Gomez, Nestor Mas and Tamm, Ernst R. and Strauss, Olaf (2013) Role of bestrophin-1 in store-operated calcium entry in retinal pigment epithelium. PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, 465 (4). pp. 481-495. ISSN 0031-6768, 1432-2013
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The retinal pigment epithelium (RPE) expresses bestrophin-1 where mutant bestrophin cause retinal degenerations. Overexpression of bestrophin-1 demonstrated Ca2+-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, Ca2+ imaging, and siRNA knockdown. Immunocytochemical quantification of bestrophin-1 localization demonstrated 2.5 times higher co-localization with the endoplasmic reticulum (ER) Ca2+-sensor protein, Stim-1, than with the membrane protein beta-catenin, implicating it in store-operated Ca2+ entry (SOCE). Ca2+ release from ER stores under extracellular Ca2+-free conditions using thapsigargin (1 mu M) to inhibit endoplasmic Ca2+ ATPase (SERCA) followed by re-adjustment of extracellular Ca2+ to physiological levels activated SOCE, which was insensitive to the blocker of numerous transient receptor potential channels and voltage-dependent Ca2+ channels SKF96563 (1 mu M). SOCE was augmented at 5 mu M and inhibited at 75 mu M 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 Ca2+ 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 Ca2+ stores where it influences the amount of Ca2+ and therefore Ca2+ signals which result from activation of Orai-1 via Stim-1.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | CHANNEL ACTIVITY; MOLECULAR-BASIS; DYSTROPHY GENE; CA2+ CHANNELS; BEST-DISEASE; ION CHANNELS; CRAC CHANNEL; LIGHT PEAK; MUTATIONS; PROTEIN; Bestrophin-1; Endoplasmic reticulum Ca2+ store; Orai-1; Store-operated calcium entry |
| Subjects: | 500 Science > 570 Life sciences 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Augenheilkunde Biology, Preclinical Medicine > Institut für Anatomie > Lehrstuhl für Humananatomie und Embryologie > Prof. Dr. Ernst Tamm |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 17 Apr 2020 09:07 |
| Last Modified: | 17 Apr 2020 09:07 |
| URI: | https://pred.uni-regensburg.de/id/eprint/16888 |
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