title: A cyclic nucleotide-gated channel mutation associated with canine daylight blindness provides insight into a role for the S2 segment tri-Asp motif in channel biogenesis.
abstract: Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in 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. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1-S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1-S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.
authors.affiliation: Department of Biology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Naoto
authors.initials: N
authors.lastname: Tanaka
authors.affiliation: Institute of Computational and Molecular Science, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Lucie
authors.initials: L
authors.lastname: Delemotte
authors.affiliation: Institute of Computational and Molecular Science, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Klein
authors.affiliation: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States of America ; Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: András M
authors.initials: AM
authors.lastname: Komáromy
authors.affiliation: Department of Biology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Jacqueline C
authors.initials: JC
authors.lastname: Tanaka
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004283:Dog Diseases / Q000235:genetics* / Q000473:pathology*; D004285:Dogs; D005453:Fluorescence; D007150:Immunohistochemistry; D008957:Models, Genetic; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D017949:Retinal Cone Photoreceptor Cells / Q000473:pathology*; D012162:Retinal Degeneration / Q000235:genetics / Q000662:veterinary*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D017931:DNA Primers