Non-canonical translation start sites in the TMEM16A chloride channel

Elvira Sondo, Paolo Scudieri, Valeria Tomati, Emanuela Caci, Amelia Mazzone, Gianrico Farrugia, Roberto Ravazzolo, Luis J.V. Galietta

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)89-97
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Issue number1 PARTB
StatePublished - 2014


  • Chloride channel
  • Non-canonical start codon
  • Protein translation
  • Structure-function relationship

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology


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