Expression of an active Na+/Ca2+ exchanger isoform lacking the six C-terminal transmembrane segments.
The short isoform of the Na+/Ca2+ exchanger (67 kDa) that is produced by alternative splicing during the expression of the 6 kb canine exchanger cDNA in 293 cells was separately expressed in the same system. The protein consisted of the five N-terminal transmembrane segments and of a large portion of the main hydrophilic loop, but lacked the six C-terminal hydrophobic segments of the regular protein (108 kDa). Very high RNA levels were found after transient cell transfection with plasmid DNA encoding this truncated isoform. The RNA processing, the translation and targeting of the resulting protein to the plasma membrane appeared to be less efficient than those of the 108-kDa polypeptide produced in the same system. The Na(+)-dependent Ca(2+)-uptake activity of 293 cells expressing the short isoform was measured by an isotopic rapid filtration method, whereas the current associated with Ca2+ extrusion was measured in electrophysiological patch-clamp experiments. The results showed that the expressed isoform functioned in the typical reverse and forward Na+/Ca2+ exchange modes. In both the electrophysiological and the isotopic measurements the activity of the short isoform was 6-7-fold lower than that of the 108-kDa protein expressed in the same system. However, lower amounts of the short isoform reached the plasma membrane: its specific activity could thus be significantly higher. Possibly, the short isoform could form a dimer in which a second 67 kDa polypeptide replaces the C-terminal part of the 108-kDa protein.
Gabellini
N
N
Department of Biological Chemistry, University of Padova, Italy.
Zatti
A
A
Rispoli
G
G
Navangione
A
A
Carafoli
E
E
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D004285:Dogs; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium