Metal ions contribute to fundamental biological processes, including maintenance of protein architecture, determination of enzyme catalytic activity and signal transduction. Transport mechanisms for trace metal ions are not well characterized, and no ion channel with the ability to permeate significant amounts of these ions has yet been described. Recently, a superfamily of the putative calcium-permeable cation channel family TRPC (transient receptor potential channels) has been identified. Our data characterize a novel and widely expressed ion channel of this superfamily, LTRPC7, which has an intriguing selectivity profile for trace metal ions. LTRPC7 is regulated by intracellular levels of Mg.ATP and is non-redundantly essential for cell viability. The current proposal characterizes this novel ion channel using electrophysiological and molecular biology techniques.
Specific Aim 1 focuses on the biophysical characterization of recombinant and native LTRPC7. First, the channel?s selectivity profile for trace metal ions will be analyzed. Second, since LTRPC7 is dependent on nucleotide triphosphates, these findings will be extended to include other phospho-nucleotides. Third, the involvement of phosphorylation in channel function will be tested.
Specific Aim 2 concentrates on the functional and molecular analysis of LTPRC7 and introduces targeted point mutations to evaluate the structural requirements that determine the selectivity and modulation of LTRPC7.
Specific Aim 3 addresses the physiological significance of LTRPC7 currents. It is hypothesized that endogenous LTRPC7 contributes to calcium influx pathways normally attributed to the ICRAC conductance in immune cells. Therefore, experiments have been designed addressing the dissociation of these two currents. Finally, the role of enhanced LTRPC7 expression and metal ion entry in pro-apoptotic events and cell death is assessed. The studies proposed above are based on the unique ion channel LTRPC7. By virtue of this channel?s sensitivity to Mg ATP levels and unusual selectivity to divalent ions, LTRPC7 seems to link fundamental processes that adjust plasma membrane divalent cation fluxes according to the metabolic state of the cell. Disturbing this delicate balance may induce LTRPC7-dependent cell death. These features predestine LTRPC7 to be a prominent player in pathophysiological situations mediating the detrimental divalent ion entry in ischaemic events. Detailed understanding of LTRPC7 physiological function will open new and fascinating possibilities to influence cell survival.
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