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Qun-Fang Wan, Heidelberger Ruth; Steady-state Ca modulates rod bipolar cell exocytosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6163.
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Loss of the synaptic vesicle protein SV2B results in altered rod bipolar cell (RBC) intraterminal Ca signaling and dramatic changes in neurotransmitter release. The latter were reversed upon restoration of Ca signaling and were partially reproduced in wild-type RBCs by short-term Ca elevation. Here, we further explore the role of intraterminal Ca in RBC neurotransmitter release.
RBCs were acutely isolated from the retinae of adult mice. Exocytosis, evoked by a pulse train that probes the state of the rapidly-releasing vesicle pool, was tracked using membrane capacitance measurements. Intraterminal Ca and Ca entry were measured with bis-fura-2 and by ICa. Intraterminal Ca was regulated by manipulating Ca extrusion mechanisms or via internal dialysis with Ca-EGTA buffered internal recording solutions.
Intraterminal steady-state Ca was increased from ≈50nM to ≈85nM for 10-60 minutes using the reverse mode of the Na/Ca exchanger. Stimulus-evoked Ca signaling remained comparable to that of control RBCs. However, the rate of rise of the cumulative capacitance increase was enhanced relative to controls, decreasing the extent of synaptic depression. This enhancement was not attributable to an initial decrease in the rapid pool, as the response to the first pulse in the train was similar between test and control conditions. When steady-state intraterminal Ca was elevated to ≈180nM, total exocytosis did not increase further. However, within the 50-180nM range of steady-state Ca, early enhancement of exocytosis, measured as the initial slope in the cumulative capacitance plot, was linear with respect to the steady-state Ca. Regardless of the method of Ca elevation, increased steady-state intraterminal Ca failed to decrease the apparent Ca-sensitivity of release.
Elevation of the steady-state intraterminal Ca resulted in an early and robust enhancement of RBC exocytosis. This enhancement occurred without a change in the apparent Ca sensitivity of release, despite tens of minutes of elevated Ca. This suggests that the decrease in the apparent Ca-sensitivity of release observed in SV2-deficiency reflects a specific effect of SV2 deletion or of longer-term Ca elevation than that studied here. Our data support a role for bulk intraterminal Ca in the regulation of vesicle recruitment and demonstrate that modest changes in steady-state Ca can significantly impact RBC synaptic output.
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