Regulation of K-dependent Na/Ca-exchanger subtype 4, NCKX4

钙信号调节从受精、分化的生理功能到细胞死亡的广泛的细胞过程。启动钙信号的膜通道已被广泛研究。然而，完整的钙稳态依赖于挤出途径，而这一点我们知道的要少得多。在极化细胞中，如神经元和上皮细胞，钙离子通量高，外流主要由钾依赖的钠/钙交换器(NCKX)家族成员介导。NCKX家族由5个基因组成，分别为slc24a1-SLC24A5，编码NCKX1至NCKX5蛋白。我的实验室的总体研究重点是了解每种NCKX亚型的不同生理作用。这个NSERC研究项目的长期目标是了解每个NCKX蛋白是如何以不同的方式整合钙信号和动态平衡的。这项建议的短期目标是了解调控NCKX4亚型表达、亚细胞定位和活性的机制。我们目前的假设是：1)棕榈酰化调节NCKX4的活性和定位；2)血清和糖皮质激素调节的激酶-1(SGK1)的磷酸化调节NCKX4的活性和定位。我们将以以下两个具体目标来解决这些假说：1)棕榈酰化是膜蛋白定位的一个重要决定因素。初步数据显示NCKX4是通过棕榈酰化修饰的。为了了解这一发现的意义，我们将使用突变和质谱仪来鉴定NCKX4上的棕榈酰化位点。邻近连接试验将被用来确定棕榈酰化NCKX4在细胞内的位置。棕榈酰化和脱氨酰化的抑制剂以及突变结构将被用来检测棕榈酰化是否影响NCKX4的运输和表面递送。最后，类似的方法将被用来解决棕榈酰化对NCKX4活性的功能意义。2)SGK1是已知的多种组织离子转运蛋白的调节者。NCKX4在其附近有一个典型的SGK1磷酸化位点，该位点也受CaM结合和PKC磷酸化的调节。野生型或突变的NCKX4与SGK1的结构活性或激酶死亡变体共表达将被用来检测NCKX4上的磷酸化位置及其对转运蛋白位置和活性的影响。我们将研究SGK1磷酸化与其他NCKX4调节方式之间的相互作用。这些研究将为调控NCKX4的表达、定位和活性的新机制提供重要信息。这项工作将进一步加深我们对K依赖的Na/Ca~(2+)交换家族的各个亚型如何作用于细胞信号事件的不同生物学后果下的整合的Ca~(2+)稳态的理解。

Calcium (Ca2+) signals regulate a wide range of cellular processes from fertilization through differentiated physiological functions to cell death. The membrane channels that initiate Ca2+ signals have been studied extensively. Integrated Ca2+ homeostasis, however, depends on extrusion pathways, about which much less is known. In polarized cells, such as neurons and epithelia, where Ca2+ fluxes are high, efflux is predominantly mediated by members of the family of K+-dependent Na+/Ca2+-exchangers (NCKX). The NCKX family comprises five genes, slc24a1 - slc24a5, encoding the NCKX1 to NCKX5 proteins. The overall research concern of my laboratory is to understand the distinct physiological roles for each NCKX isoform. The long-term objective for this NSERC research program is to understand how each NCKX protein is regulated to integrate Ca2+ signals and homeostasis in distinct ways. The short-term goal of this proposal is to understand the mechanisms that regulate expression, subcellular localization, and activity of the NCKX4 isoform. Our current hypotheses are: 1) palmitoylation modulates NCKX4 activity and localization; and 2) phosphorylation by serum- and glucocorticoid-regulated kinase-1 (SGK1) regulates NCKX4 activity and localization. We will address these hypotheses with the following two specific aims: 1) Palmitoylation is known to be an important determinant of membrane protein localization. Preliminary data reveal that NCKX4 is modified by palmitoylation. To understand the significance of this finding, we will use mutagenesis and mass spectrometry to identify the site of palmitoylation on NCKX4. The proximity ligation assay will be used to determine where within the cell palmitoylated NCKX4 resides. Inhibitors of palmitoylation and depalmitoylation, as well as mutant constructs, will be used to examine if palmitoylation influences trafficking and surface delivery of NCKX4. Finally, similar approaches will be used to address the functional significance of palmitoylation on NCKX4 activity. 2) SGK1 is a known modulator of ion transporters in various tissues. NCKX4 has a canonical SGK1 phosphorylation site in the vicinity of sites also modulated by CaM binding and PKC phosphorylation. Co-expression of wild type or mutated NCKX4 with either constitutively active or kinase dead variants of SGK1 will be used to examine the site of phosphorylation on NCKX4 and its influence on location and activity of the transporter. The interaction between SGK1 phosphorylation and other modes of NCKX4 regulation will be investigated. These studies will provide important information regarding novel mechanisms that regulate expression, location, and activity of NCKX4. Insights from this work will further our understanding of how individual isoforms of the K+-dependent Na+/Ca2+-exchanger family function to contribute to integrated Ca2+ homeostasis underlying the distinct biological consequence of cell signaling events.