A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70kDa NH(2)-terminal protein.
The Na/Ca exchanger (NCX) is an ubiquitous transporter that plays an important role in regulating cellular Ca(2+) balance. On gel electrophoresis, the NCX1 protein migrates as two major bands of 120 and 70kDa. While the 120kDa is thought to represent the native protein, the nature of the 70kDa protein remains unclear. In this report, we describe a new NCX1 splicing pattern, identified during the cloning of NCX1 isoforms from human eye. The insertion of a newly identified sequence upstream exons B and D of the NCX1.3 isoform, generates a stop codon in frame with the NCX1 coding sequence, that should lead to a truncated Na/Ca exchanger (that we called NCX1.33) comprising only the N-terminal portion of the exchanger and a shortened intracellular loop. Insulin-secreting cells were stably transfected with NCX1.33. Overexpression was assessed at the mRNA and protein level, the truncated exchanger migrating as a70kDa band. Appropriate targeting to the plasma membrane was assessed by microfluorescence and by the increase in Na/Ca exchange activity. The results of the present study constitute a clear piece of evidence indicating that the Na/Ca exchanger 70kDa protein corresponds to the N-terminal portion of the exchanger, and is functionally active.
Van Eylen
F
F
Laboratory of Pharmacology, Brussels University School of Medicine, Route de Lennik, 808-Bâtiment GE, B-1070 Brussels, Belgium.
Kamagate
A
A
Herchuelz
A
A
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics; D005123:Eye / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D017403:In Situ Hybridization; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D020033:Protein Isoforms; D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium