Cyclic-nucleotide-gated channels mediate synaptic feedback by nitric oxide

Abstract

Cyclic-nucleotide-gated (CNG) channels in outer segments of vertebrate photoreceptors generate electrical signals in response to changes in cyclic GMP concentration during phototransduction 1 . CNG channels also allow the influx of Ca 2+ , which is essential for photoreceptor adaptation 2 . In cone photoreceptors, cGMP triggers an increase in membrane capacitance indicative of exocytosis, suggesting that CNG channels are also involved in synaptic function 3 . Here we examine whether CNG channels reside in cone terminals and whether they regulate neurotransmitter release, specifically in response to nitric oxide (NO), a retrograde transmitter that increases cGMP synthesis and potentiates synaptic transmission in the brain [4][5][6] . Using intact retina, we show that endogenous NO modulates synapses between cones and horizontal cells. In experiments on isolated cones, we show directly that CNG channels occur in clusters and are indirectly activated by S-nitrosocysteine (SNC), an NO donor. Furthermore, both SNC and pCPTcGMP, a membrane-permeant analogue of cGMP, trigger the release of transmitter from the cone terminals. The NO-induced transmitter release is suppressed by guanylate cyclase inhibitors and prevented by direct activation of CNG channels, indicating that their activation is required for NO to elicit release. These results expand our view of CNG channel function to include the regulation of synaptic transmission and mediation of the presynaptic effects of NO.Patch-clamp experiments were performed on acutely dissociated cones from lizard retina, used because they possess large (5-10 μm diameter) presynaptic terminals (labelled T in Fig. 1a). By varying the duration of proteolytic enzyme treatment and trituration, we observed either intact cones or cones devoid of outer segments and/or presynaptic terminals. To investigate whether CNG channels are present in the terminals we applied the whole-cell patch-clamp configuration to cones containing terminals and cones lacking terminals. All the cones selected for use in these experiments were devoid of outer segments, to eliminate their contribution to the whole-cell CNG current. To ensure that the cone terminals were intact and functional, we applied depolarizing voltage pulses to assay for the presence of a voltage-gated Ca 2+ current, which has been previously characterized in cones with healthy terminals 3,7,8 .The results of these experiments are shown in Fig. 1b. All cones exhibiting a voltage-gated Ca 2+ current also exhibited an inward current when the membrane-permeant cGMP analogue pCPT-cGMP (8-para-chlorothio-cGMP) was applied (9 of 9 cells). In contrast, none of the cones without terminals exhibited either the voltage-gated Ca 2+ current or pCPT-cGMP-activated current (0 of 8 cells). To confirm that the pCPT-cGMP-activated current is specifically localized to the terminal, 'whole-terminal' recordings were obtained after isolating the terminal from the inner segment by transecting the axon with a glass probe. Isolated terminals exhibited bot...

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