Modulation of mouse rod response decay by rhodopsin kinase and recoverin.

Light isomerizes 11-cis-retinal in a retinal rod and produces an active form of rhodopsin (Rh*) that binds to the G-protein transducin and activates the phototransduction cascade. Rh* is turned off by phosphorylation by rhodopsin kinase [G-protein-coupled receptor kinase 1 (GRK1)] and subsequent binding of arrestin. To evaluate the role of GRK1 in rod light response decay, we have generated the transgenic mouse RKS561L in which GRK1, which is normally present at only 2-3% of rhodopsin, is overexpressed by ∼12-fold. Overexpression of GRK1 increases the rate of Rh* phosphorylation and reduces the exponential decay constant of the response (τ(REC)) and the limiting time constant (τ(D)) both by ∼30%; these decreases are highly significant. Similar decreases are produced in Rv(-/-) rods, in which the GRK1-binding protein recoverin has been genetically deleted. These changes in response decay are produced by acceleration of light-activated phosphodiesterase (PDE*) decay rather than Rh* decay, because light-activated PDE* decay remains rate limiting for response decay in both RKS561L and Rv(-/-) rods. A model incorporating an effect of GRK1 on light-activated PDE* decay rate can satisfactorily account for the changes in response amplitude and waveform. Modulation of response decay in background light is nearly eliminated by deletion of recoverin. Our experiments indicate that rhodopsin kinase and recoverin, in addition to their well-known role in regulating the turning off of Rh*, can also modulate the decay of light-activated PDE*, and the effects of these proteins on light-activated PDE* decay may be responsible for the quickening of response recovery in background light.

Effect of the ILE86TER mutation in the γ subunit of cGMP phosphodiesterase (PDE6) on rod photoreceptor signaling.

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and ß subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and ß by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.

Night blindness and the mechanism of constitutive signaling of mutant G90D rhodopsin.

The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.

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

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.

Opsin activation of transduction in the rods of dark-reared Rpe65 knockout mice.

Rpe65 knockout mice (Rpe65-/-) are unable to synthesize the visual pigment chromophore 11-cis retinal; however, if these animals are reared in complete darkness, the rod photoreceptors accumulate a small amount of 9-cis retinal and its corresponding visual pigment isorhodopsin. Suction-electrode recording of single rods from dark-reared Rpe65-/- mice showed that the rods were about 400 times less sensitive than wild-type control rods and that the maximum responses were much smaller in amplitude. Spectral sensitivity measurements indicated that Rpe65-/- rod responses were generated by isorhodopsin rather than rhodopsin. Sensitivity and pigment concentration were compared in the same mice by measuring light responses from rods of one eye and pigment concentration from the retina of the other eye. Retinas had 11-35% of the normal pigment level, but the rods were of the order of 20-30 times less sensitive than could be accounted for by the loss in quantum catch. This extra desensitization must be caused by opsin-dependent activation of the visual cascade, which leads to a state equivalent to light adaptation in the dark-adapted rod. By comparing the sensitivity of dark-reared Rpe65-/- rods to that produced in normal rods by background light, we estimate that Rpe65-/- opsin is of the order of 2.5x10(-5) as efficient in activating transduction as photoactivated rhodopsin (Rh*) in WT mice. Dark-reared Rpe65-/- rods are less desensitized than rods from cyclic light-reared Rpe65-/- mice, have about 50% more photocurrent and degenerate at a slower rate. Retinas sectioned after 9 months in darkness show a larger number of photoreceptor nuclei in dark-reared animals than in cyclic light-reared animals, though both have fewer nuclei than in cyclic light-reared wild-type retinas. Both also have shorter outer segments and a lower free-Ca2+ concentration. These experiments provide the first quantitative measurement of opsin activation in physiologically responding mammalian rods.

Light-induced Ca2+ release in the visible cones of the zebrafish.

We used suction-pipette recording and fluo-4 fluorescence to study light-induced Ca2+ release from the visible double cones of zebrafish. In Ringer, light produces a slow decrease in fluorescence which can be fitted by the sum of two decaying exponentials with time constants of 0.5 and 3.8 s. In 0Ca2+-0Na+ solution, for which fluxes of Ca2+ across the outer segment plasma membrane are greatly reduced, light produces a slow increase in fluorescence. Both the decrease and increase are delayed after incorporation of the Ca2+ chelator BAPTA, indicating that both are produced by a change in Ca2+. If the Ca2+ pool is first released by bright light in 0Ca2+-0Na+ solution and the cone returned to Ringer, the time course of Ca2+ decline is much faster than in Ringer without previous light exposure. This indicates that the time constants of 0.5 and 3.8 s actually reflect a sum of Na+/Ca2+-K+ exchange and light-induced release of Ca2+. The Ca2+ released by light appears to come from at least two sites, the first comprising 66% of the total pool and half-released by bleaching 4.8% of the pigment. Release of the remaining Ca2+ from the second site requires the bleaching of nearly all of the pigment. If, after release, the cone is maintained in darkness, a substantial fraction of the Ca2+ returns to the release pool even in the absence of pigment regeneration. The light-induced release of Ca2+ can produce a modulation of the dark current as large as 0.75 pA independently of the normal transduction cascade, though the rise time of the current is considerably slower than the normal light response. These experiments show that Ca2+ can be released within the cone outer segment by light intensities within the physiological range of photopic vision. The role this Ca2+ release plays remains unresolved.

Light stimulates a transducin-independent increase of cytoplasmic Ca2+ and suppression of current in cones from the zebrafish mutant nof.

Transducins couple visual pigments to cGMP hydrolysis, the only recognized phototransduction pathway in vertebrate photoreceptors. Here we describe a zebrafish mutant, no optokinetic response f(w21) (nof), with a nonsense mutation in the gene encoding the alpha subunit of cone transducin. Retinal morphology and levels of phototransduction enzymes are normal in nof retinas, but cone transducin is undetectable. Dark current in nof cones is also normal, but it is insensitive to moderate intensity light. The nof cones do respond, however, to bright light. These responses are produced by a light-stimulated, but transducin-independent, release of Ca2+ into the cone cytoplasm. Thus, in addition to stimulating transducin, light also independently induces release of Ca2+ into the photoreceptor cytoplasm.

P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium.

PURPOSE: To identify and characterize P2 purinergic receptors and their signaling pathways in the epithelial cells of the rabbit ciliary body. METHODS: Real-time fluorescence ratio imaging of the intact fura-2-loaded nonpigmented ciliary body epithelial (NPE) cells of rabbit were used to record changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to a number of purinergic agonists and antagonists. The effects of some of these drugs on the inositol phosphate (IP) levels in ciliary processes were also examined. RESULTS: Adenosine diphosphate (ADP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) dose dependently increased the [Ca(2+)](i) and IP levels. The [Ca(2+)](i) increases induced by ADP and UTP were distinguishable, both kinetically and pharmacologically. The effect of ADP on [Ca(2+)](i) was mimicked by a number of P2Y(1)-selective agonists, and was blocked by three P2Y(1)-receptor-specific antagonists. The [Ca(2+)](i) increases elicited by ADP (or its analogs) and UTP were additive. CONCLUSIONS: Rabbit ciliary body epithelium possesses both P2Y(1) and P2Y(2) metabotropic purinergic receptor subtypes, which differentially use the IP(3)/Ca(2+) second-messenger pathway.