GAP-independent termination of photoreceptor light response by excess gamma subunit of the cGMP-phosphodiesterase

Abstract

We have generated a mouse with rod photoreceptors overexpressing the gamma inhibitory subunit (PDE6gamma) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6gamma overexpression decreases the rate of rise of the rod response at dim intensities, indicating a reduction in the gain of transduction that may be the result of cytoplasmic PDE6gamma binding to activated transducin alpha GTP (Talpha-GTP) before the Talpha-GTP binds to endogenous PDE6gamma. Excess PDE6gamma also produces a marked acceleration in the falling phase of the light response and more rapid recovery of sensitivity and circulating current after prolonged light exposure. These effects are not mediated by accelerating GTP hydrolysis through the GAP (GTPase activating protein) complex, because the decay of the light response is also accelerated in rods that overexpress PDE6gamma but lack RGS9. Our results show that the PDE6gamma binding sites of PDE6 alpha and beta are accessible to excess (presumably cytoplasmic) PDE6gamma in the light, once endogenous PDE6gamma has been displaced from its binding site by Talpha-GTP. They also suggest that in the presence of Talpha-GTP, the PDE6gamma remains attached to the rest of the PDE6 molecule, but after conversion of Talpha-GTP to Talpha-GDP, the PDE6gamma may dissociate from the PDE6 and exchange with a cytoplasmic pool. This pool may exist even in wild-type rods and may explain the decay of rod photoresponses in the presence of nonhydrolyzable analogs of GTP.

Figure 1.

Transgenic overexpression of the 11 kDa PDE6γ subunit (PDEγ). A, Immunoblot screening of PDEγ in ROSs of C57BL/6 (control) and indicated transgenic mouse lines normalized to 150 pmol rhodopsin content, with a polyclonal antibody recognizing the N-terminal part of the PDE6γ subunit. Transgenic PDE6γ is expressed in the Pdeg6^tm1/+ genetic background. Lane 1, Transgenic line wt6H; lane 2, transgenic line wt6I; lane 3, transgenic line wt6J; lane 4, transgenic line A4 (+/+); lane 5, adult Pdeg6^tm1/Pdeg6^tm1; lane 6, B6+/+ WT C57BL/6 control; lane 7, P13 Pdeg6^tm1/Pdeg6^tm1; lane 8, transgenic line wt6A; lane 9, transgenic line wt6B; lane 10, transgenic line wtC6; lane 11, transgenic line wt6D; lane 12, transgenic line wt6E; lane 13, B6+/+ WT C57BL/6 control; lane 14, P13 Pdeg6^tm1/Pdeg6^tm1. B, Overexpression (OX; wt6C) of PDE6γ in the wt6C transgenic line, expressed in the Pdeg6^tm1/Pdeg6^tm1genetic background. The indicated amount of protein extracts from age-matched wt6C and WT mouse retinas were analyzed by immunoblot for RGS9–1, Gβ5, and PDE6γ simultaneously. Size markers are indicated at left. The PDE6γ level is approximately twofold higher in wt6C retina, whereas RGS9–1 and Gβ5 levels are similar to those of the control. C, Immunoblot analysis of the PDE6α (Pα) and PDE6β (Pβ) catalytic subunits in control and transgenic ROSs normalized for rhodopsin content as in A. Immunoblot incubated with the MOE polyclonal antibody recognizing all of the subunits of the PDE6 in B6 control and wt6C transgenic retinas is shown.

Figure 2.

Absence of light-dependent movement of PDE6γ in wt6C transgenic mice. Fluorescent and bright-field images of retinal localization of PDE6γ (PDEγ; top panels), arrestin (Arr; middle panels), and transducin α subunit (Tα; bottom panels) in dark-adapted and light-adapted conditions were examined by immunohistochemistry (see Materials and Methods). For the light-adapted condition, mice were exposed to continuous room light (60 lux) for 6 h; for the dark-adapted condition, mice were killed after 12 h in darkness under infrared illumination. Although Tα and arrestin redistribute in opposite directions in and out of the outer segment (OS) layer, PDE6γ is found exclusively in the OS regardless of light. IS, Inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 25 μm.

Figure 3.

Responses of wt6C and WT rods to light. A, WT rod, 20 ms flashes of 500 nm light at flash intensities of 17, 43, 160, 450, 1120, and 4230 photons μm⁻². The traces are averages of two to four flashes at each intensity. B, Typical responses from a wt6C rod to the same flash intensities. Each trace was averaged from three to eight flashes. C, Response amplitude versus flash intensity averaged from 34 WT and 20 wt6C rods. Flash intensity required to elicit a response of half-maximal amplitude was shifted to higher intensities by ∼0.3 log units in the wt6C rods (see Table 1).

Figure 4.

Response rise time and recovery in WT and wt6C rods. A, Superimposed averaged responses of WT (black traces) and wt6C (red traces) rods at a flash intensity of 17 photons μm⁻². B, As in A but at 160 photons μm⁻². C, As in A but at 1120 photons μm⁻². Data were filtered at 30 Hz (eight-pole Bessel) and sampled at 100 Hz. The data in A–C were averaged from 31–34 WT and 18–20 wt6C rods after normalizing to the maximum photo current for each rod. The inset in each panel shows the rising phases of the responses on an expanded time scale with SE at 10 ms intervals. D, Pepperberg plots of WT and wt6C rods. Time in saturation, T_sat, was estimated from the time for recovery to 25% of a dark circulating current after presentation of a bright flash. The dominant time constant for response turnoff was estimated from the best-fitting slope for intensities from 450 to 3250 photons μm⁻².

Figure 5.

Averaged single-photon responses for WT and wt6C rods from 47 WT and 18 wt6C rods. Individual single-photon responses were calculated by dividing the mean response to 40–60 dim light-flash responses by the number of photoisomerizations per flash, which was estimated by the scaling factor needed to adjust the individual rising phases of the mean responses to the rising phases of the ensemble variances of the means. Inset, Rising phases of single-photon responses on an expanded time scale with the SE at 10 ms intervals.

Figure 6.

Responses of rods lacking the RGS9–1 complex are accelerated by excess PDE6γ. A, Responses of a Rgs9^−/− rod to flash intensities of 4, 17, 43, and 160 photons μm⁻². B, Responses of rods from wt6C mice on an Rgs9^−/− background. See Results. Flash intensities are 4, 17, 43, 160, and 450 photons μm⁻². The responses are averages of 5–20 flashes each.

Figure 7.

Recovery from bright-light exposure (4 min, 2830 photons μm⁻² s⁻¹). The adapting light was turned off at t = 0. The pigment bleached was <0.4%. A, Rgs9^−/− rod. The break in the abscissa shows eventual recovery of Rgs9^−/− rod response 50–60 s after the light was extinguished. B, wt6C rod on Rgs9^−/− background. Flashes of 20 ms at 437 photons μm⁻² were presented every 5 s after turning off the adapting light. C, WT rod with flashes as in B.