Calcium stores and store–operated calcium channels in photoreceptors

Abstract

Abstract: : Purpose:To characterize the components that support cycling of calcium between the plasma membrane and intracellular calcium stores. In the light [Ca²⁺]_i is low and Ca stores are likely to be empty. We tested the hypothesis that Ca stores could be refilled via Ca entry through store–operated calcium channels (SOCs). We characterized expression of TRPCs (a subclass of SOCs) in salamander and mouse retinae. Methods: For immunofluorescence, isolated cells or slices were incubated overnight with the primary antibody and for 2 hours in a secondary IgGs. Antibodies that recognize the RyR, IP₃R and TRPC channel isoforms were used along with other retinal markers, to localize proteins in tiger salamander and mouse retinae. High–resolution calcium imaging was performed using a cooled digital CCD camera in cells loaded with the high–affinity calcium indicator dye fura–2. Results:Our results suggest that the predominant classes of intracellular store Ca release channels in the salamander photoreceptors are the RyR isoform 2 and IP₃ receptor isoform 2 (IP₃R2) receptors. We also found that TRPC6 is strongly expressed in cell bodies and synaptic terminals of photoreceptors as seen by colocalization with the synaptic marker SV2. TRPC6 was also prominent in bipolar cells. In mouse, TRPC6 antibody prominently labeled cell bodies of rod photoreceptors in the outer nuclear layer but was excluded from synaptic terminals. Salamander photoreceptors also expressed TRPC4 and possibly TRPC3 isoforms, whereas TRPC5 was detected postsynaptically in the outer plexiform layer. To test directly for expression of SOCs we recorded fluorescent signals from fura–2 – loaded photoreceptors. Ca stores were emptied irreversibly by an exposure to 0 Ca/thapsigargin solution. Upon return to Ca–containing solution, cells exhibit a pronounced overshoot. The overshoot was not blocked by dihydropyridine blockers of L–type voltage–gated Ca channels. However, we were able to significantly reduce the overshoot with 2 mM Ni²⁺, as well as with SKF 96365, both blockers of store–operated Ca entry. Conclusions: Our data suggests that photoreceptors express all elements necessary to sustain Ca cycling between Ca stores and the plasma membrane. This includes voltage–gated Ca channels and store–operated Ca channels in the plasma membrane as well as RyR and IP₃ receptors responsible for Ca release from the stores. We found that photoreceptors express several classes of SOC channels. These channels may play a role in stimulation of Ca influx into light adapted cells.

Keywords: calcium • photoreceptors • receptors: pharmacology/physiology