Effects of phospholamban transmembrane mutants on the calcium affinity, maximal activity, and cooperativity of the sarcoplasmic reticulum calcium pump.
Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to interactions between their respective transmembrane domains. In spite of numerous mutagenesis and kinetic studies, we still do not have a clear mechanistic picture of how PLB influences the calcium transport cycle of SERCA. Herein, we have created alanine mutants for each residue in the transmembrane domain of PLB, we have co-reconstituted these mutants with SERCA into proteoliposomes, and we have performed kinetic simulations of the calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a variable effect on the calcium affinity, maximal activity, and cooperativity of SERCA, such that a range of values was observed. Kinetic simulations using a well-established reaction scheme for SERCA then allowed us to correlate the effects on SERCA activity with changes in the reaction scheme rate constants. Only three steps in the reaction scheme were affected by the presence of PLB, namely, binding of the first calcium ion, a subsequent conformational change in SERCA, and binding of the second calcium ion. The ability of wild-type and mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated with a decreased rate of binding of the second calcium ion. In addition, the ability of wild-type and mutant forms of PLB to alter the maximal activity of SERCA correlated with a change in the forward rate constant for the slow conformational change in SERCA following binding of the first calcium ion.
Trieber
Catharine A
CA
Department of Biochemistry and National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Afara
Michael
M
Young
Howard S
HS
D000409:Alanine / Q000235:genetics; D000818:Animals; D001693:Biological Transport, Active / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000502:physiology*; D000099211:Phospholamban
D000409:Alanine; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C020485:proteoliposomes