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T. Szikra, D. Krizaj; The Role of Voltage–Operated Ca Channels, Endoplasmic Reticulum and Mitochondria in Compartmentalization of Ca Homeostasis in the Photoreceptor Inner Segment . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2265.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To quantify the relative contribution of plasma membrane Ca influx and extrusion mechanisms and intracellular organelles to the spatiotemporal pattern of Ca homeostasis in photoreceptor terminal, cell body and ellipsoid regions.Methods:Calcium imaging was performed in cells loaded with the calcium indicator dye fura–2 AM. For immunofluorescence (IF), slices were fixed in 4% paraformaldehyde, incubated overnight with the primary antibody raised against L–type Ca channels and plasma membrane Ca ATPases (PMCAs).Results:Depolarization with 30 mM KCl evoked a 2100±321 nM increase in terminal [Ca2+]i; increases in the cell body and the ellipsoid were 1047±140 and 664±147 nM, respectively. In isolated ellipsoids, high KCl triggered a 356±42 nM elevation in [Ca2+]i. The rise time and decays of depolarization evoked [Ca2+]i transients were the fastest in the synaptic terminal (375±181 and 184±68 nM/s,respectively); the responses were slower in the cell body (61± 20 nM/s and 31± 10) and in the ellipsoid (22±8 and 21±6 nM/s). The [Ca2+]i kinetics in the IS compartments corresponded to the density of voltage–gated calcium channels and Ca pumps observed with IF. Inhibition of SERCA pumps decreased the amplitude of KCl–evoked Ca responses in the cell body by ∼40%, but had little effect on ellipsoid [Ca2+]i. In contrast, blocking mitochondrial Ca sequestration with antimycin/oligomycin caused minor decrease in the cell body. To establish the amount of Ca sequestered into the ER and mitochondria, we exposed cells to ionomycin. Ionomycin released 247±22 nM Ca from control ellipsoids and 200±27 from cell bodies; following treatment with antimycin 77±10 nM was released from the ellipsoid and 68±13 from the cell body. Finally, SERCA blockade further decreased the release in the ellipsoid and the cell body, respectively. Conclusions:We analyzed plasma membrane Ca fluxes, Ca diffusion and sequestration/release from intracellular Ca stores in the ISs of rods and cones. The kinetics of Ca signals corresponded to the relative density of voltage–activated Ca channels and pumps. While about 50% Ca enters ellipsoid via resident voltage–gated Ca channels, ∼50% Ca entry is likely to occur via diffusion from the cell body. Both endoplasmic reticulum and mitochondria play a major role in shaping the spatiotemporal pattern of Ca responses in the IS. Whereas the ER plays a major role in cell body, mitochondria dominate both the cell body and the ellipsoid Ca homeostasis. Our experiments demonstrate an extraordinary compartmentalization of Ca signaling in different regions of the photoreceptor IS.
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