title: Non-canonical translation start sites in the TMEM16A chloride channel.
abstract: TMEM16A is a plasma membrane protein with voltage- and calcium-dependent chloride channel activity. The role of the various TMEM16A domains in expression and function is poorly known. In a previous study, we found that replacing the first ATG of the TMEM16A coding sequence with a nonsense codon (M1X mutation), to force translation from the second ATG localized at position 117, only had minor functional consequences. Therefore, we concluded that this region is dispensable for TMEM16A processing and channel activity. We have now removed the first 116 codons from the TMEM16A coding sequence. Surprisingly, the expression of the resulting mutant, Δ(1-116), resulted in complete loss of activity. We hypothesized that, in the mutant M1X, translation may start at a position before the second ATG, using a non-canonical start codon. Therefore, we placed an HA-epitope at position 89 in the M1X mutant. We found, by western blot analysis, that the HA-epitope can be detected, thus demonstrating that translation starts from an upstream non-ATG codon. We truncated the N-terminus of TMEM16A at different sites while keeping the HA-epitope. We found that stepwise shortening of TMEM16A caused an in parallel stepwise decrease in TMEM16A expression and function. Our results indicate that indeed the N-terminus of TMEM16A is important for its activity. The use of an alternative start codon appears to occur in a naturally-occurring TMEM16A isoform that is particularly expressed in human testis. Future experiments will need to address the role of normal and alternative amino-terminus in TMEM16A structure and function.
authors.affiliation: U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italy.
authors.forename: Elvira
authors.initials: E
authors.lastname: Sondo
authors.forename: Paolo
authors.initials: P
authors.lastname: Scudieri
authors.forename: Valeria
authors.initials: V
authors.lastname: Tomati
authors.forename: Emanuela
authors.initials: E
authors.lastname: Caci
authors.forename: Amelia
authors.initials: A
authors.lastname: Mazzone
authors.forename: Gianrico
authors.initials: G
authors.lastname: Farrugia
authors.forename: Roberto
authors.initials: R
authors.lastname: Ravazzolo
authors.forename: Luis J V
authors.initials: LJ
authors.lastname: Galietta
mesh_terms: D000075369:Anoctamin-1; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D018408:Patch-Clamp Techniques; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013737:Testis / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D001426:Bacterial Proteins; D018118:Chloride Channels; D002712:Chlorides; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; C054562:yellow fluorescent protein, Bacteria; D002118:Calcium
keywords: Ca(2+)-activated Cl(−) channel; CaCC; Chloride channel; HS-YFP; Non-canonical start codon; Protein translation; Structure–function relationship; halide-sensitive yellow fluorescent protein