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A. Mercer, K. Rabl, W. B. Thoreson; Calcium-Dependence of Calcium-Activated Chloride Channels in Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1009.
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Calcium-activated chloride channels are localized to synaptic terminals of rods. Chloride efflux through Ca2+-activated chloride channels inhibits L-type Ca2+ channels. We examined the Ca2+-dependence of Ca2+-activated chloride channels and their proximity to Ca2+ channels in the photoreceptor terminal.
Whole cell recordings were obtained from rods in the tiger salamander retinal slice preparation. ECl in the pipette solutions was -20 mV. Intracellular Ca2+ was measured on a confocal microscope using Oregon Green 488 BAPTA-6F introduced into the cell through the patch pipette.
Flash photolysis of DM-nitrophen abruptly elevated Ca2+and evoked rapidly activating inward currents in rods that exhibited a Kd for Ca2+ of 437 nM (N=31). Depolarizing steps evoked long-lasting tail currents that could be inhibited by niflumic acid and reversed near ECl, indicating that they were due to activation of Ca2+-activated chloride channels. To analyze the distance from Ca2+ channels to Ca2+-activated chloride channels, we compared effects on tail currents of fast (5 mM BAPTA) and slow (0.5 or 5 mM EGTA) Ca2+ buffers introduced into the photoreceptor terminal through the patch pipette. After waiting >10 min. for chelators to diffuse to the terminal, tail currents evoked by 500 ms steps to -10 mV averaged 429.0 ± 49.1 pA with 0.5 mM EGTA (N=11), 187.4 ± 41.1 pA with 5 mM EGTA (N=5), and 80.25 ± 25.4 pA with 5 mM BAPTA (N=9). By comparing these results with the profile of free Ca2+ predicted to surround a Ca2+ channel in the presence of the different buffers, we estimate that Ca2+-activated Cl- channels are an average of ~360 nm from Ca2+ channels.
We find that Ca2+-activated Cl- channels can be activated by submicromolar Ca2+ levels and are located a few hundred nm from Ca2+ channels. By comparison, effects of presynaptic BAPTA and EGTA on post-synaptic currents evoked in horizontal cells by presynaptic stimulation of rods suggest that synaptic release sites are less than 100 nm from Ca2+ channels.
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