Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum
Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum
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DOI：
10.1074/jbc.273.23.14238
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发表时间：
1998-06-05
影响因子：
4.8
通讯作者：
MacLennan, DH
中科院分区：
文献类型：
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作者：
Kimura, Y;Asahi, M;MacLennan, DH
在早期的一项研究中（Kimura， Y., Kurzydlowski， E., Tada， M., and MacLennan， D. H. (1997) J. Biol.），对形成磷蛋白（PLN）细胞质结构域Ib的氨基酸21-30进行丙氨酸扫描诱变，揭示了Asn(27), Gln（29）和Asn（30）的Ala突变可获得抑制功能。化学，272,15061-15064)，PLN跨膜结构域II突变体的功能获得与五聚体不稳定相关，导致提出PLN单体是活性抑制物种，PLN五聚体的解离是PLN抑制功能的一个决定因素，PLN心脏sarco（内do）质Ca2+- atp酶异构体（SERCA2a）复合物的解离是第二个决定因素。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都含有正常的五聚体与单体的比例，因此功能的获得一定是由五聚体结构不稳定以外的机制引起的。单个双突变体N30A/I40A提供了结构域Ib和结构域II突变体通过不同位点和不同机制起作用的证据，其中每个单突变体的增强抑制功能是加性的。在对双突变体N27A/N34A的研究中获得了PLN/SERCA2a异源二聚体稳定性改变的证据，该双突变体通过结合功能域Ib突变和功能域II突变获得抑制功能，这些结果支持了PLN抑制SERCA2a的建议，首先是PLN的解聚，其次是单体PLN与SERCA2a之间形成抑制相互作用。
Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib in phospholamban (PLN), revealed that mutation to Ala of Asn(27), Gln(29), and Asn(30) results ins gain of inhibitory function, In an earlier study (Kimura, Y., Kurzydlowski, E., 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 mononomeric 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.