title: Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide-gated channel.
abstract: The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore-forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease-associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37 degrees C revealed nonfunctional homo- and heteromeric channels in all five mutants, but incubation at 27 degrees C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K.
authors.affiliation: Molecular Genetics Laboratory, Institute for Ophthalmic Research, Tuebingen, Germany. Koeppen@Dartmouth.edu
authors.forename: Katja
authors.initials: K
authors.lastname: Koeppen
authors.forename: Peggy
authors.initials: P
authors.lastname: Reuter
authors.forename: Thomas
authors.initials: T
authors.lastname: Ladewig
authors.forename: Susanne
authors.initials: S
authors.lastname: Kohl
authors.forename: Britta
authors.initials: B
authors.lastname: Baumann
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Jacobson
authors.forename: Astrid S
authors.initials: AS
authors.lastname: Plomp
authors.forename: Christian P
authors.initials: CP
authors.lastname: Hamel
authors.forename: Andreas R
authors.initials: AR
authors.lastname: Janecke
authors.forename: Bernd
authors.initials: B
authors.lastname: Wissinger
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003117:Color Vision Defects / Q000235:genetics*; D006152:Cyclic GMP / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: C518238:CNGA3 protein, human; D054815:Cyclic Nucleotide-Gated Cation Channels; D050505:Mutant Proteins; D006152:Cyclic GMP; D002118:Calcium