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

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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.