Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

Abstract

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.Detailed structural information about the plasma membrane calcium pump (PMCA) 2 is currently lacking. This pump is an integral part of the Ca 2ϩ signaling mechanism (1) and is thus a crucial component of cell function. It is highly regulated by calmodulin (CaM), which activates this protein by binding to an auto-inhibitory region (2) and changes the conformation of the pump from an inhibited state to an activated one (2, 3).Information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between the membrane domain of the PMCA and surrounding phospholipids under different experimental conditions that lead to known conformations. It has been previously demonstrated that both the conformation and the activity of the pump protein are preserved in either solubilized or reconstituted purified preparations compared with the native pump located in the erythrocyte (4).In recent work, we assessed changes in the overall exposure of PMCA to surrounding lipids by quantifying the extent of protein labeling by the photoactivatable phosphatidylcholine analog [125 I]TID-PC/16 under different conditions (5). This showed that labeling of ...

Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction

“…This effect is analogous to the behavior observed for other activators of the pump such as CaM and phosphatidic acid (72,73).…”

“…We have previously reported that treatments that lead to an activation of PMCA are related to a decrease in the accessible area of the membrane domain of the pump to the surrounding lipids, strongly suggesting the involvement of a conformational change in the transmembrane segments (49,72,73).…”

Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

“…Using the same experimental approach, we were also able to measure equilibrium constants for different ligands through the change of transmembrane conformations in PMCA (22). In order to obtain a complete understanding of the physical processes that involve ATP hydrolysis and Ca 2ϩ transport, it is necessary to know more about the structure of the enzyme.…”

“…Labeling-A photolabeling assay was carried out as described by Mangialavori et al (21,22). A dried film of the photoactivatable reagent was suspended in PC/C 12 E 10 mixed micelles containing 10 g/ml of the membrane protein.…”

The Plasma Membrane Calcium ATPasesand Their Role as Major New Players in Human Disease

“…The plasma membrane calcium ATPase (PMCA) has since been classified as a member of the P-type transport ATPase family, due to the covalent intermediate state that is formed upon phosphorylation of a highly conserved aspartate residue during its catalytic cycle (285). The pumps are high affinity (0.2-0.5 M under optimal conditions) Ca 2ϩ extruders, binding a single intracellular Ca 2ϩ ion which is then deposited to the extracellular space following conformational changes which first allow transport of the ion through the plasma membrane and finally dissociation from the pump through lowering its Ca 2ϩ affinity (44,225). This means that compared with the related ATPase of the sarco(endo)plasmic reticulum (SERCA), the PMCA has a lower capacity for Ca 2ϩ clearance, removing only one Ca 2ϩ ion per ATP molecule hydrolyzed as opposed to two (143).…”