An investigation of the mechanism of inhibition of the Ca2+-ATPase by phospholamban

Abstract

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum has been reconstituted with peptides corresponding to the hydrophobic domain of phospholamban (PLB) with or without the three Cys residues replaced by Ala, and with PLB with the three Cys residues replaced by Ala [PLBcys-(1-52)]. Reconstitution with the hydrophobic domain of PLB[PLB(25-52)] was found to decrease the apparent affinity of the ATPase for Ca2+ with no effect on the maximal rate of ATP hydrolysis observed at saturating concentrations of Ca2+. Reconstitution with PLBCys-(1-52) decreased both the apparent affinity for Ca2+ and the maximal activity; the effect on maximal activity followed from a decrease in the rate of the Ca2+ transport step (E1PCa2-->E2P) as observed with the hydrophilic domain PLB(1-25). The concentration dependences of the effects of the hydrophobic domain and of the whole PLB molecule were very similar, suggesting that the hydrophilic domain made little contribution to the affinity of the ATPase for PLB. The effect of PLB on the ATPase was dependent on the molar ratio of phospholipid to ATPase, suggesting partition of the PLB between its binding site on the ATPase and the bulk lipid phase in the membrane. Neither PLB nor its hydrophobic domain affected the rates of phosphorylation or dephosphorylation of the ATPase. Despite their effects on the apparent affinity of the ATPase for Ca2+, neither PLB nor its hydrophobic domain had any effect on the true affinity of the ATPase for Ca2+, as measured from changes in the tryptophan fluorescence of the ATPase. The effects of PLB on the activity of the ATPase are the sum of the effects of its hydrophilic and hydrophobic domains.

Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase

“…A number of factors appear to be important including lipid to protein ratios and the exact PLB analogue that is used (34 -38). Previous work done on a PLB analogue similar to the one used here demonstrated a similar reduction in V max the one (35), and in the present work this result has been consistent across a number of separate assays. The important point here is that, in the presence of PLB, the K m is increased as expected, and this is shown more clearly when the data for the 500:20:1 membranes are normalized (Fig.…”

Polymorphic ventricular tachycardia and abnormal Ca2+handling in very-long-chain acyl-CoA dehydrogenase null mice

“…7D). Although several studies show that phospholamban asserts its inhibitory function by binding to SERCA in its monomeric form (2,24,25) there is some evidence that the pentameric form of phospholamban may directly interact with SERCA2a, causing a decrease in the affinity of the pump for Ca 2ϩ (45,52). However, the mechanism of this interaction is not fully understood.…”

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…. 3 E 2 P (Hughes et al, 1994(Hughes et al, , 1996 and 2) lowers its apparent affinity for Ca 2ϩ , without affecting the true chemical affinity, through interactions with its transmembrane domain (James et al, 1989;Sasaki et al, 1992a;Cantilina et al, 1993). Its phosphorylation causes its dissociation from SERCA (Tada, 1992) and increases SERCA apparent affinity for Ca 2ϩ by reducing the activation energy for a slow transition triggered by Ca 2ϩ binding in the Ca 2ϩ pumping cycle (Fig.…”