Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel.
OBJECTIVE
Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology.


METHODS
The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology.


RESULTS
All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication.


CONCLUSIONS
The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.
Center for Biotechnology, Temple University, Philadelphia, Pennsylvania 19122, USA.
Kirti A
KA
Patel
Kristen M
KM
Bartoli
Richard A
RA
Fandino
Anita N
AN
Ngatchou
Gustaw
G
Woch
Jannette
J
Carey
Jacqueline C
JC
Tanaka
D000595:Amino Acid Sequence; D003117:Color Vision Defects / Q000235:genetics* / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006152:Cyclic GMP