through a 480 nm cut-on filter, followed by recording successive spectra until no further absorbance decrease was observed. For samples that bleach immediately to 380 nm maximum with an unprotonated MII after exposure to light (such as WT, T94I and A295V), the rate of MII decay is measured by taking into consideration that 11-cis retinal binds faster than MII decays. The rhodopsin in the sample was selectively activated in the presence of excess 11-cis retinal, and the rate of regeneration of the photopigment was monitored. The MII decay was 8-fold slower for T94I (Gross et al. 2003) and 1.7-fold slower for A295V (Zeitz et al. 2008). However, slower MII decay is unlikely to affect the in vivo night blindness phenotype because the signal termination by phosphorylation of the receptor and arrestin binding occurs faster than MII decays (Ng and Henikoff 2001).

30.2.3 Activity of CSNB Mutants

The ability of the rod visual system to detect single photons requires that rhodopsin remains dormant in the dark; indeed this is the case. The half-life for spontaneous activation of rhodopsin is 49 years (Baylor et al. 1980). However, because each rod contains 108 rhodopsin molecules, thermal activation results in spontaneous fluctuations of activity that resemble single-photon events, on the order of every 100–200 s in dark-adapted rods (Baylor et al. 1984). Dark noise measurements indicate that rod noise limits behavioral sensitivity and sets the limit for absolute sensitivity of vision. If rhodopsin thermal activity were increased only a small amount (Barlow 1988) or if a genetic mutation resulted in constitutively active opsins (Rao et al. 1994), the aberrant signals generated within the rod would compete with dim external stimuli and desensitize night vision. The mechanism by which the four known rhodopsin mutations cause the underlying pathophysiology in CSNB patients remains the subject of some controversy, but one hypothesis is that their ability to activate the phototransduction cascade without bound all-trans retinal plays an important role. The highly amplified nature of this cascade means that low levels of activity may have significant physiological consequences.

30.2.3.1 In Vitro Assays of CSNB Mutants

One striking common phenotype of the four known CSNB rhodopsin mutations is that they constitutively activate transducin, the G-protein coupled to rhodopsin, in vitro. When 11-cis retinal is bound, all of the CSNB rhodopsin mutants activate transducin with kinetics similar to that of WT rhodopsin (Dryja et al. 1993; Rao et al. 1994; Gross et al. 2003; Zeitz et al. 2008); i.e., there is no detectable activity in the dark, but very high levels upon photoisomerization to all-trans retinal. However, a difference is observed in the absence of chromophore. While WT opsin does not activate transducin under the conditions of the assay, all of the known rhodopsin CSNB mutants activate transducin in the absence of chromophore and light, a property referred to as constitutive activation. There is a range of constitutive activation among the mutants: A292E > G90D ≈ A295V > T94I.

30.2.3.2 Electrophysiological Studies on Transgenic Animal Models

Mice heterozygous for G90D rhodopsin (expressing G90D as a transgene on a heterozygous knock-out background, G+/–, R+/–) exhibited considerable loss of rod sensitivity. The desensitization of the photoresponse increased with the number of G90D alleles expressed (Sieving et al. 2001). G90D formed a pigment that supported normal photoactivation of transduction leading to rod responses in vivo. Therefore the desensitization seen in these mice could not be explained simply by a decreased quantal catch or by an inability to generate a photoresponse.

In the dark the membrane current noise arises from fluctuations in the cGMP concentrations [cG] which themselves reflect the balance of the local activities of PDE and guanylyl cyclase. Thermal activation of rhodopsin, producing a true R , results in a single photon event whereas spontaneous activation of transducin or PDE would result in a smaller, shorter local decrease in [cG] and contribute to the continuous noise component of the rod photoreceptor dark noise (Rieke and Baylor 1996). Single photon events themselves vary in amplitude in different species, large (0.6–1.0 pA) in frog, toad and monkey, moderate 0.3 pA in rodents and much smaller in the salamander. Mutations in rhodopsin that destabilize the ground state could increase the probability of a thermal isomerization or result in a meta-stable or partially activated rhodopsin enzyme that flips back and forth resulting in low levels of transducin activation. The discovery that bleached opsin itself activates transducin as well as PDE (Cornwall and Fain 1994; Melia et al. 1997) adds another possible explanation for rod desensitization: excess free opsin.

