PM Ca2+-ATPase

Fluorescein 5 -isothiocyanate (FITC) Lys515 SR and PM Ca2+-ATPases Na+-K+-ATPase H+-K+-ATPase... 

Catecholamines Molecular turnover Protein kinase mediated phosphorylation of phospholamban (SR Ca2+-ATPase) or autoinhibitory domain (PM Ca2+-ATPase) relieving negative influence on kinetic parameters... 

The PM Ca2+-ATPase is activated by the Ca2+—calmodulin complex (calmodulin being a key Ca2+-binding regulatory protein) and the ER Ca2+-ATPase is stimulated by the... 

In this equation the subscripts on the concentration and enhancement terms denote total Ga3+ (T), free Gd3+ (F), and Gd3+ bound at site 1 (Bl) or Gd3+ bound at site 2 (B2). In this paper we will define site 1 as the site which binds Gd3+ more tightly. The data of Figure 11 are consistent with an Eg- of 9.4 and an Eg2 of 5.4. When solutions containing 10 or 50 pM Gd3+ were titrated with Ca2+-ATPase, the enhancement increased as the enzyme concentration increased. The reciprocal of the observed enhancement was plotted against the reciprocal of the total ATPase concentration (Figure 12) yielding a linear behavior, except at high levels of enzyme where a sharp increase in the observed enhancement is found. This behavior is consistent with two environments for bound Gd3+ ion on the Ca2+-... 

Figure 12. Titration of GdCl with Ca2 -ATPase (24). The solutions contained 0.05M TMA-Pipes buffer, pH 7.0, 24.4 pM GdCl, (A) or 49pM GdCl, (B), and the noted concentrations of Ca2 -ATPase. The value of t obtained by extrapolation of the solid line to the infinite protein concentration is the average of the enhancements at Gd3 sites 1 and 2, denoted by c , and iBi, respectively, in the text.

Figure 10. Experiment demonstrating consecutive dissociation of two calcium ions from the uptake sites of the SR Ca2+-ATPase in the unphosphorylated E, state. 45Ca2+ bound from the cytoplasmic side at the two high-affinity sites on the enzyme can be seen to dissociate back to the medium on the cytoplasmic side in two distinct phases upon dilution of the radioactivity (A). The first phase is rapid and independent of the concentration of Ca2+ in the medium. The second phase is also rapid in the absence of Ca2+ (presence of EGTA) in the buffer medium (open triangles) but slow in the presence of nonradioactive Ca2+ in the buffer medium (closed triangles, 10 pM Ca2+ open circles, 30 pM Ca2+ closed circles, 100 pM Ca2+ open squares, 300 pM Ca2+ closed squares 1 mM Ca2+). The inset shows the rate constant for dissociation of the second Ca2+ as function of the Ca2+ concentration in the medium. The lower panel (B) explains these observations in terms of steric hindrance of the dissociation of the deeper ion by the presence of a more superficial ion at its binding site (closed circles, radioactive 45Ca2+ open circles, nonradioactive 40Ca2+ from the medium). Reproduced from Orlowski and Champeil, 1991a with permission from The American Chemical Society.

Figure 17. Analysis of phosphoenzyme intermediates of SR Ca2+-ATPase mutants with alterations to carboxylate-containing residues in the transmembrane sector. Wild-type or mutant Ca2+-ATPases expressed in the endoplasmic reticulum membranes of COS-1 cells were phosphorylated with [y-32P] ATP (panel a) or [32P]P (panels b and c). Following acid-quench of the phosphorylated intermediate, the samples were subjected to SDS-polyacrylamide gel electrophoresis under acid pH conditions and the dried gels were autoradiographed to visualize the radioactivity associated with the covalently bound phosphate. Panel a shows the Ca2+-concentration dependence of phosphorylation from ATP. The Glu309- Lys mutant is unable to phosphorylate, even at 12.5 mM Ca2+. In the wild-type Ca2+-ATPase the phosphorylation reaction is fully saturated at 10 pM Ca2+. Panel b shows lack of Ca2+ inhibition of backdoor phosphorylation from P in the mutants. E indicates the presence of ECTA to chelate Ca2+ (normally a requirement for phosphorylation by the backdoor route). C indicates the...

As outlined in Chapter 3, cell excitability can in part be determined by the maintenance of gradients of Na+, K+ and CP ions. Differential plasma membrane (PM) permeabilities to such ions and the gradients of ion concentration contribute to the transmembrane potential difference (t tm), which is typically about —0.1 volt (V) (inside with respect to the outside). In addition, the cytosolic free concentration of Ca2+ is extremely low (0.1 pM in resting cells and about 10 pM in excited cells) as compared to concentrations of Na+, CP and K+ of about 10, 10 and 100 mM, respectively, in the cytosol and about 100, 100 and 10 mM, respectively, in the extracellular milieu. These huge ion gradients are maintained through the operation of ion pumps such as the adenosine 5 -triphosphate (ATP)-energized Ca2+ pump (Ca2+-ATPase) and the Na+ and K+ pump (Na+, K+-ATPase). 

Ca2+ pumps (Ca2+-ATPases) are located on the PM and on the ER membrane and pump Ca2+ out of the cell or into the lumen of the ER, respectively. This process is driven by the hydrolysis of ATP and involves the successive phosphorylation and dephosphorylation of an aspartyl residue of the Ca2+-ATPase. In the dephosphorylated state (state 1), the pump binds Ca2+ tightly at a site oriented towards the cytosol but in the phosphorylated state (state 2) this site has a lower affinity for Ca2+ and is oriented towards the other side of the membrane (i.e. towards the outside of the cell or towards the ER lumen). Ca2+ is accordingly released in state 2, the pump reverts to state 1 through dephosphorylation and the cycle continues. 

An estimate of the relative importance of Ca2+-ATPase and Na+-Ca2+ exchange has been made in cultured rat aortic smooth muscle cells by Furukawa et al. 1988). At [Ca2+]i below 1 pM, the contribution of the former was significantly greater than that of the latter. 

Our knowledge on the structure of Ca2+-ATPases is mainly based on studies of the Ca + pump of the SR of fast skeletal muscle (a SERCAl gene product). However, the general structural model that emerged from these studies is probably also valid for the other SERCAs as well as for the PM Ca + pump. The predicted structure of the Ca2+-transport ATPase incorpo-... 

In smooth muscle cells a phosphorylation of the PM Ca2+-pump protein has been clearly demonstrated only for PKC. The regulation of the PMCA ATPase by cGK does not require concomitant phosphorylation of the pump. Although a modulation by cAK of the PM Ca2+ pump of erythrocytes and cardiac cells has been reported, such an effect could not be demonstrated for the ATPase from smooth muscle (see the following). 

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