Calcium-Dependence of Calcium-Activated Chloride Channels in Rod Photoreceptors

Abstract

Purpose: : 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.

Methods: : Whole cell recordings were obtained from rods in the tiger salamander retinal slice preparation. E_Cl 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.

Results: : Flash photolysis of DM-nitrophen abruptly elevated Ca²⁺and evoked rapidly activating inward currents in rods that exhibited a K_d for Ca²⁺ of 437 nM (N=31). Depolarizing steps evoked long-lasting tail currents that could be inhibited by niflumic acid and reversed near E_Cl, 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.

Conclusions: : 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.

Keywords: photoreceptors • ion channels • synapse