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

钙（Ca 2+）信号调节从受精到分化的生理功能到细胞死亡的广泛的细胞过程。启动Ca 2+信号的膜通道已被广泛研究。然而，整合的Ca 2+稳态依赖于挤出途径，关于这一点知之甚少。在极化细胞中，如神经元和上皮细胞，其中Ca 2+通量高，流出主要由K+依赖性Na+/Ca 2+交换剂（NCKX）家族的成员介导。 NCKX家族包括5个基因slc 24 a1-slc 24 a5，编码NCKX 1至NCKX 5蛋白。我实验室的总体研究关注的是了解每个NCKX亚型的不同生理作用。这个NSERC研究计划的长期目标是了解每个NCKX蛋白如何以不同的方式调节整合Ca 2+信号和稳态。该提案的短期目标是了解NCKX 4亚型的表达，亚细胞定位和活性的调节机制。我们目前的假设是：1）棕榈酰化调节NCKX 4活性和定位;和2）通过血清和糖皮质激素调节的激酶-1（SGK 1）的磷酸化调节NCKX 4活性和定位。我们将解决这些假设与以下两个具体目标：1）棕榈酰化是已知的膜蛋白定位的一个重要决定因素。初步数据显示NCKX 4通过棕榈酰化修饰。为了理解这一发现的意义，我们将使用诱变和质谱法来鉴定NCKX 4上棕榈酰化的位点。邻位连接试验将用于确定棕榈酰化NCKX 4在细胞内的位置。棕榈酰化和脱棕榈酰化的抑制剂以及突变体构建体将用于检查棕榈酰化是否影响NCKX 4的运输和表面递送。最后，类似的方法将用于解决棕榈酰化对NCKX 4活性的功能意义。2)SGK 1是各种组织中离子转运蛋白的已知调节剂。NCKX 4在也受CaM结合和PKC磷酸化调节的位点附近具有典型的SGK 1磷酸化位点。野生型或突变的NCKX 4与SGK 1的组成型活性或激酶死亡变体的共表达将用于检查NCKX 4上的磷酸化位点及其对转运蛋白的位置和活性的影响。将研究SGK 1磷酸化和NCKX 4调节的其他模式之间的相互作用。这些研究将提供有关调节NCKX 4表达、定位和活性的新机制的重要信息。从这项工作的见解将进一步我们的理解，如何个别亚型的K+依赖的Na+/Ca 2+交换家庭的功能，以促进综合钙稳态的基础上的细胞信号事件的独特的生物学后果。

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.