Investigating the molecular mechanism of phospholamban regulation of the Ca²-pump of cardiac sarcoplasmic reticulum

Abstract

The Ca2+ pump or Ca2+-ATPase of cardiac sarcoplasmic reticulum, SERCA2a, is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the apparent Ca2+ affinity of the enzyme. We propose that PLB decreases Ca2+ affinity by stabilizing the Ca2+-free, E2·ATP state of the enzyme, thus blocking the transition to E1, the high Ca2+ affinity state required for Ca2+ binding and ATP hydrolysis. The purpose of this dissertation research is to critically evaluate this idea using series of cross-linkable PLB mutants of increasing inhibitory strength (N30C-PLB < PLB3 < PLB4). Three hypotheses were tested; each specifically designed to address a fundamental point in the mechanism of PLB action. Hypothesis 1: SERCA2a with PLB bound is catalytically inactive. The catalytic activity of SERCA2a irreversibly cross-linked to PLB (PLB/SER) was assessed. Ca2+-ATPase activity, and formation of the phosphorylated intermediates were all completely inhibited. Thus, PLB/SER is entirely catalytically inactive. Hypothesis 2: PLB decreases the Ca2+ affinity of SERCA2a by competing with Ca2+ for binding to SERCA2a. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca2+-ATPase activity and phosphoenzyme formation were measured, and correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca2+ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca2+ for binding to SERCA2a. This was confirmed with the Ca2+ pump mutant, D351A, which is catalytically inactive but retains strong Ca2+ binding. Increasingly higher Ca2+ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB competes with Ca2+ for binding to the Ca2+ pump. Hypothesis 3: PLB binds exclusively to the Ca2+-free E2 state with bound nucleotide (E2·ATP). Thapsigargin, vanadate, and nucleotide effects on PLB cross-linking to SERCA2a were determined. All three PLB mutants bound preferentially to E2 state with bound nucleotide (E2·ATP), and not at all to the thapsigargin or vanadate bound states. We conclude that PLB inhibits SERCA2a activity by stabilizing a unique E2·ATP conformation that cannot bind Ca2+.