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Tunde Molnar, Eerik Elias, Amber M. Frye, Wei Xing, Ning Tian, David Krizaj; TRPC1 and 3 Channels Mediate Store-operated Calcium Entry in Mouse Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5340.
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To characterize the properties of store-operated calcium entry (SOCE) in mouse retinal ganglion cells (RGCs). We also assessed whether SOCE in mouse RGCs is mediated by TRPC1 and/or TRPC3 channels which were shown to function as store-operated channels in non-excitable cells.
Fura-2 calcium imaging was performed in dissociated mouse RGCs prepared from wild type, transgenic Thy1:CFP and Trpc1/Trpc3 (TRPC1/3-/-) double knockout retinas. Whole-cell patch-clamp recordings were made from RGCs in whole mount retinas prepared from wild type mice. Store-operated signals were evoked with prolonged depletion of intracellular Ca2+ stores within the endoplasmic reticulum (ER). Immunohistochemistry and RT-PCR was performed using primary antibodies and primers for mouse SOC channel candidates.
Cell-type specific RT-PCR showed expression of Trpc1-7, Stim1-2, Orai1-3 transcripts in wild type mouse RGCs. Resting [Ca2+]i levels in dissociated wild type RGCs were 53 ± 7 nM. Glutamate and high K+ -containing saline elevated [Ca2+]i to ~1 µM, indicating maintained excitability of dissociated RGCs. Depletion of ER stores in Ca2+-free saline supplemented with cyclopiazonic acid (CPA) induced SOCE (187 ± 7 nM) which was manifested as [Ca2+]i overshoots following the return to control Ca2+ -containing saline in the presence of L-type Ca channel blockers. SOCE was reduced in mice lacking TRPC1 and 3 isoforms and was further diminished by SOC channel blockers SKF96365 and Gd3+ (98 ± 12 nM). Depletion of intracellular stores with CPA induced an inward current of ~5-10 pA in Ca2+-free saline and in the presence of TTX & NMDA, AMPA/kainate and voltage-gated Ca2+ channel blockers. The store depletion-evoked current was antagonized by SKF96365 and Gd3+.
We found that store depletion elicits cation influx and [Ca2+]i increases in mouse retinal ganglion cells. The suppression of depletion-evoked Ca2+ signals in double KO mice indicating that TRPC1 and/or TRPC3 channels contribute to RGC SOCE. Our data suggests SOCE is prominently expressed in mouse RGCs, highlighting a role for ER stores in RGC excitability and retinal output.
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