Developmental expression of retinal cone cGMP-gated channels: evidence for rapid turnover and trophic regulation

Abstract

The cyclic GMP-gated cationic channels of vertebrate photoreceptors are essential for visual phototransduction. We have examined the developmental regulation of cGMP-gated channels in morphologically identified cones in the chick retina. Expression of cone-type cGMP-gated channel mRNA can be detected at embryonic day 6 (E6), but expression of functional channels, as accessed by patch-clamp recordings, cannot be detected until E8. Plasma membrane channels in embryonic cones have a high turnover rate because inhibition of protein synthesis or disruption of the Golgi apparatus causes an almost complete loss of functional cGMP-gated channels within 12 hr. Different subpopulations of cones begin to express functional channels at different developmental stages, but all cones express channels by E10. Expression of cGMP-gated channels in at least one cone subpopulation appears to require one or more soluble differentiation factors, which are presumably present in the normal microenvironment of the developing retina. Application of chick embryo extract (CEE), a rich source of trophic factors, causes marked stimulation of cGMP-gated channel expression in chick cones at E8, but not at E6. Inhibition of MAP kinase (Erk) signaling using PD98059, or inhibition of PI3 kinase signaling by LY294002, blocked the stimulatory effects of CEE on E8 cones. Several recombinant trophic factors were also tested, but none could mimic the stimulatory effects of CEE on channel expression. In summary, the developmental expression of cGMP-gated cationic channels in embryonic cones appears to be regulated by epigenetic factors. The ability of cones to respond to these epigenetic factors is also developmentally regulated.

Fig. 1.

Two different Hoffman optics photomicrographs of embryonic chick cones isolated at E6 and maintained in culture for 5 d. Arrows indicate oil droplets, the principle criterion for identification of this cell type in dissociated cell culture. Scale bar, 10 μm.

Fig. 2.

Developmental expression of cone-type cGMP-activated channel mRNA in embryonic chick retina.A, Detection of cGMP-activated channel transcripts by RT-PCR. Total RNA was isolated at the indicated developmental stages, a constant amount was reverse-transcribed, and channel cDNA was amplified using matched primers designed to obtain a 284 bp product (arrow). No product was obtained in the negative control lane in which the reverse transcription step was omitted (RT−). Other developmental stages are indicated above the lanes, and PH indicates 2 d after hatching.B, Quantitative analysis of developmental expression of channel transcripts by RNase protection assay. Top panelshows representative data showing signal obtained for channel transcripts, or β-actin loading controls, as indicated. Bottom panel summarizes data obtained from three replications of this experiment. The ordinate is the relative mean channel/β-actin transcript ratios, and error bars represent SEM. Both methods indicate that cone-type cGMP-gated channel transcripts are present as early as E6 and increase during embryonic development.

Fig. 3.

Developmental expression of functional cGMP-gated channels in developing cones of embryonic chick retina.A, Typical currents evoked by bath application of various concentrations cGMP to an excised inside-out patch from an E10 cone. Fully closed states are indicated by dotted line,and cGMP concentrations are indicated to the right of each trace. Note presence of multiple cGMP-gated channels in this patch and increasing response amplitude with concentration. B, Concentration–response curve constructed from a different E10 inside-out patch. Data points are shown with superimposed least-squares fit to the Hill equation, with K_D and Hill slope as indicated. C, Mean currents evoked by bath application of 100 μm cGMP to inside-out patches excised from cone photoreceptors at the developmental stages indicated. No current was detected in the vast majority of patches excised from E6 cells. The mean current levels at E8 and E10 are significantly (p < 0.05) different from each other and from E6 (Kruskal-Wallis test). D, Distribution of current amplitudes from data shown in C. Data obtained at E6 and E10 are not significantly different from a normal distribution. In contrast, data obtained at E8 are significantly (p < 0.05) different from a normal distribution (Kolmogorov–Smirnov one-sample test) and appear to be bimodal, suggesting existence of multiple cell populations.

Fig. 4.

Evidence for epigenetic regulation of cGMP-gated channel expression in developing cones. A, Summary of mean currents evoked by bath application of 100 μm cGMP to inside-out patches excised from cells isolated at E6, E8, or E10 and then maintained in culture in the dark for various periods of time after dissociation. Cells isolated at E6 did not express channels 1 d after isolation (E6 + 1) but expressed an intermediate level of channels 3 d (E6 + 3) or 5 d (E6 + 5) after isolation. Channel expression in E6 + 3 is similar to that observed in age-matched E8 + 1 cells. In contrast, channel expression in E6 + 5 cells is significantly (p < 0.05) less than that observed in age-matched E10 + 1 cells, indicating the need for a factor not present in these culture conditions. B, Distribution of response amplitudes in E6 + 5 cells and E10 + 1 cells from data shown in A. Note significantly (p < 0.05) non-normal and apparently bimodal distribution of response amplitudes in E6 + 5 cells and normal distribution of response amplitudes in E10 + 1 cells, suggesting that populations of cells differ in their ability to express functional channels in these culture conditions.

Fig. 5.

Chick embryo extracts (CEE) stimulate functional expression of cGMP-gated channels in cones developing in vitro. The effects of CEE depend on the stage at which cells are removed from the retinal microenvironment.A, E6 + 5 cells were cultured for all 5 d in the presence of basal medium (no CEE), in medium containing 10% CEE, or in 10% heat-inactivated CEE (H-CEE). None of these treatments had a significant effect on the expression of functional channels in cones isolated at E6. B, Application of 10% CEE to E8 + 1 cells for 12 hr caused a robust stimulation of channel expression, whereas heat-inactivated CEE (H-CEE) had no effect.

Fig. 6.

Effects of protein synthesis inhibitors and kinase inhibitors on channel-stimulatory effects of chick embryo extract (CEE) in developing chick cones. All experiments were performed on E8 + 1 cells. A, Application of CEE for 12 hr caused a robust stimulation of functional channel expression compared with control cells cultured in basal media. Channel expression was markedly reduced when CEE was applied in the presence of either the translational inhibitor anisomycin (0.1 mg/ml) or brefeldin A (1 μg/ml), an agent that causes disruption of the Golgi apparatus. Mean current amplitudes in anisomycin- or brefeldin A-treated cells were significantly lower than those observed in either control or CEE-treated cells, suggesting that functional plasma membrane channels turn over rapidly. B, The stimulatory effects of CEE are completely blocked by PD98059 (50 μm), an inhibitor of Erk MAP kinase, or LY294002 (50 μm), an inhibitor of PI3 kinase. Current amplitudes in drug-treated cells are not significantly different from controls cultured in the absence of CEE. These drugs do not produce direct blockade of the channels.