The effects of mutation on the regulatory properties of phospholamban in co-reconstituted membranes.
Reconstitution into proteoliposomes is a powerful method for studying calcium transport in a chemically pure membrane environment. By use of this approach, we have studied the regulation of Ca(2+)-ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Co-reconstitution of PLB and Ca(2+)-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca(2+)-ATPase (K(Ca)) and unexpected effects of PLB on maximal activity (V(max)). Wild-type PLB, six loss-of-function mutants (L7A, R9E, I12A, N34A, I38A, L42A), and three gain-of-function mutants (N27A, L37A, and I40A) were evaluated for their effects on K(Ca) and V(max). With the loss-of-function mutants, their ability to shift K(Ca) correlated with their ability to increase V(max). A total loss-of-function mutant, N34A, had no effect on K(Ca) of the calcium pump and produced only a marginal increase in V(max). A near-wild-type mutant, I12A, significantly altered both K(Ca) and V(max) of the calcium pump. With the gain-of-function mutants, their ability to shift K(Ca) did not correlate with their ability to increase V(max). The "super-shifting" mutants N27A, L37A, and I40A produced a large shift in K(Ca) of the calcium pump; however, L37A decreased V(max), while N27A and I40A increased V(max). For wild-type PLB, phosphorylation completely reversed the effect on K(Ca), but had no effect on V(max). We conclude that PLB increases V(max) of Ca(2+)-ATPase, and that the magnitude of this effect is sensitive to mutation. The mutation sensitivity of PLB Asn(34) and Leu(37) identifies a region of the protein that is responsible for this regulatory property.
Trieber
Catharine A
CA
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada.
Douglas
Jennifer L
JL
Afara
Michael
M
Young
Howard S
HS
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D004285:Dogs; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D008081:Liposomes; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin / Q000737:chemistry; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D008081:Liposomes; D008565:Membrane Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin; D000099211:Phospholamban; C020485:proteoliposomes