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
Genre
Journal ArticleDate
2014-02-21Author
Tanaka, NDelemotte, L
Klein, ML
Komáromy, AM
Tanaka, JC
Subject
Amino Acid MotifsAmino Acid Sequence
Animals
Base Sequence
Cloning, Molecular
Computational Biology
Cyclic Nucleotide-Gated Cation Channels
DNA Primers
Dog Diseases
Dogs
Fluorescence
Immunohistochemistry
Models, Genetic
Molecular Dynamics Simulation
Molecular Sequence Data
Mutation, Missense
Retinal Cone Photoreceptor Cells
Retinal Degeneration
Sequence Alignment
Sequence Analysis, DNA
Sequence Homology
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http://hdl.handle.net/20.500.12613/5093
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10.1371/journal.pone.0088768Abstract
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. © 2014 Tanaka et al.Citation to related work
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http://dx.doi.org/10.34944/dspace/5075
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