Calcium-independent, cGMP-mediated light adaptation in invertebrate ciliary photoreceptors

Abstract

Calcium is thought to be essential for adaptation of sensory receptor cells. However, the transduction cascade of hyperpolarizing, ciliary photoreceptors of the scallop does not use IP3-mediated Ca release, and the light-sensitive conductance is not measurably permeable to Ca2+. Therefore, two typical mechanisms that couple the light response to [Ca]i changes seem to be lacking in these photoreceptors. Using fluorescent indicators, we determined that, unlike in their microvillar counterparts, photostimulation of ciliary cells under voltage clamp indeed evokes no detectable change in cytosolic Ca. Notwithstanding, these cells exhibit all of the hallmarks of light adaptation, including response range compression, sensitivity shift, and photoresponse acceleration. A possible mediator of Ca-independent sensory adaptation is cGMP, the second messenger that regulates the light-sensitive conductance; cGMP and 8-bromo cGMP not only activate light-dependent K channels but also reduce the amplitude of the light response to an extent greatly in excess of that expected from simple occlusion between light and chemical stimulation. In addition, these substances accelerate the time course of the photocurrent. Tests with pharmacological antagonists suggest that protein kinase G may be a downstream effector that controls, in part, the cGMP-triggered changes in photoresponse properties during light adaptation. However, additional messengers are likely to be implicated, especially in the regulation of response kinetics. These observations suggest a novel feedback inhibition pathway for signaling sensory adaptation.

Figure 1.

The light response is not accompanied by changes in cytosolic calcium. Simultaneous recording of membrane current and fluorescence in a voltage-clamped ciliary photoreceptor. The cell was dialyzed for ≈4 min with an internal solution containing the Ca²⁺ indicator Calcium Green-2 (83 μm). The epi-illumination beam from a xenon arc source was turned on for 500 ms (λ_max = 480 nm; bandwidth, 40 nm). Top trace, The outward photocurrent elicited by the excitation light, measured at a holding potential of -30 mV. Bottom trace, The emitted fluorescence (λ_max = 535 nm; bandwidth, 50 nm), collected from a 10 × 10 μm region that included the light-sensitive ciliary appendages of the cell. The optical signal jumped abruptly after the shutter was opened, but no additional change in fluorescence occurred during the light response. Inset, A similar experiment conducted in a microvillar (depolarizing) cell from the same preparation. In this case, the photocurrent was inwardly directed, and the fluorescence signal measured from the light-sensitive lobe indicated a pronounced Ca²⁺ increase coincident with the light response. cps, Counts per second.

Figure 2.

Stimulation of the light-sensitive conductance by cGMP antagonizes the light response. A, Left, Outward potassium current activated by internal perfusion with 20 μm 8-Br-cGMP in the dark. The trace begins at the time the membrane patch was ruptured to gain access to the intracellular compartment. Right, After equilibration, a 100 ms flash of saturating intensity was delivered and elicited a photoresponse of only ≈500 pA. B, Summary of the results pooled from five control cells and four cells treated with 8-Br-cGMP: the bar on the left represents the sum of the cGMP-evoked current (filled portion) and the residual photocurrent (open portion); the total falls significantly short of the maximum amplitude of the light-evoked current measured in control cells (right). Error bars indicate SD.

Figure 3.

cGMP decreases light sensitivity. A, Left, Intensity series recorded from a control photoreceptor in the dark-adapted (D.A.) state (top) and in the presence of an adapting background light (4.2 × 10¹⁴ photons · cm^-2 · s^-1). The peak amplitudes of the photocurrent are plotted as a function of test flash intensity on the right. The 20 ms unattenuated flash delivered 1.62 × 10¹⁴ photons × cm^-2 (photon flux, 1.29 log relative to the background). L.A., Light-adapted. B, Effect of 8-Br-cGMP on the intensity-response curve. The top family of traces was measured in a control cell, and the bottom family was measured in a different photoreceptor internally dialyzed with 20 μm 8-Br-cGMP. Right, Average peak amplitude of the photocurrents measured under the two conditions, plotted as a function of light intensity (n = 4 and 5, respectively); error bars indicate SD. Unattenuated test flash, 1.38 × 10¹⁴ photons ×cm^-2. C, Left, Maximum amplitude of the photocurrent measured with control intracellular solution and with increasing concentrations of 8-Br-cGMP (n = 6, 5, 4, and 4). Right, Magnitude of the membrane currents evoked in the dark by internal perfusion of the same concentrations of 8-Br-cGMP.

