Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):858-63. doi: 10.1073/pnas.1013642108. Epub 2010 Dec 27.
P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn(2+) from the cytosol into the Golgi. We developed an intra-Golgi Mn(2+) sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn(2+) pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn(2+) transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn(2+) transport by expression of SPCA1-Q747A increased cell viability upon Mn(2+) exposure, supporting the therapeutic potential of increased Mn(2+) uptake by the Golgi in the management of Mn(2+)-induced neurotoxicity.
P 型 ATP 酶转运多种离子，调节多种细胞过程，并与许多人类疾病有关。然而，这些普遍存在的蛋白质增加离子转运的机制尚不清楚。SPCA1 是一种 P 型泵，可将 Mn(2+)从细胞质转运到高尔基体。我们开发了一种高尔基体内部的 Mn(2+)传感器，并根据其预测结构，对 SPCA1 进行了突变筛选，这些突变可能会增加其 Mn(2+)泵浦活性。值得注意的是，一个预测会增加其离子渗透腔大小的点突变（Q747A）增强了传感器的响应，而恢复腔原始大小的补偿性突变则消除了这种效应。体内和体外 Mn(2+)转运实验证实了 SPCA1-Q747A 的高活性。此外，通过表达 SPCA1-Q747A 增加高尔基体 Mn(2+)转运，可增加 Mn(2+)暴露时的细胞存活率，这支持了高尔基体摄取更多 Mn(2+)在管理 Mn(2+)-诱导的神经毒性方面的治疗潜力。
