RESUMO
Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca(2+) channels, which are in turn regulated by Cl(-) moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca(2+) channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca(2+) buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca(2+) channels. Comparing Cl(Ca) currents with predicted Ca(2+) diffusion profiles suggested that Cl(Ca) and Ca(2+) channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca(2+) channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca(2+)](i)) elevation through flash photolysis of DM-nitrophen exhibited EC(50) values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca(2+)](i) in photoreceptor terminals. Consistent with control of exocytosis by [Ca(2+)] nanodomains near Ca(2+) channels, average submembrane [Ca(2+)](i) remained below the vesicle release threshold (â¼ 400 nM) over much of the physiological voltage range for cones. Positioning Ca(2+) channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca(2+) influx at one site to influence relatively distant Ca(2+) channels.