Photopigment quenching is Ca2+ dependent and controls response duration in salamander L-cone photoreceptors

Abstract

The time scale of the photoresponse in photoreceptor cells is set by the slowest of the steps that quench the light-induced activity of the phototransduction cascade. In vertebrate photoreceptor cells, this rate-limiting reaction is thought to be either shutoff of catalytic activity in the photopigment or shutoff of the pigment's effector, the transducin-GTP-phosphodiesterase complex. In suction pipette recordings from isolated salamander L-cones, we found that preventing changes in internal [Ca(2+)] delayed the recovery of the light response and prolonged the dominant time constant for recovery. Evidence that the Ca(2+)-sensitive step involved the pigment itself was provided by the observation that removal of Cl(-) from the pigment's anion-binding site accelerated the dominant time constant for response recovery. Collectively, these observations indicate that in L-cones, unlike amphibian rods where the dominant time constant is insensitive to [Ca(2+)], pigment quenching rate limits recovery and provides an additional mechanism for modulating the cone response during light adaptation.

Figure 1.

Effect on the bright flash response in a salamander L-cone of superfusion with 0 Ca²⁺/0 Na⁺ solution. (A) Superimposed responses to bright flashes in Ringer's solution (heavy trace) and on exposure to 0 Ca²⁺/0 Na⁺ solution from 1 s before the flash until progressively increasing times thereafter (light traces). Top traces represent solution change and flash monitors. Suction pipette currents include a junction current resulting from the liquid junction potential between the dissimilar solutions in pipette and bath. Traces are the average of four responses in Ringer's solution and two responses in 0 Ca²⁺/0 Na⁺ solution; measurements were bracketed symmetrically in time. The bright flash delivered 1.22 × 10⁶ photons µm⁻² at 578 nm. In 0 Ca²⁺/0 Na⁺ solution, sodium ions have been replaced with choline; the virtual absence of external permeant ions abolished the inward dark current, but unlike the situation in rods, little outward potassium current was recorded, perhaps as a result of inner segment hyperpolarization (Matthews, 1995, 1996; Lyubarsky et al., 1996). (B) Recovery phases of the responses from A after subtraction of the junction current obtained upon the return to Ringer's solution after a 2-s exposure to 0 Ca²⁺/0 Na⁺ solution during saturating light at the end of the experiment. For each trace, the junction current has been offset in time to coincide with the return to Ringer's solution. Data have been digitally low-pass filtered at 20 Hz. Heavy trace denotes response in Ringer's solution. (C) Dependence of response duration on the time spent in 0 Ca²⁺/0 Na⁺ solution after the flash. Response duration was measured as the time taken after the flash for the response to recover 25% of the original dark current in Ringer's solution (interrupted line in B); times of solution changes measured from the half-relaxation time of the junction current. Regression line of slope 0.52 fitted using a least-squares algorithm.

Figure 2.

Collected data illustrating the dependence of response duration on the time spent in 0 Ca²⁺/0 Na⁺ solution after a bright flash delivered in darkness, as in Fig. 1. Response duration was measured as the time taken after the flash for the recovery of 25% of the dark current in Ringer's solution. Mean data from nine L-cones; error bars represent SEM. Regression line of slope 0.49 ± 0.07 fitted using a weighted least-squares algorithm.

Figure 3.

Effect of prior steady illumination in Ringer's solution on the prolongation of the bright flash response in an L-cone by exposure to 0 Ca²⁺/0 Na⁺ solution. (A) Superimposed responses to bright flashes in Ringer's solution in darkness (heavy trace) or upon superfusion with 0 Ca²⁺/0 Na⁺ solution in darkness or after steady background illumination (light traces). The cone was exposed for 3 s to steady light, and then a bright flash was delivered and the background was extinguished. The solution superfusing the outer segment was rapidly changed from Ringer's solution to 0 Ca²⁺/0 Na⁺ solution 1 s before the flash, and then returned to Ringer's solution 600 ms thereafter. Each trace is the average of two responses in 0 Ca²⁺/0 Na⁺ solution and four responses in Ringer's solution; measurements were bracketed symmetrically in time. Top traces denote light and solution change monitors. Background intensities in 0 Ca²⁺/0 Na⁺ solution were 0, 6.00 × 10², 2.30 × 10³, 9.86 × 10³, and 3.96 × 10⁴ photons µm⁻² s⁻¹ at 578 nm, evoking background responses of progressively increasing amplitude and flash responses of progressively decreasing duration. Bright flashes delivered 1.22 × 10⁶ photons µm⁻² at 578 nm. In 0 Ca²⁺/0 Na⁺ solution, sodium ions have been replaced with choline; the rising phase of the flash response in this solution is barely visible due to the virtual absence of permeant ions. (B) Recovery phases of the responses from A after subtraction of the junction current obtained upon the return to Ringer's solution after exposure to 0 Ca²⁺/0 Na⁺ solution during saturating light at the end of the experiment. Data have been digitally low-pass filtered at 20 Hz. Heavy trace denotes response in Ringer's solution. (C) Dependence of response duration after exposure to 0 Ca²⁺/0 Na⁺ solution on the circulating current during prior steady illumination (filled circles). Circulating current was measured in Ringer's solution just before the solution change. Response duration was measured as the time taken after the flash for the response to recover 25% of the original dark current in Ringer's solution (interrupted line in B); times of solution changes measured from the half-relaxation time of the junction current. Regression line fitted using a least-squares algorithm. Filled square represents time for 25% recovery of the flash response in Ringer's solution without prior background exposure.

