Replacement of Val(674) by pro increases the sensitivity of the plasma membrane Ca2+ pump to inhibition by Mg2+

A cDNA encoding a plasma membrane Ca2+ pump mutant (VP)-P-674(ct120) was constructed and expressed in COS-1 cells. Immunoblots of transfected COS-1 membranes showed that the (VP)-P-674(ct120) and the wild-type hPMCA4b(ct120) proteins were expressed at similar levels. The change of Val(674) to Pro reduced the activity of the hPMCA4b(ct120) to an extent similar to that observed previously in the full-length Ca2+ pump (Adamo et al. (1995) J. Biol. Chem. 270, 30111-30114). Despite its lower activity, the apparent affinity for Ca2+ of the (VP)-P-674(ct120) enzyme was at least as high as that of hPMCA4b(ct120), indicating that substitution of Val(674) by Pro did not impair the interaction of the enzyme with Ca2+. The sensitivity of the (VP)-P-674(ct120) enzyme to inhibition by vanadate was not significantly different from that of the hPMCA4b(ct120), supporting the idea that the mutation did not alter the equilibrium between E(2)-E(1). The study of the dependency of the Ca2+ transport showed that the (VP)-P-674(ct120) mutant reached maximum activation at 100 mu M Mg2+ in contrast with 500 mu M in the hPMCA4b(ct120). Furthermore, while at 2 mM Mg2+ the hPMCA4b(ct120) showed no sign of inhibition, the activity of the mutant decreased to less than 50% of the maximum activity observed at 100 mu M Mg2+ These results indicate that the decrease in the activity observed upon substitution of Val(674) by Pro was due to a higher sensitivity to Mg2+ as inhibitor.