Hp. Adamo et Me. Grimaldi, FUNCTIONAL CONSEQUENCES OF RELOCATING THE C-TERMINAL CALMODULIN-BINDING AUTOINHIBITORY DOMAINS OF THE PLASMA-MEMBRANE CA2-TERMINUS( PUMP NEAR THE N), Biochemical journal, 331, 1998, pp. 763-766
A mutant of the plasma membrane Ca2+ pump (PMCA) called (nCI)hPMCA4b(c
t120) in which the C-terminal regulatory segment including the calmodu
lin-binding autoinhibitory domains C and I had been relocated near the
N-terminus, has been expressed in COS-1 cells. The measurements of Ca
2+ transport in microsomal preparations showed that the rearranged enz
yme was functional. The activity of the (nCI)hPMCA4b(ct120) mutant was
compared with those of the wild-type hPMCA4b and the fully active cal
modulin-insensitive mutant hPMCA4b(ct120). In the absence of calmoduli
n the activity of (nCI)hPMCA4b(ct120) was higher than that of hPMCA4b
but only 45% of that of hPMCA4b(ct120). Mutant (nCI)hPMCA4b(ct120) exh
ibited an apparent affinity for Ca2+ similar to that of hPMCA4b, typic
al of the inhibited state of the enzyme. Calmodulin at concentrations
that fully activated hPMCA4b increased the activity of(nCI)hPMCA4b(ct1
20) to 68% of that of hPMCA4b(ct120). The lower maximal activity of (n
CI)hPMCA4b(ct120) was not due to a lower affinity for calmodulin becau
se the concentration of calmodulin required for half-maximal activatio
n of (nCI)hPMCA4b(ct120) was equal to that of the wild-type hPMCA4b. T
hese results indicate that (1) the disturbance of the N-terminal regio
n of the PMCA by the insertion of the C-terminal segment decreased the
ability of the pump to transport Ca2+, and (2) the calmodulin-binding
autoinhibitory domain was still able to access its acceptor site from
the N-terminal end of the molecule. However, although the calmodulin-
binding and inhibitory functions of the C-domain were fully preserved,
the I domain at its new position seemed less effective at inhibiting
the pump.