lysine 296 in the seventh transmembrane helix. While there are over 100 distinct mutations in the rhodopsin gene that are found in patients with the degenerative disease autosomal dominant retinitis pigmentosa (ADRP), there are only four known rhodopsin mutations found in patients with the dysfunction congenital stationary night blindness (CSNB). All four of these display a dominant pattern of inheritance (adCSNB). CSNB patients have a much less severe phenotype than those with ADRP; the patients only lose rod function which affects their vision under dim light conditions, whereas their cone function remains relatively unchanged. The known rhodopsin CSNB mutations are found clustered around the site of retinal attachment. Two of the mutations encode replacements of neutral amino acids with negatively charged ones (A292E and G90D), and the remaining two are neutral amino acid replacements (T94I and A295V). All four of these mutations have been shown to constitutively activate the apoprotein in vitro. The mechanisms by which these mutations lead to night blindness are still not known with certainty and remain the subject of some controversy. The dominant nature of these genetic defects, as well as the relative normalcy of photopic vision in individuals with half the complement of wild type (WT) rhodopsin (Dryja et al. 1993; Sieving et al. 1995; al-Jandal et al. 1999; Zeitz et al. 2008), suggest that it is an active property of the mutant opsin proteins that leads to defective rod vision rather than a loss of some needed function. Biochemical studies of the mutant rhodopsins have been carried out on recombinant proteins expressed in cultured mammalian cells, and electrophysiology has been carried out on transgenic animals expressing them. In addition, results from electroretinography studies (ERG) of rod-driven responses in patients add to our understanding of how these proteins function in the human rods. Herein, we review the known biochemical and electrophysiological data for the four known rhodopsin mutations found in patients with CSNB.

30.2 Properties of Rhodopsin CSNB Mutants

30.2.1 Spectral and Photochemical Properties

All vertebrate rod pigments share the same basic structure: a 40 kDa apoprotein arranged in seven transmembrane (TM) helices, with a chromophore attached via a protonated Schiff base bond on a conserved Lys residue on TM7. The positive charge on the Schiff base nitrogen is stabilized through an electrostatic interaction, or salt bridge, with the carboxylate on the counterion Glu113 (Sakmar et al. 1989; Nathans 1990; Zhukovsky et al. 1992). This negative charge perturbs the pKa of the Schiff base to 16 (Steinberg et al. 1993), so that it is always protonated under physiological conditions. Upon absorption of a photon, 11-cis retinal isomerizes to all-trans retinal, causing a series of conformational and spectrophotometrically detectable changes to occur, ultimately leading to the active conformation of photoexcited rhodopsin, metarhodopsin II (MII). To reset rhodopsin back to its dark state, the Schiff base between Lys296 and all-trans retinal is hydrolyzed, and the

chromophore dissociates, allowing a new molecule of 11-cis retinal to bind to the active site and covalently attach to Lys296.

While opsin alone does not absorb light in the visible region, 11-cis retinal free in solution has a λmax of 380 nm, and an 11-cis retinal protonated Schiff base (PSB) molecule, such as an ‘acid-trapped’ acid-denatured rhodopsin, has a λmax of 440 nm. In rods, dark rhodopsin absorbs maximally at 500 nm. The difference in the λmax values of the pigment from that of the PSB free in solution has been termed the ‘opsin-shift’ (Nakanishi et al. 1980). The active conformation of the receptor, MII, absorbs maximally at 380 nm, characteristic of an unprotonated Schiff base.

The absorbance spectrum of A292E rhodopsin is similar to that of WT rhodopsin (Dryja et al. 1993). However, the absorption maxima of the other three CSNB mutants are shifted towards the blue: G90D absorbs maximally at 483 nm (Rao et al. 1994), T94I at 478 nm (Gross et al. 2003), and A295V at 482 nm (Zeitz et al. 2008). The slight blue-shift in the absorption spectrum indicates the retinal binding interaction has been perturbed slightly in these mutants. All four rhodopsin CSNB mutants are photobleachable with visible light to yield a MII species that absorbs maximally at 380 nm, indicative of an unprotonated Schiff base attachment.

Another indication that the structure of G90D rhodopsin is significantly different from that of WT rhodopsin is shown through hydroxylamine treatment. Dark WT rhodopsin is resistant to chemical bleach by hydroxylamine treatment, whereas G90D rhodopsin reacts with hydroxylamine, yielding a retinal oxime with a = 367 nm (Sieving et al. 2001). This result implies that the retinal binding pocket is more accessible to hydroxylamine in the G90D mutant, and therefore may

be structurally more similar to MII.

30.2.2 Retinal Binding Kinetics of Rhodopsin CSNB Mutants

Another measurable property of rhodopsin that distinguishes the CSNB mutants from one another is the kinetics of regeneration. Using stopped-flow spectrophotometry on detergent solubilized and purified rhodopsin mutants in the context of a stabilizing mutation N2C, D282C which is known to significantly increase the stability of the apoprotein (Xie et al. 2003), the rate of the Schiff base formation upon addition of 11-cis retinal to opsin was found to be 80-fold slower for the G90D mutant than for WT opsin, but not for the A292E or T94I mutants (Gross et al. 2003). This effect is likely due to interference with the salt bridge between Lys296 and the counterion Glu113 that holds the protein in an inactive conformation. Even though the acidic amino acid glutamate is introduced at position 292, it does not seem to interfere with the salt bridge in the same manner.

Another property important for rhodopsin function that can be measured spectrophotometrically is the rate of MII decay, which must precede regeneration in functioning rods. For A292E and G90D that generate a protonated MII upon exposure to light, the rate of MII decay was determined after exposure to light passed