in the Xenopus model of the same disorder (Fig. 30.1a), and effective in reversing sensitivity losses in an RPE65–/– mouse model (Fig. 30.1b). Similar results were obtained in rods from G90D+/–, R+/– that mimic more closely the heterozygous state of this autosomal dominant disease (Dizhoor et al. 2008).

There are at least three mechanisms that can alter the dominant time constant and saturation time of the light responses of rods to bright light flashes: (1) Continuous presence of background light; (2) Recent history of background light (novel form of adaptation) (Krispel et al. 2003); (3) molecular adaptations or manipulations that increase the content or effective concentration of RGS9 (Krispel et al. 2006). The presence of background light leads to reduced photocurrent and lower internal calcium along with the concomitant change in basal levels of PDE and guanylyl cyclase. The other two mechanisms need not necessarily change calcium levels to affect their influences on light response.

One cannot properly conclude equivalence to background light without specifically comparing the effects of background light to reproduce both the sensitivity and kinetic changes in the dim light response as well as the saturating light responses. Noise analysis of the membrane current or photoreceptor voltage can separate out the single photon vs. continuous noise provided the single photon response is large and may be able to address the issue of spontaneous PDE activation vs. partially activated rhodopsin as well.

30.3 Proposed Mechanisms of CSNB Mutations

Multiple studies have confirmed that rod cells of human CSNB patients and of animal models of CSNB are desensitized as if there was a consistent low basal stimulation that confounds vision under dim-light conditions. It is as though the rods are partially light adapted. The desensitization has been proposed to be due to constitutively active mutant opsins (Dryja et al. 1993; Jin et al. 2003), or thermal isomerization of the chromophore to cause activation of mutant rhodopsin without light (Sieving et al. 1995) or to activate rhodopsin.

30.3.1 Desensitization Due to Mutant Opsin Activity in Xenopus

One proposed model of CSNB is that constitutive activation of the A292E opsin causes rod cell desensitization (Dryja et al. 1993). At any given time there would be some opsin not bound to either 11-cis retinal or all-trans retinal that could theoretically activate the phototransduction cascade in the absence of light as observed in vitro. This model is further supported by the fact that constitutive activation of the phototransduction cascade is a common phenotype of all four known rhodopsin CSNB mutations in vitro. In addition, there are CSNB mutations of genes that encode other proteins of the phototransduction cascade including the β subunit of

rod cGMP phosphodiesterase 6 (Gal et al. 1994) and the alpha subunit of transducin (Szabo et al. 2007), that both appear to be constitutively active.

The mechanism for constitutive activation is thought to be disruption of the salt bridge between Glu113 and Lys296 that holds the protein in an inactive conformation. For A292E and G90D the introduced negative residues compete with Glu113 for interaction with Lys296. For the other two, it is likely the position of the mutation that affects salt bridge formation. The location of the T94I mutation must be important because replacing threonine at position 94 with eight different amino acids all resulted in constitutively active proteins (Gross et al. 2003). For A295V, it was proposed that the introduction of the non-polar, β-branched amino acid valine may restrict movement of the polypeptide in the vicinity of the salt bridge (Zeitz et al. 2008). This model was tested in a study on transgenic Xenopus laevis tadpole models of CSNB mutations mentioned above (Jin et al. 2003). The addition of 11- cis retinal restored WT sensitivity of rods expressing G90D (Fig. 30.1a), A292E or T94I (Jin et al. 2003). Therefore, the results support the model that constitutively active free opsin is causing the reduced sensitivity of rod cells.

30.3.2 Proposed Dark-Active Rhodopsin in Mouse

In contrast to the findings discussed above, loss of rod cell sensitivity in G90D transgenic mouse rods is not rescued by the addition of 11-cis retinal (Fig. 30.1b) (Dizhoor et al. 2008). These data do not support the model of active free opsin causing desensitization of rod cells expressing G90D. Data from the same study does not support the proposed ‘dark-light’ model in which the CSNB mutations reduce rod sensitivity due to thermal isomerization of the chromophore in mutant rhodopsin; that is to say it does not appear to be photon-like noise (Sieving et al. 1995). Noise levels of WT and G90D+/–, R+/– were very similar, and noise for WT cells exposed to the estimated equivalent light produced by G90D in the dark was higher (Dizhoor et al. 2008). Because the noise in the G90D expressing rods was not higher than that of WT rods, a highly active G90D rhodopsin is likely not the cause of the apparent ‘light’ causing the rod cells to desensitize. Ultimately, the authors propose that the rod cell desensitization is due to an active G90D rhodopsin with low gain (Dizhoor et al. 2008).

G90D opsin is more active in Xenopus rods than in mouse rods. Perhaps the differences in results are due to model system differences as the rhodopsin expressed in the Xenopus model was bovine and the rhodopsin expressed in the mouse model was human. A mammalian rhodopsin expressed in Xenopus tadpoles can yield a different phenotype than the endogenous rhodopsin. For example, dark rearing P23H transgenic Xenopus rescued retinal degeneration due to light sensitivity when bovine or human P23H rhodopsin was expressed, but not Xenopus P23H rhodopsin (Tam and Moritz 2007).

Human patients with the G90D mutation do not recover sensitivity even after long periods of dark adaptation (Sieving et al. 1995). If active G90D opsin is the