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M. Wilson, S. Borges, A. Warrier; Calcium Released from ER Stores through IP3 but not Ryanodine Receptors Promotes Transmitter Release from Cultured Chick Amacrine Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4148.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the sources of Ca2+ responsible for transmitter release from amacrine cell synapses. Methods: Cultured amacrine cells from embryonic chick retinas were examined at embryonic day 16-18 using either perforated or whole-cell patch clamp methods. For Ca2+ imaging experiments cells were loaded with the Ca2+ indicator OGB-1 and small segments of dendrites examined in line scan mode with a confocal microscope. Results:Linescan imaging of OGB-loaded dendrites revealed that transient increases in dendritic [Ca2+] evoked by 2 s high K+ depolarizations were diminished in duration with the drugs, 2-APB (2-aminoethoxydiphenylborate, 50 µM) and Ry (Ryanodine, 4-40 µM). Most likely, these drugs act on IP3 receptors (IP3R) and Ryanodine receptors (RyR) respectively. Consistent with this, we found that Ry also suppressed the transient [Ca2+] increase caused by 20mM caffeine, a RyR agonist. 2-APB is known to have inhibitory effects on store-operated Ca2+ entry, however in our experiments it failed to suppress a small, La3+-sensitive, inward current that we tentatively identify with Trp channels, suggesting that 2-APB is not acting on store-operated channels. 2-APB had no effect on voltage-dependent Ca2+ currents, nor did Ry in a majority of cells (15 of 19), arguing against non-specific effects of these drugs on voltage-gated Ca2+ channels. Further support for the idea that these drugs suppress Ca2+ release from internal stores comes from experiments in which we have shown that the depolarization-induced depletion of ER Ca2+ stores is reduced, when both 2-APB and Ry are present. To understand the role of stored Ca2+ in synaptic transmission, autaptic miniature postsynaptic currents (minis) were elicited by 5 s depolarizations from -70 mV to 0 mV. 2-APB reduced the frequency of minis observed during the depolarizing step (p<0.05) but surprisingly Ry had no effect (p>0.2). Consistent with the lack of effect for Ry we found that 20mM caffeine failed to increase the frequency of minis. Conclusions: These results imply that IP3R gated stores, perhaps in spatial proximity to vesicular release sites, promote transmitter release by allowing efflux of Ca2+ from ER stores. RyRs, though also present on the ER, appear not to be directly involved in the mechanism of transmitter release.
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