Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes (original) (raw)
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Biophysical Journal, 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]
Journal of Biological Chemistry, 2011
The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [ 125 I]TID-PC/16 to the protein. In the presence of Ca 2؉ both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [ 125 I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [ 125 I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [ 125 I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains.
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.
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 (
PMCA Differential Exposure of Hydrophobic Domains after Calmodulin and Phosphatidic Acid Activation
Biophysical Journal, 2011
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 highaffinity 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 autoinhibited 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.
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 (
Journal of Biological Chemistry, 2008
The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca 2؉ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2 2؉ . This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca 2؉ and calmodulin decreases by approximately the same amount. However, incubation with Ca 2؉ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [ 125 I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E 1 conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca 2؉ ؉ C28 for SERCA and Ca 2؉ alone for PMCA), and one in which the enzyme is fully active (Ca 2؉ for SERCA and Ca 2؉ -calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.
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.