Rhodopsin kinase and recoverin modulate phosphodiesterase during mouse photoreceptor light adaptation

Abstract

Light stimulates rhodopsin in a retinal rod to activate the G protein transducin, which binds to phosphodiesterase (PDE), relieving PDE inhibition and decreasing guanosine 3',5'-cyclic monophosphate (cGMP) concentration. The decrease in cGMP closes outer segment channels, producing the rod electrical response. Prolonged exposure to light decreases sensitivity and accelerates response kinetics in a process known as light adaptation, mediated at least in part by a decrease in outer segment Ca(2+). Recent evidence indicates that one of the mechanisms of adaptation in mammalian rods is down-regulation of PDE. To investigate the effect of light and a possible role of rhodopsin kinase (G protein-coupled receptor kinase 1 [GRK1]) and the GRK1-regulating protein recoverin on PDE modulation, we used transgenic mice with decreased expression of GTPase-accelerating proteins (GAPs) and, consequently, a less rapid decay of the light response. This slowed decay made the effects of genetic manipulation of GRK1 and recoverin easier to observe and interpret. We monitored the decay of the light response and of light-activated PDE by measuring the exponential response decay time (τREC) and the limiting time constant (τD), the latter of which directly reflects light-activated PDE decay under the conditions of our experiments. We found that, in GAP-underexpressing rods, steady background light decreased both τREC and τD, and the decrease in τD was nearly linear with the decrease in amplitude of the outer segment current. Background light had little effect on τREC or τD if the gene for recoverin was deleted. Moreover, in GAP-underexpressing rods, increased GRK1 expression or deletion of recoverin produced large and highly significant accelerations of τREC and τD. The simplest explanation of our results is that Ca(2+)-dependent regulation of GRK1 by recoverin modulates the decay of light-activated PDE, and that this modulation is responsible for acceleration of response decay and the increase in temporal resolution of rods in background light.

Figure 1.

Reduction of transducin GAP level in GAPux mouse retinas. As shown previously by Keresztes et al. (2004), inactivating one copy of the R9AP gene leads to a noticeable reduction of transducin GAP level. (A) Representative immunoblot simultaneously probed for RGS9-1, Gβ5-L, Gβ5-S, Gβ1, and GAPDH in 10 µg of retinal extracts derived from WT, R9AP heterozygous knockout (Het), and compound R9AP and RGS9-1 heterozygous (ux) mice. (B) Representative immunoblot simultaneously probed for RGS9-1, PDE6β, and GAPDH in WT and ux retinal extracts. (C) Quantification of RGS9-1 level for experiments described in A, showing in Het (middle bar) and ux (right bar) mouse retinas a decrease to 51 ± 3 and 34 ± 3 (mean ± SEM) percent of WT level (left bar). GAPDH level was used for normalization. Similar degree of reduction was seen in Gβ5-L level but not in Gβ5-S or Gβ1 level (not depicted). (D) Quantification of PDE6β expression relative to GAPDH level in experiments of B showed a comparable level in ux retinal extracts to that of WT at 95 ± 7%, while RGS9-1 level dropped to 37 ± 3% (n =3). Error bars are SEMs.

Figure 2.

Comparison of mean response waveform of WT, R9AP+/−, R9AP+/−;RKS561L, and R9AP+/−;Rv−/− rods to 20-ms flashes given at t = 0 for each rod type at the following light intensities (in photons µm⁻²): (A and B) 3, 9, 23, 75, 240, and 780; and (C and D) 9, 23, 75, 240, 780, and 2,800. (A) WT, mean of 12 rods. (B) R9AP+/−, mean of seven rods. (C) R9AP+/−;RKS561L, mean of nine rods. (D) R9AP+/−;Rv−/−, mean of nine rods. Red traces are responses for each rod type to flashes of 23 photons µm⁻². Note that averaging of rod responses tends to slur the decay phases of individual photoreceptors, which vary from rod to rod, with the result that the averaged response especially at bright intensities is not representative of any one individual cell. Mean decay times averaged cell by cell are given in Figs. 4 B and 5.

Figure 3.

Comparison of mean response waveform of GAPux, GAPux;RKS561L, and GAPux;Rv−/− rods to 20-ms flashes given at t = 0 for each rod type at the same intensities (in photons µm⁻²): 3, 9, 23, 75, 130, 240, 430, 780, 1,500, and 2,800. Horizontal lines show value of current at 25% of maximum used in estimating values of Tsat in Fig. 5. (A) GAPux, mean of 16 rods. (B) GAPux;RKS561L, mean of 20 rods. (C) GAPux;Rv−/−, mean of 10 rods. Not all of the rods in Table 1 were used for this figure because responses at every flash intensity were not recorded from every rod. Red traces are responses for each rod type to flashes of 23 photons µm⁻². Note that averaging of rod responses tends to slur the decay phases of individual photoreceptors, which vary from rod to rod, with the result that the averaged response especially at bright intensities is not representative of any one individual cell. Mean decay times averaged cell by cell are given in Figs. 4 B and 5.

