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

钙（Ca2+）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。介导内流启动Ca2+信号的膜通道已被广泛研究。然而，综合Ca2+稳态依赖于挤出途径，这是知之甚少。在Ca2+通量高的可兴奋细胞中，外排主要由K+依赖性Na+/Ca2+交换器（NCKX）家族成员介导。**** NCKX家族由slc24a1 - slc24a5 5个基因编码。我实验室的总体研究计划是了解每个NCKX亚型的不同生理作用。这项NSERC研究计划的长期目标是了解NCKX蛋白亚型如何被调节，以独特的方式在表达它们的细胞中整合Ca2+信号和稳态。本研究的短期目标是了解purinergic signaling and calmodulin （CaM）调控NCKX4亚型的机制。我们目前的假设是：1)嘌呤能刺激需要蛋白激酶C （PKC）和Ca2+-钙调素依赖性蛋白激酶II （CaMKII）直接磷酸化NCKX4；2) CaM与NCKX4结合刺激交换。我们将通过以下两个具体目标来解决这些假设：****1)确定NCKX4蛋白上负责刺激的磷酸化位点。我们最近证明NCKX4被嘌呤能激动剂激活，并通过抑制PKC或CaMKII来阻止。首先，通过确定所涉及的激酶同工型来确定磷酸化位点；第二，通过在体外检测纯化组分的磷酸化；第三，通过识别细胞中使用的磷酸化位点；第四，通过测试上述确定的磷酸化位点是否负责NCKX4刺激。****2)检测CaM对NCKX4活性的影响。我们最近的初步数据表明，CaM与NCKX4的细胞内环结合。我们现在将测试，首先，如果CaM与NCXK4共表达刺激交换活性；第二，CaM是否影响NCKX4对嘌呤能激活的反应；第三，失活CaM突变体的共表达是否可以阻止活化；第四，缺乏CaM结合的突变NCKX4对CaM或嘌呤能激活的刺激不敏感。****新奇和意义。这些实验将为调控NCKX4活性的机制提供新的信息。从这些研究中获得的见解将进一步加深我们对细胞信号事件后Ca2+稳态如何以不同方式整合和控制的理解

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.**