Integration of Calcium Signaling: Regulation of K-dependent Na/Ca-exchangers

钙信号传导整合：K 依赖性 Na/Ca 交换器的调节

• 批准号: RGPIN-2015-05293
• 负责人: Lytton, Jonathan
• 金额: $ 2.19万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683495
• 关键词: Integration; Calcium; Signaling; Regulation; dependent

Calcium (Ca2+) signals regulate a wide range of cellular processes including fertilization, muscle contraction, neurotransmitter release, hormone secretion, and even cell death. The membrane channels that mediate influx to initiate Ca2+ signals have been studied extensively. Integrated Ca2+ homeostasis, however, depends on extrusion pathways, about which much less is known. In excitable cells, where Ca2+ fluxes are high, efflux is predominantly mediated by members of the family of K+-dependent Na+/Ca2+-exchangers (NCKX).****The NCKX family is encoded by five genes, slc24a1 - slc24a5. The overall research program in my laboratory is concerned with understanding the distinct physiological roles for each NCKX isoform. The long-term objectives for this NSERC research program are to understand how NCKX protein isoforms are regulated to integrate Ca2+ signals and homeostasis in unique ways in the cells where they are expressed. The short-term goal of this proposal is to understand the mechanism for regulation of the NCKX4 isoform by purinergic signaling and by calmodulin (CaM). Our current hypotheses are: 1) purinergic stimulation requires direct phosphorylation of NCKX4 by both protein kinase C (PKC) and Ca2+-calmodulin-dependent protein kinase II (CaMKII); and 2) CaM binding to NCKX4 stimulates the exchanger. We will address these hypotheses with the following two specific aims:****1) To identify the phosphorylation sites on the NCKX4 protein responsible for stimulation. We recently demonstrated that NCKX4 is activated by purinergic agonists, and prevented by inhibition of either PKC or CaMKII. Phosphorylation sites will be identified, first, by defining the kinase isoforms involved; second, by examining phosphorylation of purified components in vitro; third by identifying the phosphorylation sites used in cells; and fourth, by testing if those phosphorylation sites identified above are responsible for NCKX4 stimulation.****2) To examine the influence of CaM on NCKX4 activity. Our recent preliminary data demonstrate that CaM binds to the intracellular loop of NCKX4. We will now test, first, if co-expressing CaM with NCXK4 stimulates exchange activity; second, if CaM influences the NCKX4 response to purinergic activation; third, if co-expression of inactive CaM mutants can prevent activation; and, fourth, if mutated NCKX4 that lacks CaM binding is insensitive to stimulation by either CaM or purinergic activation.****Novelty and significance. These experiments will provide novel information about mechanisms that regulate the activity of NCKX4. The insight gained from these studies will further our understanding of how Ca2+ homeostasis is integrated and controlled in distinct ways following cell signaling events.**

钙（Ca 2+）信号调节广泛的细胞过程，包括受精，肌肉收缩，神经递质释放，激素分泌，甚至细胞死亡。介导内流以启动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Ca 2+通量高的可兴奋细胞中，外流主要由K+依赖性Na+/Ca 2+交换剂（NCKX）家族成员介导。**** NCKX家族由5个基因slc 24 a1-slc 24 a5编码。在我的实验室的整体研究计划是关注了解不同的生理作用，为每个NCKX亚型。该NSERC研究计划的长期目标是了解NCKX蛋白亚型是如何调节的，以独特的方式在表达它们的细胞中整合Ca 2+信号和稳态。该提案的短期目标是了解嘌呤能信号和钙调蛋白（CaM）调节NCKX 4亚型的机制。我们目前的假设是：1）嘌呤能刺激需要通过蛋白激酶C（PKC）和Ca 2 +-钙调蛋白依赖性蛋白激酶II（CaMKII）直接磷酸化NCKX 4;和2）CaM与NCKX 4结合刺激交换剂。我们将通过以下两个具体目标来解决这些假设：*1）鉴定NCKX 4蛋白上负责刺激的磷酸化位点。我们最近证明，NCKX 4被嘌呤能激动剂激活，并通过抑制PKC或CaMKII来阻止。磷酸化位点将首先通过定义所涉及的激酶同种型来鉴定;第二，通过检查体外纯化组分的磷酸化;第三，通过鉴定细胞中使用的磷酸化位点;第四，通过测试上述鉴定的磷酸化位点是否负责NCKX 4刺激。2)检测CaM对NCKX 4活性的影响。我们最近的初步数据表明，钙调素结合到NCKX 4的细胞内环。我们现在将测试，第一，如果共表达CaM与NCXK 4刺激交换活性;第二，如果CaM影响NCKX 4对嘌呤能激活的反应;第三，如果共表达失活的CaM突变体可以防止激活;和，第四，如果突变的NCKX 4缺乏CaM结合是不敏感的刺激CaM或嘌呤能激活。新奇和意义。这些实验将提供有关调节NCKX 4活性的机制的新信息。从这些研究中获得的见解将进一步加深我们对细胞信号传导事件后Ca 2+稳态如何以不同方式整合和控制的理解。