 2012/12/04
Volume: 465, Issue: 4, Pages: 481-495
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Abstract

The retinal pigment epithelium RPE expresses bestrophin1 where mutant bestrophin cause retinal degenerations Overexpression of bestrophin1 demonstrated Ca2+dependent Cl channel function whereas the RPE in bestrophin1 knockout or mutant bestrophin1 knockin mice showed no change in Cl− conductance To account for these apparently mutually exclusive findings we investigated the function of endogenously expressed bestrophin1 in a shorttime RPE cell culture system by means of immunocytochemistry Ca2+ imaging and siRNA knockdown Immunocytochemical quantification of bestrophin1 localization demonstrated 25 times higher colocalization with the endoplasmic reticulum ER Ca2+sensor protein Stim1 than with the membrane protein βcatenin implicating it in storeoperated Ca2+ entry SOCE Ca2+ release from ER stores under extracellular Ca2+free conditions using thapsigargin 1 μM to inhibit endoplasmic Ca2+ ATPase SERCA followed by readjustment of extracellular Ca2+ to physiological levels activated SOCE which was insensitive to the blocker of numerous transient receptor potential channels and voltagedependent Ca2+ channels SKF96563 1 μM SOCE was augmented at 5 μM and inhibited at 75 μM by 2aminoethoxydiphenyl borate which indicates the involvement Orai1 channels In confirmation SOCE was decreased by siRNA knockdown of Orai1 expression SOCE amplitude was strongly reduced by siRNA knockdown of bestrophin1 expression which was due to neither changes in Stim1/Orai1 expression nor Stim1/bestrophin1 interaction The amount of Ca2+ released by SERCA inhibition was reduced after siRNA knockdown of bestrophin1 but not of Orai1 In conclusion we found that a proportion of bestrophin1 is functionally localized to ER Ca2+ stores where it influences the amount of Ca2+ and therefore Ca2+ signals which result from activation of Orai1 via Stim1

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