Structure-function analysis of PMCA-Neuroplastin/Basigin complexes, the native Ca2+ pump(s) of the plasma membrane

钙(Ca~(2+))信号控制着多种细胞过程和反应途径，从递质的释放和收缩到酶活性和/或细胞运动的调节：这些过程通过钙离子进入胞浆而被启动，并被位于肌浆/内质网(SERCA)或质膜(PMCA)中的各种钙离子转运体(最重要的是钙-ATPase或泵)的协同作用所关闭。我们最近发现，在任何类型的细胞中，PMCA都是由两个ATPase亚基(PMCAs1-4)和两个单跨膜蛋白-神经纤溶素或basigin组成的双分子复合体。后者与PMCA蛋白的共同组装对于复合物的稳定和运输以及它们的底物运输都是必需的。事实上，神经降解素或basigin的结合使钙离子的转运速度增加了两个数量级以上，从而在10毫秒内促进了钙的清除，而不是之前假设的几秒钟。这种显著增加的运输活动的结构和功能基础，以及两个辅助亚基在天然PMCA的细胞生理中的作用目前尚不清楚。这项建议旨在全面了解PMCA-神经活化素/basigin复合体的形成和运行的结构和功能基础。为此，我们将追求以下密切相关的目标和实验方法/步骤：(1)用冷冻-EM方法阐明PMCA_2-神经纤溶蛋白和PMCA_4-basigin复合体的三维结构，以及PMCA_2处于辅助亚基自由‘apo’状态下的三维结构；(2)对PMCA-神经纤溶素/basigin复合体中的钙转运进行结构-功能分析；(3)解开天然PMCA的相互作用组，重点是通过神经纤溶酶和basigin的Ig结构域相互作用。

Calcium (Ca2+) signaling controls a broad variety of cellular processes and reaction pathways ranging from transmitter release and contraction to regulation of enzymatic activities and/or cell motility: These processes are switched on by influx of Ca2+ into the cytosol, and they are switched off by the concerted action of various Ca2+ transporters, most importantly Ca2+-ATPases (or pumps) that are located in the sarco/endoplasmic reticulum (SERCA) or the plasma membrane (PMCA). We have recently identified PMCAs in any type of cell as bimolecular complexes assembled from two ATPase subunits (PMCAs1-4) and two single-span membrane proteins neuroplastin or basigin. Co-assembly of the latter with the PMCA proteins is obligatory for both stabilization and trafficking of the complexes, as well as for their substrate transport. In fact, binding of neuroplastin or basigin increases the rates of Ca2+-transport by more than two orders of magnitude, thus promoting Ca2+-clearing within 10s of milliseconds rather than seconds, as previously assumed. The structural and functional basis of this profoundly increased transport activity, as well as the role of the two auxiliary subunits for the cell-physiology of native PMCAs are currently unknown. This proposal aims at a comprehensive understanding of the structural and functional basis for the formation and operation of PMCA-neuroplastin/basigin complexes. For this purpose we will pursue the following closely interrelated aims and experimental approaches/steps: (1) Elucidate 3D-structures of PMCA2-neuroplastin and PMCA4-basigin complexes, and of PMCA2 in auxiliary subunit-free ‘apo’ state by cryo-EM, (2) Perform structure-function analyses of the Ca2+-transport in PMCA-neuroplastin/basigin complexes, (3) Unravel the interactome of native PMCAs with emphasis on interactions via the Ig-domains of neuroplastin and basigin.