INVESTIGADORES
DE TEZANOS PINTO Felicitas
artículos
Título:
Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A MUTANT WITH HIGH AFFINITY FOR CA2+ RESEMBLING THE ACIDIC LIPID-ACTIVATED ENZYME
Autor/es:
DE TEZANOS PINTO, F. Y ADAMO, H. P.
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
American Society for Biochemistry and Molecular Biology
Referencias:
Lugar: Bethesda; Año: 2002 vol. 277 p. 12784 - 12789
ISSN:
0021-9258
Resumen:
The C-terminal segment of the loop between transmembrane helices 2 and 3 (AL region) of the plasma membrane Ca2 pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca2 pump isoform h4xb with deletions in the AL region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296?349 had full Ca2 transport activity, but deletions involving the segment of amino acids 350?356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the Vmax of mutant d296?349 was similar to that of the recombinant wild type pump, but its K0.5 for Ca2 was about 5-fold lower. The addition of calmodulin increased the Vmax and the apparent Ca2 affinity of both the wild type and d296?349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca2 and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296?349. Taken together these results suggest that (a) deletion of residues 296? 349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the AL region; (b) the AL region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca2 affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the AL region is independent of the autoinhibition by the C-terminal calmodulin-binding region.
