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

Irene Mangialavori,Mariela Ferreira-Gomes,María F Pignataro,Emanuel E Strehler,Juan Pablo F.C Rossi

doi:10.1074/jbc.m109.076679

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.

Highlights

JANUARY 1, 2010 VOLUME 285 NUMBER 1 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]
We assessed changes in the overall exposure of PMCA to surrounding lipids by quantifying the extent of protein labeling by the photoactivatable phosphatidylcholine analog [125I]TID-PC/16 under different conditions [5]. This showed that labeling of PMCA incubated with Ca2ϩ and calmodulin decreases by 25% and incubation with Ca2ϩ alone increases labeling by more than 50% compared with control labeling of the PMCA in the absence of Ca2ϩ and CaM. These results suggest that the PMCA assumes two different E1 conformations: one that is auto-inhibited and in which the membrane domain is in contact with a higher amount of lipids and one in which the enzyme is fully active and exhibits a more compact transmembrane arrangement with lesser exposure to lipids
The other two conformers are E1-Ca conformation (E1I), which is obtained by incubating the enzyme in the presence of Ca2ϩ and binds the maximum concentration of [125I]TID-PC/16 (180% at optimal concentration of Ca2ϩ, Fig. 1) and E1A, a conformation attainable in the presence of Ca2ϩ and CaM or with a CaM-like effector such as phosphatidic acid, oleic acid or after removal of the C terminus of PMCA

Summary

Introduction

JANUARY 1, 2010 VOLUME 285 NUMBER 1 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]. We assessed changes in the overall exposure of PMCA to surrounding lipids by quantifying the extent of protein labeling by the photoactivatable phosphatidylcholine analog [125I]TID-PC/16 under different conditions [5] This showed that labeling of PMCA incubated with Ca2ϩ and calmodulin decreases by 25% and incubation with Ca2ϩ alone increases labeling by more than 50% compared with control labeling of the PMCA in the absence of Ca2ϩ and CaM. These results suggest that the PMCA assumes two different E1 conformations: one that is auto-inhibited and in which the membrane domain is in contact with a higher amount of lipids (incubating with Ca2ϩ alone, E1I) and one in which the enzyme is fully active (incubating with Ca2ϩ-calmodulin, E1A) and exhibits a more compact transmembrane arrangement with lesser exposure to lipids.

Objectives

Methods

Results