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

Pflugers Arch. 2013 Apr;465(4):481-95. doi: 10.1007/s00424-012-1181-0. Epub 2012 Dec 4.


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.

MeSH terms

  • Animals
  • Calcium / metabolism
  • Calcium Channel Blockers / pharmacology
  • Calcium Signaling*
  • Chloride Channels / genetics
  • Chloride Channels / metabolism*
  • Endoplasmic Reticulum / metabolism
  • Eye Proteins / genetics
  • Eye Proteins / metabolism*
  • Membrane Glycoproteins / metabolism
  • Protein Binding
  • RNA, Small Interfering
  • Retinal Pigment Epithelium / metabolism*
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / antagonists & inhibitors
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism
  • Swine
  • beta Catenin / metabolism


  • Calcium Channel Blockers
  • Chloride Channels
  • Eye Proteins
  • Membrane Glycoproteins
  • RNA, Small Interfering
  • beta Catenin
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Calcium