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Role of bestrophin-1 in store-operated calcium entry in retinal pigment epithelium

  • Ion channels, Receptors and Transporters
  • Published:
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Abstract

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 β-catenin, implicating it in store-operated 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 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 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 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.

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  1. Experimental Ophthalmology, Eye Hospital, University Medical Center Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany

    Néstor Más Gómez & Olaf Strauβ

  2. Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany

    Ernst R. Tamm

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Gómez, N.M., Tamm, E.R. & Strauβ, O. Role of bestrophin-1 in store-operated calcium entry in retinal pigment epithelium. Pflugers Arch - Eur J Physiol 465, 481–495 (2013). https://doi.org/10.1007/s00424-012-1181-0

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