Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum.
Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib in phospholamban (PLN), revealed that mutation to Ala of Asn27, Gln29, and Asn30 results in gain of inhibitory function. In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M. , and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), gain of function in PLN transmembrane domain II mutants was correlated with pentamer destabilization, leading to proposals that the PLN monomer is the active inhibitory species, that dissociation of the PLN pentamer is one determinant of PLN inhibitory function and that dissociation of the PLN.cardiac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a second determinant. Because each of the new domain Ib mutants contained a normal ratio of pentamer to monomer in SDS-polyacrylamide gel electrophoresis, gain of function must have resulted from mechanisms other than destabilization of pentameric structure. Evidence that domain Ib and domain II mutants act through different sites and different mechanisms was provided by a monomeric double mutant, N30A/I40A, in which the enhanced inhibitory function of each single mutant was additive. Evidence for an alteration in stability of the PLN/SERCA2a heterodimer was obtained in a study of double mutant N27A/N34A in which inhibitory function was regained by combining a gain of function, domain Ib mutation with a loss of function domain II mutation. These results support the proposal that PLN inhibition of SERCA2a involves, first, depolymerization of PLN and, second, the formation of inhibitory interactions between monomeric PLN and SERCA2a.
Kimura
Y
Y
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Asahi
M
M
Kurzydlowski
K
K
Tada
M
M
MacLennan
D H
DH
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000196:embryology; D008565:Membrane Proteins / Q000737:chemistry; D016296:Mutagenesis / Q000235:genetics; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection / Q000235:genetics; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D000099211:Phospholamban