Location of Release Sites and Calcium-activated Chloride Channels Relative to Calcium 1 Channels at the Photoreceptor Ribbon Synapse. 2

23 Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca 24 channels which are in turn regulated by Cl moving through calcium-activated chloride 25 (Cl(Ca)) channels. We assessed the proximity of Ca channels to release sites and 26 Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast 27 (BAPTA) and slow (EGTA) intracellular Ca buffers. BAPTA did not fully block synaptic 28 release, indicating some release sites are <100 nm from Ca channels. Comparing 29 Cl(Ca) currents with predicted Ca diffusion profiles suggested that Cl(Ca) and Ca 30 channels average a few hundred nanometers apart, but the inability of BAPTA to block 31 Cl(Ca) currents completely suggested some channels are much closer together. Diffuse 32 immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A 33 supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic 34 terminal, in contrast with clustering of Ca channels near ribbons. Cl(Ca) currents 35 evoked by [Ca]i elevation through flash photolysis of DM-nitrophen exhibited EC50 36 values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, 37 respectively. These relationships were used to estimate average submembrane [Ca]i 38 in photoreceptor terminals. Consistent with control of exocytosis by [Ca] nanodomains 39 near Ca channels, average sub-membrane [Ca]i remained below vesicle release 40 threshold (~400 nM) over much of the physiological voltage range for cones. Positioning 41 Ca channels near release sites may improve fidelity in converting voltage changes to 42 synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca influx at one 43 site to influence relatively distant Ca channels. 44 45