Hannah Rahamimoff | PubMed Health

PubMed2002

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.

Amino Acid Sequence↗

PubMed2001

Transport activity and surface expression of the Na+-Ca2+ exchanger NCX1 are inhibited by the immunosuppressive agent cyclosporin A and by the nonimmunosuppressive agent PSC833.

Cyclosporin A (CsA) treatment of HEK 293 cells expressing the rat heart RHE-1 (NCX1.1, EMBL accession number ) or the rat brain RBE-2 (NCX1.5, GenBank(TM) accession number ) Na(+)-Ca(2+) exchanger inhibited their transport activity in a concentration-dependent manner. The inhibition was detectable at 2 microm CsA, and exposure of the cells to 20 microm CsA resulted in a decrease of the Na(+)-dependent Ca(2+) uptake to about 20% relative to that of untreated cells. Determination of the surface expression of the exchanger protein revealed a parallel concentration-dependent reduction in the amount of the immunoreactive protein. No reduction was detected in the amount of total immunoreactive exchanger protein in CsA-treated cells relative to untreated ones. Among the different drugs tested, only PSC833, an analog of cyclosporin D, mimicked the effects of CsA. Exposure of the transfected cells to the chemically related cyclosporin D and macrolide drugs (FK506 or rapamycin) had no effect on the transport activity or the surface expression of the Na(+)-Ca(2+) exchanger. Co-expression of the human multidrug transporter P-glycoprotein (of which both drugs are modulators) with the cloned Na(+)-Ca(2+) exchanger revealed that transport activity and surface expression of each transporter in the co-transfected system were similar to those of each transporter alone in both the presence and absence of CsA or PSC833. CsA and PSC833 inhibited the surface expression of the NCX1 protein but did not alter the surface expression of P-glycoprotein. Unlike some P-glycoprotein endoplasmic reticulum-retained mutants (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709-712), CsA did not rescue RBE-2/F913-->Stop, an endoplasmic reticulum-retained function-competent mutant of the Na(+)-Ca(2+) exchanger (Kasir, J., Ren, X., Furman, I., and Rahamimoff, H. (1999) J. Biol. Chem. 274, 24873-24880) and did not induce its kinesis to the surface membrane, further demonstrating molecular differences between P-glycoprotein and NCX1 mutants for interaction with CsA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