The Plasma Membrane Calcium ATPases and Their Role as Major New Players in Human Disease.

The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease

A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes

Spectroscopic analysis of halothane binding to the plasma membrane Ca2+-ATPase.

The intrinsic tryptophan (Trp) fluorescence of the plasma membrane Ca2+-ATPase (PMCA) is significantly quenched by halothane, a volatile anesthetic common in clinical practice. It has been proposed that halothane inhibition of the Ca2+-ATPase activity results from conformational changes following anesthetic binding in the enzyme. We have investigated whether the observed quenching reflects halothane binding to PMCA. We have shown that the quenching is dose dependent and saturable and can be fitted to a binding curve with an equilibrium constant K(Hal) = 2.1 mM, a concentration at which the anesthetic approximately half-maximally inhibits the Ca2+-ATPase activity. The relatively low sensitivity of halothane quenching of Trp fluorescence to the concentration of phosphatidylcholine and detergent in the PMCA preparation concurs with the quenching resulting from anesthetic binding in the PMCA molecule. Analysis of the Trp fluorescence quenching by acrylamide indicates that the Trp residues are not considerably exposed to the solvent (Stern-Volmer quenching constant of 2.9 M(-1)) and do not differ significantly in their accessibility to halothane. Other volatile anesthetics, diethyl ether and diisopropyl ether, reduce the quenching caused by halothane in a dose-dependent manner, suggesting halothane displacement from its binding site(s). These observations indicate that halothane quenching of intrinsic Trp fluorescence of PMCA results from anesthetic binding to the protein. The analysis, used as a complementary approach, provides new information to the still rudimentary understanding of the process of anesthetic interaction with membrane proteins

Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca2+ dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K-aet 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K-aet than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca2+-ATPases. The plant Ca2+-ATPase was activated maximally by both isoforms, while the erythrocyte Ca2+-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K-aet and an approx. 25 % reduction in V-max. Importantly, SCaM isoforms showed a distinct Ca2+ concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca2+] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca2+] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca2+ transients.close606