However, free opsin can be quenched by the addition of excess exogenous chromophore. In Xenopus just this case was elegantly demonstrated by Jin et al. (2003) where the dramatic changes in kinetics and sensitivity of rods from three CNSB mutants (G90D, T94I, and A292E) were completely restored by the addition of 11- cis retinal. WT or mutant bovine rhodopsin with EGFP followed by a repeat of the last eight C-terminal amino acids of rhodopsin (1D4) fused to the C terminus was introduced under the Xenopus opsin promoter. The authors performed suction micropipette recordings on individual WT rods or those expressing A292E, G90D or T94I before and after incubation with 11-cis retinal. The sensitivity curve of rods expressing G90D was shifted to the right compared with WT rods (Fig. 30.1a). After incubation with 11-cis retinal, the sensitivity of G90D rods was rescued to WT levels. If desensitization were due to thermal isomerization of the chromophore, then the addition of 11-cis retinal would have no effect. Similar results were seen for rods expressing T94I as well as those expressing A292E.

In the G90D mouse (G+/–, R+/+) single cell recordings showed a doubling of the stimulus strength required to evoke a 50% maximal response with a 0.1 log unit (20%) reduction of rhodopsin content compared to WT mice. And for dim light responses the time-to-peak was 25% faster in the G90D expressing rods (Sieving et al. 2001). In the same study, the G+/–, R+/– genotype showed about a 25% higher rhodopsin content. The rod responses of this genotype were studied as a massed receptor potential by pharmacologically eliminating bipolar responses with 2-amino-4-phosphonobutyrate (APB) and blocking the slow PIII with barium.

Fig. 30.1 Activity of G90D in transgenic rods. a. Top: Intensity-response curves of isolated transgenic Xenopus rods expressing wild type (WT) rhodopsin (squares), G90D rhodopsin (triangles) or G90D rhodopsin after treatment with 11-cis retinal (grey filled circles). Bottom: Dim-flash kinetics of isolated rods expressing WT or G90D rhodopsin with and without addition of 11-cis retinal (adapted from Jin et al 2003). b. Top: Intensity-response curves from isolated transgenic mouse rods. Data from isolated WT rods (filled circles), from D+/+ rods before treatment with 11-cis retinal (open squares) and from D+/+ rods after addition of 11-cis retinal (open circles). Bottom: Intensity-response curves from D+/–; R–/– rods before (filled squares) and after (filled triangles) incubation with 11-cis retinal, and from D+/–; R–/–; Rpe65–/– rods before (open squares) and after (open circles) incubation with 11-cis retinal (adapted from Dizhoor et al. 2008)

Those results showed a rod response in the G+/–, R+/– and the R+/– genotypes that appeared more similar to the light adapted R+/+ than the massed receptor potential of the dark-adapted R+/+ strain.

Earlier, Makino recorded from the rods of mice with only 50% of the normal rhodopsin content and found the expected reductions of sensitivity (about 70%). However, they also found a small acceleration in the time to peak of the response (14%) and a greater reduction of the integration time (almost 30%) indicating more profound effects on the inactivation phase of the phototransduction cascade (Lem et al. 1999). Thus the loss of 50% of the functioning rhodopsin can by itself alter the phototransduction cascade.

Sieving and colleagues further investigated their mouse model of CSNB, measuring single cell responses in their D+ (G+/–, R–/–) and D+/+ (G+/+, R–/-) lines. The

ERG desensitization was confirmed and the integration time, dim flash sensitivity and dominant time constants were measured and found to be substantially reduced compared to WT rod responses. Moreover, these changes were not reversed by prior bathing in lipid vesicle containing 11-cis retinal (Fig. 30.1b), a treatment effective