title: A newly cloned ClC-3 isoform, ClC-3d, as well as ClC-3a mediates Cd-sensitive outwardly rectifying anion currents.
abstract: BACKGROUND
  ClC-3, a member of the ClC family, is predicted to have six isoforms, ClC-3a to -3f, with distinct N- and C-terminal amino acid sequences. There have been conflicting reports on the properties of ClC-3a (also known as the N-terminal short form of ClC-3) and ClC-3b (the N-terminal long form of ClC-3) as plasmalemmal Cl(-) channels. Meanwhile, little is known about other isoforms. The amino acid sequence of ClC-3d (a C-terminal variant of the short form) listed in the NCBI database was derived from the genomic sequence, but there has been no experimental evidence for the mRNA.
  
  
  METHODS
  PCR-cloning was made to obtain the full coding region of ClC-3d from mouse liver. Its molecular expression on the plasma membrane was microscopically examined in HEK293T cells transfected with GFP-tagged ClC-3d. Its functional plasmalemmal expression and the properties of currents were studies by whole-cell recordings in the cells transfected with ClC-3d.
  
  
  RESULTS
  The cloned ClC-3d was found to be the only isoform which has an N-terminal amino acid sequence identical to ClC-3a. When introduced into HEK293T cells, a minor fraction of exogenous ClC-3d proteins was detected at the plasma membrane, and activation of anion currents was observed at neutral pH under normotonic conditions. The properties of ClC-3d currents were found to be shared by ClC-3a-mediated currents. Also, both ClC-3d and -3a currents were found to be sensitive to Cd(2+). ClC-3d overexpression never affected the endogenous activity of acid- or swelling-activated anion channels.
  
  
  CONCLUSIONS
  We thus conclude that plasmalemmal ClC-3d, like ClC-3a, mediates Cd(2+)-sensitive outwardly rectifying anion currents and that ClC-3d is distinct from the molecular entities of acid- and volume-sensitive anion channels.
authors.affiliation: Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Okada
authors.forename: Tenpei
authors.initials: T
authors.lastname: Akita
authors.forename: Kaori
authors.initials: K
authors.lastname: Sato-Numata
authors.forename: Md Rafqul
authors.initials: MR
authors.lastname: Islam
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Okada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D018118:Chloride Channels / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018118:Chloride Channels; C115376:ClC-3 channel; D020033:Protein Isoforms