In this competitive renewal, we propose to dissect and characterize the in vitro and in vivo role of the TRPM4 ion channel in lymphocytes and mast cells. Increases in intracellular Ca2+, calcium flux, is a fundamental mechanism of cellular signaling, controlling a plethora of cellular responses. In cells of the immune system, calcium flux is required for gene transcription programs, cytokine production, proliferation, apoptosis, cell motility and exocytosis. In fact, the central role of calcium as a vital signaling molecule in cells of the immune system is illustrated by clinical utility of potent immunosuppressive drugs such as cyclosporin and tacrolimus (FK506) that target the calcium influx-sensitive phosphatase calcineurin. But, what are the channels that control and regulate calcium influx in immune system cells? Indeed, elegant physiological techniques have established the existence of and provided characterization for several currents that are required for the multitude vital processes downstream of cellular activation. Remarkably, the channels and molecular mechanisms that regulate such currents have remained obscure. This is now changing with the cloning and subsequent characterization of novel ion channels. We have cloned and characterized three new members of the TRPM family of ion channels. Of these, TRPM4 displays unique properties that place it as a fundamental regulator of calcium influx in non-excitable cells, including cells of the immune system. TRPM4 is a Ca2+-activated nonselective (CAN) cation channel that mediates cell membrane depolarization, which decreases the driving force for calcium flux. Furthermore, it is activated through cell surface receptors. Our hypothesis is that TRPM4 regulates a key mechanism that controls the magnitude of calcium entry after antigen-receptor engagement. We propose to dissect TRMPM4's role in lymphocytes and mast cells.
In Aim 1, we will determine the function of two recently cloned variants of TRPM4. Furthermore, we will fully characterize its regulation and association domains.
In Aim 2, we will perform an in vitro structure/function analysis of TRPM4 in T cell and mast cell lines. In addition, we will assess its role in downstream signaling events using a dominant negative approach. Finally, in Aim3, we will investigate the in vivo role of TRPM4 in T cell and mast cell populations in conventional and conditional TRPM4-deficient mice. All together, the proposed experiments will provide a comprehensive characterization of a new TRPM family member and an assessment of a new mechanism that impacts calcium entry in lymphocytes and mast cells. ? ? ? ?
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