Differences in the Reaction Cycle among PMCA Isoforms

描述(由申请人提供)： 质膜钙泵(PMCA)是维持和恢复细胞内低钙浓度的重要机制之一。在哺乳动物中，有四个基因编码这种泵的不同亚型，这种可变性通过选择性剪接进一步增强。总体而言，PMCA有20多种变体。这些PMCA亚型在参与细胞类型特异性钙调节的不同组织中有不同的表达。与许多其他阳离子转运ATPase一样，PMCA的反应周期包括磷酸化中间体的合成和水解。然而，人们对不同PMCA之间反应周期的差异知之甚少。此外，在阳离子跨膜传输期间，可以区分中间步骤，在该中间步骤中，传输离子不能到达膜的任何一侧。这种现象被称为离子遮挡，到目前为止还没有在PMCA中被表征。为了表征不同PMCA之间反应周期的差异，包括钙封闭分析，有必要结合PMCA的分子生物学和其反应周期的动力学分析方面的专业知识。明尼苏达州罗切斯特梅奥诊所的伊曼纽尔·E·斯特雷勒博士的实验室与阿根廷布宜诺斯艾利斯大学胡安·P·罗西博士的实验室之间拟议的合作为研究这一重要课题提供了一个独特的机会。这项研究将作为美国国立卫生研究院#RO1GM28835号拨款的延伸。

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.