Cyclic-nucleotide-gated channels mediate synaptic feedback by nitric oxide - PubMed (original) (raw)

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

A Savchenko et al. Nature. 1997 Dec.

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. CNG channels also allow the influx of Ca2+, which is essential for photoreceptor adaptation. In cone photoreceptors, cGMP triggers an increase in membrane capacitance indicative of exocytosis, suggesting that CNG channels are also involved in synaptic function. 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. 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 pCPT-cGMP, 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.

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Figures

Figure 1

Figure 1

Responses of cone terminals to pCPT–cGMP. a, Acutely dissociated cone photoreceptor from Anolis carolinensis retina. Note the outer segment (OS), oil droplet (OD), inner segment (IS), and presynaptic terminal, or ‘pedicle’, of the cone (T). Scale bar, 10 μm. b, Records from a cone containing a terminal (left) and from a cone lacking a terminal (right). Only the cone with the terminal exhibits an inward current in response to 100 μM pCPT–cGMP (top traces; holding potential = −60 mV). Similarly, only the cone with the terminal exhibits voltage-gated Ca2+ currents in response to 20 mV incremental depolarizations from −60 mV to +40 mV (bottom traces). c, An isolated terminal exhibits current in response to 100 μM pCPT–cGMP and 10 mV incremental depolarizations from −60 to +30 mV. Tail currents result from Ca2+-activated Cl− channels,, owing to the presence of 140 mM Cl− in the pipette, rather than 30 mM Cl− plus 110 mM aspartate, as in b.

Figure 2

Figure 2

Steady-state I–V curves of the pCPT–cGMP-activated and voltage-gated Ca2+ currents. a, I–V curve in normal saline (control) and after application of 100 μM pCPT–cGMP. b, I–V curve in normal saline and after application of 1 μM nifedipine. c, I–V relation of the pCPT–cGMP-activated conductance obtained by subtraction of the I–V curve in control saline from the I–V curve in pCPT–cGMP, both obtained in the absence (circles) or presence (squares) of 1 μM nifedipine. All currents were measured at the end of 200-ms voltage pulses from a holding potential of −60 mV.

Figure 3

Figure 3

CNG channels in cone terminals. a, Direct activation of CNG channels in an inside-out patch excised from a cone terminal. The patch contained an estimated 29 channels. b, Dose–response curves for cone terminal (t) and outer segment (os) CNG-channel activation by cGMP and pCPT–cGMP. Continuous curves show fits to the Hill equation. c, Distribution of CNG channels within cones.

Figure 4

Figure 4

Modulation of cone synapses by NO. a, Endogenous NO modulates synaptic communication between cones and horizontal cells. Voltage responses from horizontal cells incubated in normal saline (trace 1) or in 2 mM L-NNA (trace 2) to 500-ms steps of full-field illumination (2 × 10−7 μW μm−2). Control and L-NNA responses were normalized to peak amplitudes (15 and 10 mV, respectively). b, Summary of feedback response experiments, showing mean ± s.e.m. of the ‘response sag’, defined as 1 − (S/P), where P is the peak voltage and S is the voltage at the end of the light step. Recordings were made from horizontal cells in retinae super-fused for 1 h with control saline, L-NNA, 2 mM L-arginine, 2 mM L-arginine + L-NNA, or 100 μM ODQ, as indicated. Results with ODQ (P < 0.02) or L-NNA alone (P < 0.001) were statistically different from control. c, Activation of CNG current in an isolated lizard cone terminal by 50 μM SNC. d, Occlusion of the SNC response by prior application of 100 μM pCPT–cGMP.

Figure 5

Figure 5

Biosensor measurements of transmitter release from cone terminals. a, Experimental arrangement. b, Sensitivity of the glutamate response of the horizontal cell biosensor. Bath application of 1, 2.5, 5, 10 and 100 μM glutamate elicits the responses shown in the inset (vertical scale 10 pA, horizontal scale 10 s). The graph shows the Hill fit to peak responses with a _K_1/2 of 4.9 μM. c, Steady-state changes in current noise in a horizontal cell contacted by the terminal of a dissociated cone. Recordings show reversible effects of 100 μM pCPT–cGMP and 50 μM SNC applied alone, and pCPT–cGMP and SNC applied together. d, Summary of release experiments. Ratio of horizontal-cell current variance during treatment to control variance. Bars show mean variance ±s.e.m., with number of cells tested in parentheses. Results for pCPT–cGMP, SNC and SNC + pCPT–cGMP were statistically different from control (P < 0.001).

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