Figure 4.

Collected data illustrating the dependence of response duration on the circulating current during prior steady illumination according to the protocol of Fig. 3. Filled symbols, flash delivered in 0 Ca²⁺/0 Na⁺ solution, as in Fig. 2 (nine L-cones); open symbols, flash delivered in Ringer's solution (seven L-cones). Circulating current was measured in Ringer's solution immediately before the solution change or flash and has been normalized for each cell to the dark current in Ringer's solution. Response duration was measured as the time taken after the flash for the recovery of 25% of the dark current in Ringer's solution and has been normalized for each cell to the time for 25% recovery of the flash response in Ringer's solution without prior background exposure. Regression lines of slope 0.89 ± 0.05 (0 Ca²⁺/0 Na⁺ solution) and 0.68 ± 0.03 (Ringer's solution) were fitted using a least-squares algorithm.

Figure 5.

Determination of the dominant time constant from the dependence of L-cone response duration on flash intensity. (A) Flash delivered to a cone in Ringer's solution. (B) Flash delivered to another cone in 0 Ca²⁺/0 Na⁺ solution in which sodium was substituted with the permeant ion guanidinium, which does not support sodium–calcium exchange (Matthews et al., 1988; Nakatani and Yau, 1988). Dotted lines denote recovery of 25% of the dark current immediately preceding the flash in each solution. Flashes in Ringer's solution increased from 6.7 × 10⁴ to 1.2 × 10⁶ photons µm⁻² by factors of ∼2; flashes in 0 Ca²⁺/0 Na⁺ solution increased from 3.9 × 10³ to 1.2 × 10⁶ by factors of ∼4. (C) Mean data for the dependence of response duration on flash intensity in Ringer's solution (open circles, as in A; seven cells) and 0 Ca²⁺/0 Na⁺ solution (filled circles, as in B; 12 cells); error bars denote SEM. Regression lines of slopes 0.14 ± 0.04 s (Ringer's solution) and 0.56 ± 0.06 s (0 Ca²⁺/0 Na⁺ solution), fitted by a weighted least-squares algorithm.

Figure 6.

Dependence of dim flash response properties upon the anion present in the solution bathing the outer segment. Derived response per incident photon from an L-cone for 10-ms dim flashes of wavelength 600 nm (heavy trace) and 720 nm (light trace) in Ringer's solution (A), and in a Ringer's solution in which sulfate had been substituted for chloride (B). The difference in the average response amplitude per incident photon stems from the reduced sensitivity of the cone at the longer wavelength. In Ringer's solution, the average ratio of the sensitivity at 720 nm to that at 600 nm was 9.5 ± 1% (mean ± SEM; nine cells), whereas in Cl⁻-free Ringer's solution, the relative sensitivity decreased to 5.7 ± 0.9% (mean ± SEM; nine cells) at the longer wavelength. Note also that removal of external Cl⁻ substantially reduced the sensitivity and speeded the kinetics of the response. (Inset) Responses of a salamander S-cone to a dim flash of 80 photons µm⁻² at 460 nm in Ringer's solution (heavy trace) and Cl⁻-free solution (light trace). Plotted responses were averaged from five trials and normalized to their peak amplitude to compare time course. (C) Mean sensitivity at 600 and 720 nm, normalized to the sensitivity at the shorter wavelength. Filled upright triangles, Ringer's solution; open inverse triangles, Cl⁻-free solution. Spectral sensitivity curves are plotted according to the visual pigment nomogram of Govardovskii et al. (2000), with peak sensitivity at 610 nm (solid curve) and 620 nm (interrupted curve) adjusted by eye to pass through the 720-nm data point in each solution.

Figure 7.

Effect of anion substitution on the dominant time constant. Flashes delivered to two L-cones in guanidinium-substituted 0 Ca²⁺/0 Na⁺ solution containing either (A) chloride or (B) sulfate as anion. Dotted lines denote recovery of 25% of the dark current immediately preceding the flash in each solution. Flash intensities increased by factors of ∼4 from 1.3 × 10⁴ to 9.8 × 10⁵ photons µm⁻² in chloride-containing 0 Ca²⁺/0 Na⁺ solution and 5.5 × 10⁴ to 4.0 × 10⁶ photons µm⁻² in sulfate-containing 0 Ca²⁺/0 Na⁺ solution. Data have been digitally low-pass filtered at 10 Hz. (C) Mean data for the dependence of response duration on flash intensity in guanidinium-substituted 0 Ca²⁺/0 Na⁺ solution containing chloride (filled circles; 13 cells) or sulfate (open squares; seven cells) as anion. Regression lines of slopes 0.42 ± 0.05 s (chloride) and 0.25 ± 0.05 s (sulfate) fitted by a weighted least-squares algorithm. The cells in Cl⁻-containing solution exhibited a somewhat lower sensitivity and shorter dominant time constant than those of Fig. 5 C, probably reflecting their origin from a different batch of animals obtained at a later time, but within each figure the comparisons are contemporaneous.