The rate of activation by calmodulin of isoform 4 of the plasma membrane Ca2+ pump is slow and is changed by alternative splicing

A reconstitution system allowed us to measure the ATPase activity of specif ic isoforms of the plasma membrane Ca2+ pump continuously, and to measure t he effects of adding or removing calmodulin, The rate of activation by calm odulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 mu M free Ca2+) of about 1 min. The rate of inactivatio n of isoform 4b when calmodulin was removed was even slower, with a half-ti me 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 wi th 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 activ ation of 4b occurs on a time scale comparable to that of many Ca2+ spikes, this phenomenon is important to the function of the pump in living cells, T he slow response of 4b indicates that this isoform may be the appropriate o ne for cells which respond slowly to Ca2+ signals.