Loss of Autoinhibition of the Plasma Membrane Ca2+ Pump by Substitution of Aspartic 170 by Asparagine

Abstract

The plasma membrane calcium ATPase (PMCA) actively transports Ca 2؉ from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca 2؉ -calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp 170 , in the putative "A" domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca 2؉ . The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the Cterminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.

Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

“…In the presence of 1 M Ca 2ϩ the activity of the BFP-PMCA-GFP enzyme in the absence of calmodulin was ϳ40% of that attained at saturating concentrations of calmodulin. This value of basal activity is similar to that of the recombinant wild-type hPMCA4xb enzyme obtained from yeasts under similar conditions (19). These results suggest that the inhibitory function of the C-terminal regulatory domain was preserved despite the addition of the GFP.…”

“…Thus, the most important point of interest was to investigate which step of the BFP-PMCA-GFP activation was being reported by the decrease in FRET. For this purpose we took advantage of the activating effect of mutation D170N reported previously (19). As shown in Fig.…”

“…Membrane Isolation and Purification of Recombinant BFP-PMCA-GFP-Four liters of yeasts expressing the BFP-PMCA-GFP protein were grown in YNB Leu Ϫ media at 28°C in glass flasks with agitation, and, after the culture reached an approximate A 600 of 1.5, 4 liters of complete media was added, and the incubation continued for 6 h (A 600 ϭ 4.0 -5.0). Total yeast membranes were solubilized with C 12 E 10 , and the PMCA was purified by calmodulin-affinity chromatography as described previously (19). ϳ400 g of purified PMCA was obtained from one batch of yeast.…”

Domain Organization and Movements in Heavy Metal Ion Pumps

“…Similarly, in ATP7B, the fifth and sixth NMBDs connected to the Ma helix are relatively important for enzymatic property (51). In a class of PII-ATPases, including plasma membrane Ca 2ϩ -ATPase, a regulatory domain is attached to the C terminus (52). Thus, our current hypothesis is that only the NMBD directly connected to the Ma helix is prominently important for metal transport but not other metal binding domains.…”

Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

“…Moreover, the C-terminal portion of the autoinhibitory segment would be flanked by Glu 99 and Asp 170 from the transmembrane segments M1 and M2, respectively. We have previously shown that Asp 170 is a highly sensitive spot for autoinhibition and that even a conservative substitution by Asn increases the V max and the apparent affinity for Ca 2ϩ (35). Interestingly, although the basic features of the model may be shared by all autoinhibited Ca 2ϩ -ATPases, there are probably also some differences.…”