Figure 4.

cGMP analogs potentiate light-induced desensitization. Ciliary photoreceptor cells voltage clamped at -30 mV were stimulated with a sustained light stimulus sufficiently bright to induce adaptation (8.8 × 10¹⁴ photons · cm^-2 · s^-1). Left, Superimposed photocurrents measured in one control dark-adapted cell and in one dialyzed with 20 μm 8-Br-cGMP; the amplitude of the response was markedly reduced in the treated cell. Right, The same two records have been normalized with respect to the peak, highlighting the pronounced acceleration in the relaxation and the reduced steady-state plateau after administration of the cGMP analog.

Figure 5.

Acceleration of the photocurrent by 8-Br-cGMP resembles light adaptation effects. A, Left, Superimposed flash responses (4.95 × 10¹³ photons × cm^-2) in a control photoreceptor stimulated under dark-adapted conditions and subsequently during exposure to sustained background illumination (4.2 × 10¹³ photons · cm^-2 · s^-1). Right, The same two traces were normalized with respect to their peak amplitude (Norm. Ampl.), revealing a pronounced acceleration in response kinetics after light adaptation. B, A similar comparison is made between the dark-adapted responses in one control cell and one dialyzed with 20 μm 8-Br-cGMP. Again, the treatment caused not only a reduction in photocurrent amplitude but also a more rapid time course. C, Flash response speed, measured as the FWHM of the light-evoked current, in control conditions and during perfusion with increasing concentrations of 8-Br-cGMP, in separate groups of cells. Error bars denote SD.

Figure 6.

Desensitization is reduced by an inhibitor of PKG. A, The response (Resp.) to a test flash of fixed, just-saturating intensity was measured under dark-adapted (D.A.) conditions and in the presence of a steady background (Bkg) light (5.33 × 10¹² photons · cm^-2 · s^-1), which was turned on 4.8 s before the test flash (5.6 × 10¹³ photons × cm^-2). The top traces were recorded with standard intracellular solution, whereas the bottom pair were obtained from a different photoreceptor dialyzed with 3 μm KT5823. L.A., Light-adapted. B, Summary of the effects of KT5823 on background adaptation parameters. The peak amplitude and the FWHM of the incremental response, relative to dark-adapted conditions, were averaged for 11 control cells (filled bars) and nine cells dialyzed with KT5823 (cross-hatched bars). Both measurements indicate that, in each case, the PKG inhibitor partially antagonized the effects of the adapting light. The dimmer background was as in A; the brighter background was increased by 1 log unit. Error bars indicate SD. C, Effects of varying the intensity of the adapting background. The mean amplitude of the flash response (normalized with respect to that measured in dark-adapted conditions) is plotted as a function of the intensity of the background light, which was raised at 0.6 log increments (unattenuated intensity, 5.1 × 10¹⁴ photons · cm^-2 · s^-1). The experiment was conducted in five control cells and four cells treated with KT5823; error bars denote SD.

Figure 7.

Modulation of response kinetics by cAMP analogs. A, Representative intensity series obtained in one voltage-clamped control cell (left) and one dialyzed with 50 μm 8-Br-cAMP (right). Test flashes were 100 ms in duration, and the holding potential was -30 mV. B, Mean amplitude of the saturating photocurrent for control and 8-Br-cAMP-treated cells; the difference was not statistically significant. Error bars indicate SD. C, Normalized, superimposed traces of photocurrents elicited by flashes of identical, subsaturating intensity. The time course of the response obtained in the presence of 8-Br-cAMP is markedly accelerated. D, Bar graph representing the average width of the flash response at half-maximal amplitude (n = 8 and 9 for control and 8-Br-cAMP-treated cells, respectively).