Kimura Y, Asahi M, Kurzydlowski K, Tada M, MacLennan D H
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
J Biol Chem. 1998 Jun 5;273(23):14238-41. doi: 10.1074/jbc.273.23.14238.
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.
对构成受磷蛋白（PLN）胞质结构域Ib的21 - 30位氨基酸进行丙氨酸扫描诱变，结果显示，将Asn27、Gln29和Asn30突变为丙氨酸会导致抑制功能增强。在早期研究中（Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061 - 15064），PLN跨膜结构域II突变体的功能增强与五聚体不稳定相关，这导致有人提出PLN单体是具有活性的抑制性物种，PLN五聚体的解离是PLN抑制功能的一个决定因素，并且PLN与心肌肌浆（内质）网Ca²⁺ - ATP酶同工型（SERCA2a）复合物的解离是另一个决定因素。由于每个新的结构域Ib突变体在SDS - 聚丙烯酰胺凝胶电泳中五聚体与单体的比例正常，功能增强必定是由五聚体结构不稳定以外的机制导致的。一个单体双突变体N30A/I40A提供了证据，表明结构域Ib和结构域II突变体通过不同位点和不同机制起作用，其中每个单突变体增强的抑制功能是相加的。在对双突变体N27A/N34A的研究中获得了PLN/SERCA2a异二聚体稳定性改变的证据，在该双突变体中，通过将功能增强的结构域Ib突变与功能丧失的结构域II突变相结合，恢复了抑制功能。这些结果支持了以下提议：PLN对SERCA2a的抑制作用首先涉及PLN的解聚，其次涉及单体PLN与SERCA2a之间形成抑制性相互作用。
