The Plasma Membrane Ca2+ Pump Isoform 4a Differs from Isoform 4b in the Mechanism of Calmodulin Binding and Activation Kinetics: IMPLICATIONS FOR Ca2+ SIGNALING (original) (raw)
Related papers
Journal of Biological Chemistry, 2007
The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca 2؉. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca 2؉ showed a much smaller effect (2versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (
Plasma Membrane Ca2+ Pump Isoforms 2a and 2b Are Unusually Responsive to Calmodulin and Ca2+
Journal of Biological Chemistry, 1997
The full-length a and b variants of the rat plasma membrane calcium pump, isoform 2 (rPMCA2a and rPMCA2b), were constructed and expressed in COS-7 cells. To characterize these isoforms, calcium transport was determined in a microsomal fraction. Both rPMCA2a and rPMCA2b had a much higher affinity for calmodulin than the corresponding forms of hPMCA4, and rPMCA2b had the highest affinity among the isoforms that have been tested so far. When analyzed at a relatively high calmodulin concentration, rPMCA2b and, to a lesser extent, rPMCA2a showed higher apparent calcium affinity; i.e. they were more active at lower Ca 2؉ concentrations than hPMCA4b. This indicates that these two variants of rat isoform 2 will tend to maintain a lower free cytosolic Ca 2؉ level in cells where they are expressed. Both variants also showed a higher level of basal activity (in the complete absence of calmodulin) than hPMCA4b, a property which would reinforce their ability to maintain a low free cytosolic Ca 2؉ concentration. Experiments designed to determine the source of the higher apparent Ca 2؉ affinity of rPMCA2b showed that it came from the properties of the carboxyl terminus, rather than from any difference in the catalytic core.
The Biochemical journal, 1998
A mutant of the plasma membrane Ca2+ pump (PMCA) called (nCI)hPMCA4b(ct120), in which the C-terminal regulatory segment including the calmodulin-binding autoinhibitory domains C and I had been relocated near the N-terminus, has been expressed in COS-1 cells. The measurements of Ca2+ transport in microsomal preparations showed that the rearranged enzyme was functional. The activity of the (nCI)hPMCA4b(ct120) mutant was compared with those of the wild-type hPMCA4b and the fully active calmodulin-insensitive mutant hPMCA4b(ct120). In the absence of calmodulin the activity of (nCI)hPMCA4b(ct120) was higher than that of hPMCA4b but only 45% of that of hPMCA4b(ct120). Mutant (nCI)hPMCA4b(ct120) exhibited an apparent affinity for Ca2+ similar to that of hPMCA4b, typical of the inhibited state of the enzyme. Calmodulin at concentrations that fully activated hPMCA4b increased the activity of (nCI)hPMCA4b(ct120) to 68% of that of hPMCA4b(ct120). The lower maximal activity of (nCI)hPMCA4b(ct12...
Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin
Journal of Biological Chemistry, 2000
Isoform 3f of the plasma membrane Ca 2؉ pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K d of 15 nM) than the corresponding peptide of isoform 2b (K d value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.
Journal of Biological Chemistry, 1999
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 M 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 fulllength 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.
Journal of Biological Chemistry, 2010
The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca 2؉ -pump (PMCA) in the membrane region upon interaction with Ca 2؉ , calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [ 125 I]TID-PC/16, measuring the shift of conformation E 2 to the auto-inhibited conformation E 1 I and to the activated E 1 A state, titrating the effect of Ca 2؉ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca 2؉ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca 2؉ transport but does not modify the affinity for Ca 2؉ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E 1 I to the activated E 1 A conformation and thus modulating the transport of Ca 2؉ . This is reflected in the different apparent constants for Ca 2؉ in the absence of CaM (calculated by Ca 2؉ -ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca 2؉ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca 2؉ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca 2؉ binding and activation. . 2 The abbreviations used are: PMCA, plasma membrane calcium pump; [ 125 I]TID-PC/16, 1-O-hexadecanoyl-2-O-[9-[[[2-[ 125I ]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]
Interaction of calmodulin with the calmodulin binding domain of the plasma membrane calcium pump
Biochemistry, 1990
Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D N M R experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding 'This work has been made possible by the financial contributions of the Swiss Nationalfonds (Grant 3.53 1.086) and the National Institues of Health (Grant GM28835).
A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells
Journal of Biological Chemistry, 2003
The four basic isoforms of the plasma membrane Ca 2؉ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca 2؉ in the native intracellular environment. The level of [Ca 2؉ ] in the endoplasmic reticulum and the height of the [Ca 2؉ ] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca 2؉ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.