Increases in the cytosolic concentration of Ca2+ ions represent a major component of the cellular signals generated by a variety of hormones and neurotransmitters acting on receptors coupled to phospholipase C (PLC). A general feature of these Ca2+ signals is an enhanced entry of extracellular Ca2+. The focus of our research is to understand the nature of this Ca2+ entry, and its role(s) in the overall intracellular signaling mechanisms. Until recently, such entry was thought to exclusively involve so-called store-operated Ca2+ channels (or SOC channels) whose gating was dependent on the depletion of intracellular Ca2+ stores. However, in recent years we, and others, have identified a novel arachidonic acid-dependent pathway for agonist-activated Ca2+ entry that is entirely distinct from the store-operated pathway. We have now characterized the channels responsible (ARC channels, for Arachidonate-Regulated Ca2+ channels) at the whole-cell level, (and, more recently, at the single channels level), and have identified their presence in a variety of different cell types. Importantly, our studies over the past three (3) years have demonstrated that it is these ARC channels, and not the SOC channels, that provide the predominant route for the enhanced entry of Ca2+ seen at physiologically relevant levels of stimulation. Understanding the regulation and function of these novel channels is, therefore, of key importance and forms the basis of the current proposal.
In Aim 1, we examine the mechanism by which arachidonic acid activates the ARC channels at the single channel level.
Aim 2 addresses the mechanisms underlying the demonstrated regulation of the channels by phosphorylation and the kinases responsible.
Aim 3 explores the unique roles that the ARC channels play in overall cellular Ca2+ signaling, both by modulating the signals themselves, as well as by a direct influence on specific effectors by the Ca2+ entering the ARC channels. Although the molecular identity of this novel channel remains unknown, it is clear that the characterization of its key features - its properties, its regulation, and its roles - is of critical significance to our understanding of PLC dependent Ca2+ signaling in cells.
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