Differences in the Reaction Cycle among PMCA Isoforms

描述（由申请人提供）： 质膜钙泵（PMCA）是将钙排出质膜，从而保持和恢复低的胞浆钙浓度的重要机制之一。在哺乳动物中，四个基因编码这种泵的不同亚型，这种变异性通过选择性剪接进一步增强。PMCA总共有20多种变体。这些PMCA亚型在不同组织中差异表达，它们参与细胞类型特异性钙调节。与许多其他阳离子转运ATP酶一样，PMCA的反应循环包括磷酸化中间体的合成和水解。然而，很少有人知道不同的PMCAs之间的反应周期的差异。此外，在跨膜的阳离子转运期间，可以区分中间步骤，其中转运离子不能到达膜的任一侧。这种现象被称为离子吸留，到目前为止还没有在PMCA中表征。为了表征不同PMCAs之间反应周期的差异，包括钙包埋的分析，有必要将PMCAs分子生物学和反应周期动力学分析方面的专业知识联合收割机结合起来。Emanuel E.明尼苏达州罗切斯特的马约诊所的Strehler和阿根廷布宜诺斯艾利斯大学的Juan P. Rossi博士的实验室为研究这一重要课题提供了独特的机会。这项研究将作为NIH资助# RO1GM28835的延伸。

DESCRIPTION (provided by applicant): The plasma membrane calcium pump (PMCA) is one of the essential mechanisms to extrude calcium across the plasma membrane, thereby keeping and restoring a low cytosolic calcium concentration. In mammals, four genes encode separate isoforms of this pump, and this variability is further enhanced by alternative splicing. In total, there are more than 20 variants of the PMCA. These PMCA isoforms are differentially expressed in various tissues where they participate in cell type-specific calcium regulation. As is the case in many other cation-transporting ATPases, the reaction cycle of the PMCA includes the synthesis and hydrolysis of a phosphorylated intermediate. However, little is known about differences in the reaction cycle among different PMCAs. Also, during cation transport across the membrane, it is possible to distinguish an intermediate step in which the transporting ion is not accessible to either side of the membrane. This phenomenon is known as ion occlusion, and so far has not been characterized in the PMCA. To characterize the differences in the reaction cycle among different PMCAs, including the analysis of calcium occlusion, it is necessary to combine expertise both in the molecular biology of PMCAs and in the kinetic analysis of their reaction cycle. The proposed collaboration between the laboratory of Dr. Emanuel E. Strehler at the Mayo Clinic in Rochester, MN, and the laboratory of Dr. Juan P. Rossi at the University of Buenos Aires in Argentina provides a unique opportunity to study this important topic. This research will be done as an extension of NIH grant # RO1GM28835.