Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl −/− mice, using suction pipette recordings from their “inner segments” (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the α-subunit of the G-protein transducin (G tα−/−), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (λmax = 360 nm) and M- (λmax = 508 nm) cone opsins and to be maximally sensitive at 360 nm (“S-cones”); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm (“M-cones”). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was ∼70 ms, and ∼0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A ∼4 s−2) was found to be about twofold lower than in rods (A ∼8 s−2). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate “dark light.”

Mutations in rod opsin, the visual pigment protein of rod photoreceptors, account for Ϸ15% of all inherited human retinal degenerations. However, the physiological and molecular events underlying the disease process are not well understood. One approach to this question has been to study transgenic mice expressing opsin genes containing defined mutations. A caveat of this approach is that even the overexpression of normal opsin leads to photoreceptor cell degeneration. To overcome the problem, we have reduced or eliminated endogenous rod opsin content by targeted gene disruption. Retinas in mice lacking both opsin alleles initially developed normally, except that rod outer segments failed to form. Within months of birth, photoreceptor cells degenerated completely. Retinas from mice with a single copy of the opsin gene developed normally, and rods elaborated outer segments of normal size but with half the normal complement of rhodopsin. Photoreceptor cells in these retinas also degenerated but did so over a much slower time course. Physiological and biochemical experiments showed that rods from mice with a single opsin gene were Ϸ50% less sensitive to light, had accelerated f lash-response kinetics, and contained Ϸ50% more phosducin than wild-type controls.

CorrectionsNEUROBIOLOGY. For the article ''Phototransduction in transgenic mice after targeted deletion of the rod transducin ␣-subunit'' by

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

Excessive phototransduction signaling is thought to be involved in light-induced and inherited retinal degeneration. Using knockout mice with defects in rhodopsin shut-off and transducin signaling, we show that two different pathways of photoreceptor-cell apoptosis are induced by light. Bright light induces apoptosis that is independent of transducin and accompanied by induction of the transcription factor AP-1. By contrast, low light induces an apoptotic pathway that requires transducin. We also provide evidence that additional genetic factors regulate sensitivity to light-induced damage. Our use of defined mouse mutants resolves some of the complexity underlying the mechanisms that regulate susceptibility to retinal degeneration.

Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 109 copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.

Mutations in Rpe65 disrupt synthesis of the opsin chromophore ligand 11-cis-retinal and cause Leber congenital amaurosis (LCA), a severe, early-onset retinal dystrophy. To test whether light-independent signaling by unliganded opsin causes the degeneration, we used Rpe65-null mice, a model of LCA. Dark-adapted Rpe65-/- mice behaved as if light adapted, exhibiting reduced circulating current, accelerated response turn-off, and diminished intracellular calcium. A genetic block of transducin signaling completely rescued degeneration irrespective of an elevated level of retinyl ester. These studies clearly show that activation of sensory transduction by unliganded opsin, and not the accumulation of retinyl esters, causes light-independent retinal degeneration in LCA. A similar mechanism may also be responsible for degeneration induced by vitamin A deprivation.

DEP (for Disheveled, EGL-10, Pleckstrin) homology domains are present in numerous signaling proteins, including many in the nervous system, but their function remains mostly elusive. We report that the DEP domain of a photoreceptor-specific signaling protein, RGS9 (for regulator of G-protein signaling 9), plays an essential role in RGS9 delivery to the intracellular compartment of its functioning, the rod outer segment. We generated a transgenic mouse in which RGS9 was replaced by its mutant lacking the DEP domain. We then used a combination of the quantitative technique of serial tangential sectioning-Western blotting with electrophysiological recordings to demonstrate that mutant RGS9 is expressed in rods in the normal amount but is completely excluded from the outer segments. The delivery of RGS9 to rod outer segments is likely to be mediated by the DEP domain interaction with a transmembrane protein, R9AP (for RGS9 anchoring protein), known to anchor RGS9 on the surface of photoreceptor membranes and to potentiate RGS9 catalytic activity. We show that both of these functions are also abolished as the result of the DEP domain deletion. These findings indicate that a novel function of the DEP domain is to target a signaling protein to a specific compartment of a highly polarized neuron. Interestingly, sequence analysis of R9AP reveals the presence of a conserved R-SNARE (for soluble N-ethylmaleimide-sensitive factor attachment protein receptor) motif and a predicted overall structural homology with SNARE proteins involved in vesicular trafficking and fusion. This presents the possibility that DEP domains might serve to target various DEP-containing proteins to the sites of their intracellular action via interactions with the members of extended SNARE protein family.