CALCIUM CHANNELS IN NONEXCITABLE CELLS

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The long range goal of this research project is to understand the molecular mechanisms associated with agonist-induced Ca2+ signaling in mammalian non-excitable cells. In a variety of cell types, stimulation of membrane receptors causes the release of Ca2+ from internal stores and the concomitant influx of Ca2+ from the extracellular space. Although the mechanisms responsible for inositol-1,4,5-trisphosphate(Ins(1,4,5)P3)-induced Ca2+ release from internal stores are well established, the molecular mechanisms associated with Ca2+ entry remain unknown. In many cells, Ca2+ influx is secondary to the depletion of the internal Ca2+ store, i.e. the so-called capacitative Ca2+ entry (CCE) model. Although the membrane current generated by depletion of the store has been recorded, our ability to understand the biochemical events leading to activation of CCE is hampered by our lack of knowledge concerning the molecular identity of the channels involved. A clue to their identity derives from studies of Drosophila phototransduction. Stimulation of Drosophila photoreceptor cells by light causes an increase in membrane conductance that requires phospholipase C (PLC) and reflects the activity of both the transient receptor potential channel (Trp) and the Trp-like channel (TrpL). It was originally thought that an Ins(1,4,5)P3-induced Ca2+ release activated Trp and TrpL via a mechanism analogous to CCE in mammalian non-excitable cells. More recent data however, suggests that this is not the case. In the present application, we consider an alternative model for the activation of the Trp family of ion channels that requires PLC, but is independent of the internal Ca+ stores. Specifically, we propose that it's not the release of Ins(1,4,5)P3 per se, but rather the hydrolysis of PIP2 that is responsible for activation of Trp. Using a combination of biochemical, molecular biological, and electrophysiological techniques, the specific aims of this proposal will test several hypotheses concerning the structure, function and regulation of the Trp channels. The specific aims are: 1) To determine the mechanism by which the Trp family of ion channels are regulated by receptor stimulation. 2) To determine the mechanism by which Trp channels are regulated by a rise in [Ca2+]i. 3) To determine the role of the highly conserved triple proline and acidic regions that exist NH2- and COOH-terminal to the PIP2 binding domain, respectively.