Yes / Congenital achromatopsia or rod monochromatism is a rare autosomal recessive condition defined by a severe loss of cone photoreceptor function in which rods purportedly retain normal or near-to-normal function. This report describes the results of electroretinography in two siblings with CNGB3-associated achromatopsia. Full field light- and dark-adapted electroretinograms (ERGs) were recorded using standard protocols detailed by the International Society for Clinical Electrophysiology of Vision (ISCEV). We also examined rod-mediated ERGs using series of stimuli that varied over a 6 log unit range of retinal illuminances (−1.9–3.5 log scotopic trolands). Dark-adapted ERGs in achromatopsia patients exhibited severely reduced b-wave amplitudes with abnormal b:a ratios (1.3 and 0.6). In comparison, the reduction in a-wave amplitude was less marked. The rod-mediated ERG took on an electronegative appearance at high-stimulus illuminances. Although the defect that causes achromatopsia is primarily in the cone photoreceptors, our results reveal an accompanying disruption of rod function that is more severe than has previously been reported. The differential effects on the b-wave relative to the a-wave points to an inner-retinal locus for the disruption of rod function in these patients.

Light energy is converted into an electrical signal by a set of proteins in the phototransduction cascade in photoreceptors. In this work, I focus on two critical elements of the phototransduction cascade in canine, the opsin molecules and CNG channels of cone photoreceptors. Canines are dichromats possessing two types of cone photoreceptors with different opsin molecules that detect either long and medium wavelengths (L/M) or short wavelengths (S). The L/M- and S-opsin genes were cloned from an Alaskan Malamute and used to investigate key amino acids that are responsible for tuning the spectral properties of the 11-cis retinal chromophore. Cone CNG channels are composed of CNGA3 and CNGB3 subunits. I characterized antibodies to detect cone CNG channel subunits to investigate expression of mutations in CNGB3 subunit on two canine models for achromatopsia. One model contains a missense CNGB3 mutation D262N (CNGB3m/m) and the other is a complete deletion of all exons of the CNGB3 gene (CNGB3-/-). Studies presented in this thesis show CNGB3 is expressed later in cone during retinal development compared to CNGA3. It also presents evidence for the necessity of CNGB3 in cone outer segment targeting of CNGA3. / Biology

Cone cyclic nucleotide-gated (CNG) channels are located in the retinal outer segments, mediating daylight color vision. The channel is a tetramer of A-type (CNGA3) and B-type (CNGB3) subunits. CNGA3 subunits are able to form homotetrameric channels, but CNGB3 exhibits channel function only when co-expressed with CNGA3. Mutations in the genes encoding these cone CNG subunits are associated with achromatopsia, an autosomal recessive genetic disorder which causes incomplete or complete loss of daylight and color vision. A missense mutation, aspartatic acid (Asp) to asparagine (Asn) at position 262 in the canine CNGB3 subunit (cB3-D262N), results in loss of cone function and therefore daylight blindness, highlighting the crucial role of this aspartic acid residue for proper channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. We exploit the conservation of these residues in CNGA3 subunits to examine the motif using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in a loss of nucleotide-activated currents and mislocalization in heterologous expression. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 tetramers. This failure to record heteromeric currents implies that Asp/Asn mutations impact negatively both CNGA3 and CNGB3 subunits. A homology model of canine CNGA3 relaxed in a membrane using molecular dynamics simulations suggests that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3 - S4. We propose that the CNGB3-D262N mutation in daylight blind dogs results in the loss of these interactions and leads to an alteration of the electrostatic equilibrium in the S1 - S4 bundle. Because residues analogous to Tri-Asp residues in the voltage-gated Shaker K+ channel superfamily were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions might impair the monomer folding state in D262N mutant CNG channels during biogenesis. Another missesnse sense mutation, Arginine (Arg) to tryptophan (Trp) at position 424 in the canine CNGA3 subunit (cA3-R424W), also results in loss of cone function. An amino acid sequence alignment with Shaker K+ channel superfamily indicates that this R424 residue is located in the C-terminal end of the sixth transmembrane segment. A3-R424W mutant channels resulted in no cyclic nucleotide-activated currents and mislocalization with intracellular aggregates. However, the localization of cA3-R424W mutant channels was not affected as severely as the Asp/Asn mutation in S2 Tri-Asp motif, showing a lot of cells with the proper localization of Golgi-like and membrane fluorescence. Moreover, the substitution of Arg 424 to Lysine (Lys), conserving the positive charge, preserved channel function in some cells, which is different from the results of the S2 Tri-Asp motif in which the Asp/Glu substitutions, conserving the negative charge, leads to loss of cyclic nucleotide-activated currents. Even though these missense mutations are both associated with canine daylight blindness, the Arg 424 residue might not be as critical for folding as the Tri-Asp residues in the S2 Tri-Asp motif and might be more of a problem in channel structure and function. The cA3 model relaxed with MD simulations indicated a possible interaction of Arg 424 with the Glu 304 residue in the S4-S5 linker. This hypothesis is supported by electrophysiological data in which the double mutation of reversing these residues, Glu 306 to Arg and Arg 424 to Glu (E306R-R424E) preserves channel function. In the model, this salt bridge appears to contribute to stabilization of the open pore state. The R424W mutation might disrupt the salt bridge formation, leading to deforming and closing the pore region. / Biology