The rate of activation by calmodulin of isoform 4 of the plasma membrane Ca(2+) pump is slow and is changed by alternative splicing.
A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca(2+) pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 microM free Ca(2+)) of about 1 min. The rate of inactivation of isoform 4b when calmodulin was removed was even slower, with a half-time of about 20 min. Isoform 4a has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster (half time about 20 s). This was coupled with a much faster inactivation rate, consistent with its low affinity. A truncated mutant of isoform 4b also had a more rapid activation rate, indicating that the downstream inhibitory region of full-length 4b contributed to its slow activation. The results indicate that the slow activation is due to occlusion of the calmodulin-binding domain of 4b, caused by its strong interaction with the catalytic core. Since the activation of 4b occurs on a time scale comparable to that of many Ca(2+) spikes, this phenomenon is important to the function of the pump in living cells. The slow response of 4b indicates that this isoform may be the appropriate one for cells which respond slowly to Ca(2+) signals.
Caride
A J
AJ
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Elwess
N L
NL
Verma
A K
AK
Filoteo
A G
AG
Enyedi
A
A
Bajzer
Z
Z
Penniston
J T
JT
D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D016296:Mutagenesis; D020033:Protein Isoforms; D013997:Time Factors; D014162:Transfection
D002147:Calmodulin; D020033:Protein Isoforms; D000252:Calcium-Transporting ATPases