Background light produces a recoverin-dependent modulation of activated-rhodopsin lifetime in mouse rods

Abstract

The Ca(2+)-binding protein recoverin is thought to regulate rhodopsin kinase and to modulate the lifetime of the photoexcited state of rhodopsin (Rh*), the visual pigment of vertebrate rods. Recoverin has been postulated to inhibit the kinase in darkness, when Ca(2+) is high, and to be released from the disk membrane in light when Ca(2+) is low, accelerating rhodopsin phosphorylation and shortening the lifetime of Rh*. This proposal has remained controversial, in part because the normally rapid turnoff of Rh* has made Rh* modulation difficult to study in an intact rod. To circumvent this problem, we have made mice that underexpress rhodopsin kinase so that Rh* turnoff is rate limiting for the decay of the rod light response. We show that background light speeds the decay of Rh* turnoff, and that this no longer occurs in mice that have had recoverin knocked out. This is the first demonstration in an intact rod that light accelerates Rh* inactivation and that the Ca(2+)-binding protein recoverin may be required for the light-dependent modulation of Rh* lifetime.

Figure 1.

Effect on response waveform of reducing kinase expression to 40% of normal in RK+/− animals. A–D, Averaged suction-electrode recordings of currents to 20 ms flashes delivered at t = 0 for 10 rods from two WT mice (A); 22 rods from 3 RK+/− mice heterozygous for the rhodopsin kinase gene (B); 13 rods from 2 R9AP95 mice, which overexpress the GAP complex by approximately sixfold (see Materials and Methods) (C); and 19–28 rods from two mice which are both RK+/− and R9AP95 (D). Flash intensities in units of photons · μm⁻² were as follows: A: 4, 17, 43, 159, 453, 1120; B: 4, 17, 43, 159, 453, 863, 1871; C: 17, 43, 159, 453, 646, 863, 1120, 1871; D: 4, 17, 43, 159, 453, 1120.

Figure 2.

Value of τ_REC as function of flash intensity. The declining phase of the flash response was fitted cell by cell with a single exponential decay function; for responses to bright light, only that part of the response which fell below 0.5 of r_max was used to avoid saturation nonlinearly. Data points give mean ± SEM from the following mouse lines (with the number of rods in parentheses): WT (22), RK+/− (11), R9AP95 (12), RK+/−:R9AP95 (11), and the Bark/RK4 line of the RK/Bark chimera (15).

Figure 3.

Generation of BarkRK4 and BarkRK7 transgenic mouse lines. A, Transgenic construct carrying a cDNA called Bark/RK was generated as described in Materials and Methods. Bark/RK, previously named RK/GRK2 (Palczewski et al., 1995), is a mutant GRK1 with 13 aa (underlined) derived from Bark1 around its major autophosphorylation sites (asterisk). B, Determination of GRK1 levels by quantitative Western blotting in BarkRK4 (3) and BarkRK7 (2) mouse lines, versus nontransgenic wild-type control (1). Shown here is a representative Western blot image of GRK1, Bark/RK, and RGS9-1. The mutant Bark/RK has slower mobility during electrophoresis. The level of mutant GRK in BarkRK4 and BarkRK7, is ∼15% and ∼60% of the control level, respectively (n = 3).

Figure 4.

Normal outer segment morphology in Bark/RK4 mouse retinas. A, Immunohistochemistry showing that mutant GRK1 (green) is expressed only in rods and is targeted to the outer segment (OS) layer in Bark/RK4 animals. Cones, marked by cone arrestin (mCAR, red), do not express the mutant kinase. Blue signals are DAPI stains of outer nuclear layer (ONL) nuclei. IS, Inner segment; OPL, outer plexiform layer. B, Semithin retinal sections showing normal morphology in Bark/RK4 and WT animals as compared to the shortened OS layer found in RK−/− animals. All animals were reared in 12/12 cyclic light. INL, Inner nuclear layer; IPL, inner plexiform layer. Scale bars equal 10 μm.

Figure 5.

Effect of reducing kinase expression to ∼15% of normal on response waveform in Bark/RK7 animals. A, B, Averaged suction-electrode recordings of currents of 22 rods from two Bark/RK7 mice (A) and 19 rods from three Bark/RK7:R9AP95 mice (B). Intensities of 20 ms flashes in photons · μm⁻² delivered at t = 0 were as follows: A, 4, 17, 43, 159, 453, 863; and B, 4, 17, 43, 159, 453, 863, 1120. C, τ_REC as a function of light intensity (as in Fig. 2) for dark-adapted Bark/RK7 rods (•) and Bark/RK7:R9AP95 rods (■); and for Bark/RK7 rods (○) and Bark/RK7:R9AP95 rods (□) in the presence of a steady background light of 438 photons · μm⁻² · s⁻¹.

Figure 6.

Effect of background light on time course of decay of responses of rods from Bark/RK7 mice. A, B, Suction-electrode recordings of currents averaged from 22 rods of two Bark/RK7 mice in the absence (A) and presence (B) of a background light of 438 photons · μm⁻² · s⁻¹. Intensities of 20 ms flashes in photons · μm⁻² delivered at t = 0 were as follows: A, 4, 17, 43, 159, 453, 863, 1870; and B, 43, 159, 453, 863, 1871. C, Comparison of waveforms of responses from Bark/RK7 rods for flash of 159 photons · μm⁻². Responses of A and B have been normalized rod by rod to the maximum amplitude of the response and then averaged. Black trace is from data in A (dark adapted), gray trace from data in B (in presence of background). D, As in C but for flash of 453 photons · μm⁻².

Figure 7.

Effect of background light on time course of decay of responses of rods from Bark/RK7:R9AP95 mice. Left, Suction-electrode recordings of currents averaged from 19 rods of 3 Bark/RK7:R9AP95 mice. All responses are to 20 ms flashes delivered at t = 0 in absence (black traces) and presence (gray traces) of steady background light of 438 photons · μm⁻² · s⁻¹. Intensities of flashes (I_F) increased from top to bottom and are given in each panel in units of photons · μm⁻². Right, Responses in left panels have been normalized cell by cell to the peak amplitude of the response and then averaged. Note more rapid decay of gray traces recorded in background light.

Figure 8.

Effect of background light on time course of decay of responses of rods from Bark/RK7:Rv−/− mice. A, B, Suction-electrode recordings of currents averaged from 8–11 rods of four Bark/RK7:Rv−/− mice in the absence (A) and presence (B) of a background light of 438 photons · μm⁻² · s⁻¹. Intensities of 20 ms flashes in photons · μm⁻² delivered at t = 0 were as follows: A, 4, 17, 43, 159, 453, 863, 1870; and B, 17, 43, 159, 453, 863, 1870. C, Comparison of waveforms of responses from Bark/RK7:Rv−/− rods for flash of 159 photons · μm⁻². Responses of A and B have been normalized rod by rod to the maximum amplitude of the response and then averaged. D, As in C but for flash of 453 photons · μm⁻².