Figure 4.

Exponential time course of flash decay. (A) Single-photon responses calculated from the squared mean and variance as in Chen et al. (2000) and Tsang et al. (2006). Traces give means of 41 WT rods (black), 21 GAPux rods (green), 18 GAPux;RKS561L rods (red), and 15 GAPux;Rv−/− rods (blue). Fits through data (solid curves) are exponential decay functions with values of the single time constant τ_REC of 254 ms (WT), 331 ms (GAPux), 193 ms (GAPux;RKS561L), and 174 ms (GAPux;Rv−/−). (B) Mean values of τ_REC as a function of flash intensity for 12 WT rods (black), 13 GAPux rods (green), 18 GAPux;RKS561L rods (red; only six rods were used for the lowest intensity data point), and 17 GAPux;Rv−/− rods (blue). Error bars are SEMs.

Figure 5.

Tsat as a function of the natural log of the light intensity. Values of Tsat were determined rod by rod as the time from the beginning of the flash for the photocurrent to fall to 75% of its saturating value from the same rods used for Fig. 3. Data points give means and error bars give SEMs from 21 GAPux rods, 28 GAPux;RKS561L rods, and 17 GAPux;Rv−/− rods. Straight lines through data are for values of τ_D as follows: GAPux, 249 ms; GAPux;RKS561L, 176 ms; and GAPux;Rv−/−, 209 ms. See Results.

Figure 6.

Limiting time constant (τ_D) as function of light intensity and circulating current. (A) Mean values of the τ_D as a function of the intensity of the background light (I_B) in photons µm⁻² s⁻¹ for 12 GAPux (□) and 6 GAPux;Rv−/− (■) rods (only 2 GAPux;Rv−/− rods at the brightest background intensity). Curve through GAPux data points is of the form τ_D = τ_D0 + A[exp(−I_B/k)], with τ_D0, A, and k constants whose best-fitting values were τ_D0 = 69 ms, A = 160 ms, and k = 538 photons µm⁻² s⁻¹. Dashed line is linear regression for data from GAPux;Rv−/− rods with the slope constrained to be zero. The best-fitting value of τ_D was 191 ms. (B) Values of the τ_D for the same GAPux rods as in A (□) but plotted as a function of circulating current (r) normalized to its maximum value before presentation of the backgrounds (r_max). Circulating current was estimated rod by rod from the saturating value of the response to flashes in each of the backgrounds. Straight line through data are best fit of linear straight line with coefficient of determination r² = 0.76. Data for GAPux;Rv−/− rods (■) is also given as a function of circulating current, and the dashed line is again linear regression with the slope constrained to be zero and with a best-fitting value of τ_D of 191 ms. Error bars in A and B are SEMs.

Figure 7.

Adaptation to background lights. Ordinate plots sensitivity S_F in the presence of steady background light divided by sensitivity in the absence of background, $S_F^D$; abscissa gives intensity of background in photons µm⁻² s⁻¹. Sensitivity was calculated as the peak response amplitude for small-amplitude responses divided by the flash intensity in photons µm⁻². Data points give means and error bars give SEMs for 20 WT rods (●), 6 GAPux;Rv−/− rods (□), and 16 GAPux rods (◆). Means have been fitted with the Weber–Fechner equation, S_F/$S_F^D$ = I₀/(I₀ + I_B), where I₀ is a constant and I_B is the intensity of the background light. The curve in the middle is the best-fitting curve for WT rods with I₀ = 77 photons µm⁻² s⁻¹. The curve to the left is for GAPux rods with I₀ = 20 photons µm⁻² s⁻¹, and the curve to the right is for GAPux;Rv−/− rods with I₀ = 154 photons µm⁻² s⁻¹. (Inset) Superimposed normalized responses for GAPux and GAPux;Rv−/− rods to 20-ms flashes at 238 photons µm⁻² in dark-adapted rods and in the presence of various background lights. Mean responses have been calculated from 11 GAPux rods at backgrounds of 8, 21, 75, and 204 photons µm⁻² s⁻¹, and 6 GAPux;Rv−/− rods at background light intensities of 21, 75, 204, and 760 photons µm⁻² s⁻¹.