All-trans-retinal shuts down rod cyclic nucleotide-gated ion channels: a novel role for photoreceptor retinoids in the response to bright light?

Abstract

In retinal rods, light-induced isomerization of 11-cis-retinal to all-trans-retinal within rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP. Consequently, cyclic nucleotide-gated (CNG) ion channels close, generating the first electrical response to light. After isomerization, all-trans-retinal dissociates from rhodopsin. We now show that all-trans-retinal directly and markedly inhibits cloned rod CNG channels in excised patches. 11-cis-retinal and all-trans-retinol also inhibited the channels, but at somewhat higher concentrations. Single-channel analysis suggests that all-trans-retinal reduces average open probability of rod CNG channels by inactivating channels for seconds at a time. At physiological cGMP levels, all-trans-retinal inhibited in the nanomolar range. Our results suggest that all-trans-retinal may be a potent regulator of the channel in rods during the response to bright light, when there is a large surge in the concentration of all-trans-retinal.

Figure 1

In the presence of a saturating (2 mM) concentration of cGMP, rod channels are inhibited by all-trans-retinal. Data were measured from multichannel, inside-out patches of homomultimeric (CNGA1 only) rod channels. The families of cGMP-activated currents were recorded in response to voltage jumps ranging from −100 to +100 mV in steps of 50 mV, from a holding potential of 0 mV. Currents measured in the absence of cGMP were subtracted from all traces. (A) Current families demonstrating inhibition at saturating cGMP: control, 0.4 μM all-trans-retinal (40% inhibition), and 1.4 μM all-trans-retinal (87% inhibition). (B) Dose-response relation for inhibition by all-trans-retinal in saturating cGMP. Steady-state, cGMP-activated currents were measured at +100 mV from several patches at increasing concentrations of all-trans-retinal added to the solution bathing the “inside” surface of the patch. Averaged data were fit with the Hill equation, IN/IN_MAX = [all-trans-retinal]ⁿ/(IC₅₀ⁿ + [all-trans-retinal]ⁿ), where IN is percent inhibition, IN_MAX is maximal inhibition, IC₅₀ is the concentration of all-trans-retinal required to achieve half maximal inhibition, and n is the Hill coefficient. Data points are averaged values from 5 patches, and plotted with SD (error bars). IN_MAX = 100%; IC₅₀ = 0.35 μM; and n = 1.5.

Figure 2

Rod channels are inhibited by other retinoids less potently than by all-trans-retinal. (A) Dose–response relation for inhibition by 11-cis-retinal in saturating cGMP. Points are mean values from 2 to 4 patches along with SD (error bars). IN_MAX = 100%; IC₅₀ = 0.88 μM; and n = 1.36. (B) Dose-response relation for inhibition by all-trans-retinol in saturating cGMP. Points are mean values from 2 to 4 patches along with SD (error bars). IN_MAX = 100%; IC₅₀ = 0.99 μM; and n = 1.77.

Figure 3

Inhibition of rod channels by all-trans-retinal is more pronounced at low concentrations of cGMP. Recordings shown in A were made as those shown in Fig. 1, except that the bath concentration of cGMP was far below saturating, eliciting only ≈8% of the maximal current evoked by a saturating (2 mM) cGMP concentration. (A) Current families demonstrating inhibition at low (15 μM) cGMP: control, 40 nM all-trans-retinal (62% inhibition), and 140 nM all-trans-retinal (99% inhibition). (B) Dose–response relation for inhibition by all-trans-retinal in low cGMP. Measurements were made in a manner similar to those described in Fig. 1. Data were fit with the Hill equation as in Fig. 1. Data points with error bars (SD) are averaged values from 2 patches; other points are from a single patch. Experiments with intermediate subsaturating cGMP concentrations yielded intermediate IC₅₀s for inhibition by all-trans-retinal. IN_MAX = 100%; IC₅₀ = 35 nM; and n = 1.5.

Figure 4

Inhibition of the olfactory channel suggests that all-trans-retinal is a closed-state inhibitor. Steady-state currents were measured from patches of homomultimeric (CNGA2 only) olfactory channels at +100 mV. Data obtained at saturating cGMP (open circles) are the average of 2–4 patches with SD (error bars) and were fit with the Hill equation with the following parameters: IN_MAX = 10.7%; IC₅₀ = 0.27 μM; and n = 2.90. Data obtained at low cGMP (filled triangles) are from a single patch and were fit with the Hill equation with IN_MAX = 91.3%; IC₅₀ = 0.12 μM; and n = 2.47. These data are consistent with those from other experiments of this type with different subsaturating cGMP concentrations. The open triangle represents the recovery of much of the cGMP-activated current following the experiment at low cGMP through the addition of a saturating amount of cGMP. This finding demonstrates a reversibility of the inhibition without removal of the retinoid.

Figure 5

Single-channel analysis reveals a dramatic decrease in open probability by all-trans-retinal. Raw current traces in A–D were recorded from a single inside-out patch containing 2 homomultimeric rod channels at a holding potential of +80 mV. Sampling rate was 25 kHz after filtering at 5 kHz. The line labeled c represents the zero-current level when both channels were closed. The upper two lines represent the current when one or both channels were open as determined from the fits to the histograms. Patches were bathed in the low divalent sodium solution (see Experimental Procedures) without cGMP (A), with saturating cGMP (B), and with two different all-trans-retinal concentrations at saturating cGMP as designated (C and D). Each amplitude histogram on the right was constructed from four 2.2-s traces of continuous recording. The application of 0.4 μM all-trans-retinal markedly decreases channel open probability, so that there are no simultaneous openings of two channels for the duration of this recording. There is no channel activity during the recording obtained in 1 μM all-trans-retinal. Histograms in A and D were fit with a Gaussian distribution. The histogram in B was fit by a sum of two Gaussian functions constrained so that the opening of the channels is described by a binomial distribution with the number of open channels n = 2; open probability P_O = 0.97; single-channel current i = 2.49 pA; and standard deviation σ = 0.60 pA. The histogram in C was fit by similar distributions, with n = 1; P_O = 0.96; i = 2.50 pA; and σ = 0.48 pA.

Figure 6

Model for the role of retinoid inhibition of rod CNG channels. The visual transduction pathway in photoreceptors, including two modes of positive feedback involving the all-trans-retinal liberated from rhodopsin following photoisomerization. Evidence suggests that all-trans-retinal can reenter the binding pocket in the opsin protein and stimulate transduction through a noncovalent interaction (30). We suggest that the inhibitory effect of all-trans-retinal on the cGMP-gated channel may also contribute to channel closure under certain circumstances, perhaps during the response to bright light.