We have generated a mouse with rod photoreceptors overexpressing the ␥ inhibitory subunit (PDE6␥) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6␥ 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 PDE6␥ binding to activated transducin ␣ GTP (T ␣ -GTP) before the T ␣ -GTP binds to endogenous PDE6␥. Excess PDE6␥ 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 PDE6␥ but lack RGS9. Our results show that the PDE6␥ binding sites of PDE6 ␣ and ␤ are accessible to excess (presumably cytoplasmic) PDE6␥ in the light, once endogenous PDE6␥ has been displaced from its binding site by T ␣ -GTP. They also suggest that in the presence of T ␣ -GTP, the PDE6␥ remains attached to the rest of the PDE6 molecule, but after conversion of T ␣ -GTP to T ␣ -GDP, the PDE6␥ 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. Key words: rod; phototransduction; retina; phosphodiesterase; G-protein; RGS protein IntroductionPhotoexcited rhodopsin in a vertebrate rod binds to and activates the G-protein transducin, facilitating the exchange of GTP for GDP on the transducin ␣ subunit (T ␣ or GNAT1). The T ␣ -GTP then binds to the inhibitory ␥ subunit (PDE6␥) of the phosphodiesterase effector enzyme (PDE6), relieving the inhibition of the PDE6 ␣ and ␤ catalytic subunits. Activated PDE6 hydrolyzes cGMP, leading to the closing of the cGMP-gated channels in the outer segment. This produces the hyperpolarizing light response that signals the detection of the light to the rest of the nervous system .The turnoff of the photoreceptor response and reopening of the channels requires the inactivation of rhodopsin by phosphorylation and subsequent binding of arrestin, as well as the return of the PDE6 to its dark resting level by hydrolysis of T ␣ -GTP back to T ␣ -GDP. The intrinsic rate of transducin GTP hydrolysis is slow but is facilitated by interaction of transducin with other proteins . The first of these to be identified was PDE6␥, which was initially thought to act by itself to accelerate GTP hydrolysis (Arshavsky and Bownds, 1992) but was later shown to have no effect on the rate of hydrolysis in isolation (Angleson and Wensel, 1993;Antonny et al., 1993) and to require additional components, subsequently identified as RGS9 -1 (He et al., 1998), G␤5 (Makino et al., 1999, and a membrane anchor protein, R9AP (Hu and Wensel, 2002). These together form a GTPase activating protein (GAP) complex that is essential for the rapid conve...

The 9-methyl group of 11-s-retinal plays a crucial role in photoexcitation of the visual pigment rhodopsin. A hydrogen-substituted analogue, 11-cis-9-desmethylretlnal, combines with opsin to form a pigment that produces abnormal photoproducts and diinished activation of the GTP-binding protein transducin in vitro. We have measured the formation of this analogue pigment in bleached salamander rods and determined the size and shape of its quantal response. In addition, we have characterized the influence of opsin and newly formed analogue pigment on the quantal response to native porphyropsin. We find that, as 11-cis-9-desmethylretinal combines with opsin in bleached rods, the amplitude of the quantal response from residual native pigment is elevated by 74.5-fold to 0.15 ± 0.09 pA, a value close to the amplitude of the quantal response before bleach (0.31 ± 0.10 pA). When activated by light, the new analogue pigment produces a quantal response that is ==30-fold smaller and decays ""5 times more slowly than that ofnative pigment in unbleached cells. We conclude that the 9-methyl group of retinal is not critical for conversion of opsin to its nondesensitizing state but that it is critical for the normal processes of activation and deactivation of metarhodopsin that give rise to the quantal response.Photoisomerization of li-cis-retinal (Fig. 1, structure 1) initiates an intramolecular rearrangement of rhodopsin that results in a catalytically active state of rhodopsin, R * (1-5). Deactivation of R* requires phosphorylation by rhodopsin kinase (6, 7) and the subsequent binding of arrestin (3,(7)(8)(9). In isolated photoreceptors, pigment activation and deactivation produce a discrete electrical response with a characteristic amplitude and time course (10,11). In an examination of the steric interactions between the apoprotein opsin and its chromophore, Ganter et al. (12) reported that 11-cis-9-desmethylretinal ( Fig. 1, structure 3) produced abnormal photoproducts and transducin activation that was 8% of the rhodopsin control. Here we examine the influence of the 9-methyl group of retinal on the amplitude and shape of the quantal response in isolated rods.To provide access to the ligand binding pocket ofopsin, the native chromophore (13) 11-cis-3,4-dehydroretinal (Fig. 1, structure 2) was removed by bleaching. Bleaching reduces the sensitivity of a cell by depleting the supply of native pigment and by reducing the amplitude of the quantal response from the residual pigment (14)(15)(16)(17). In the absence of li-cis-retinal, this desensitization persists indefinitely (15,18) and is unresponsive to the addition of all-trans-retinal (15,19) or its removal from opsin by hydroxylamine (20,21). We refer to the persistent component of desensitization that results from response attenuation and does not require the presence of a retinal-containing photoproduct as opsin desensitization. Taken together with the loss of sensitivity resulting from pigment depletion, the total loss of sensitivity is commonly referred to as ...