NCX1 surface expression: a tool to identify structural elements of functional importance.
The rat Na(+)/Ca(2+) exchanger isoforms of the NCX1 gene have 14 cysteine residues. Each of these cysteines can be mutated individually to alanine or serine without loss of functional expression in transfected HEK293 cells. Yet sequential exchange starting from the amino terminal end of three or more cysteines results in reduced transport activity and surface expression. As more and more cysteines are replaced, transport activity and surface expression decrease in parallel, and the cysteineless mutant exhibits only traces of Na(+)/Ca(2+) exchange activity and surface expression. No significant differences are detected in the amount of total cell exchanger protein between the wild-type exchanger and its functional or nonfunctional cysteine mutants. Reduced surface expression of the Na(+)/Ca(2+) exchanger NCX1 is also observed when HEK293 cells expressing the transporter are treated with cyclosporin A (CsA) or with PSC833. The reductions in transport activity and surface expression are concentration dependent and parallel. No reduction is obtained in the total amount of exchanger protein by CsA or PSC833 treatment, suggesting that the effects of these drugs on NCX1 expression are posttranslational. FK506 and rapamycin treatment of HEK293 cells expressing rat NCX1 isoforms has no effect on transport activity, surface expression, or the total amount of exchanger protein in the transfected cells.
Rahamimoff
Hannah
H
Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. hannah@cc.huji.ac.il
Ren
Xiaoyan
X
Kimchi-Sarfaty
Chava
C
Ambudkar
Suresh
S
Kasir
Judith
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000502:physiology; D002460:Cell Line; D002463:Cell Membrane Permeability; D003545:Cysteine; D006321:Heart / Q000502:physiology; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000502:physiology*; D014162:Transfection
D020033:Protein Isoforms; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D003545:Cysteine