Little is known of calcium channels underlying flux in lymphocytes and other non-excitable cells. I have recently cloned a novel ion channel, designated LTRPC7, that is expressed in many types of non-excitable cells including all immune system tissues. This channel is a member of the LTRPC TRP sub-family and is unique in that it contains a catalytic kinase domain homology to eEF-2K at its C-terminus. Preliminary data indicate that this protein is bi-functional: it is a cation channel that is permeable to calcium and magnesium, and it has kinase activity. We have observed that the ion permeability of LTRPC7 is sensitive to and suppressed by Mg-ATP. These data suggest that the kinase activity and/or changes in conformation of the kinase domain upon phosphorylation could be critical to the gating and/or modulation of this channel. Experiments are proposed to fully characterize the enzymatic potential of this domain (Aim 1). In addition, experiments are proposed to explore the regions of LTRPC7 that mediate and influence the Mg- ATP suppression effect (Aim 2). Finally, preliminary functional characterization of LTRPC7 demonstrates that over-expression of it in HEK293 cells is toxic and/or induces growth arrest. In addition, production of a double allele LTRPC7 knockout cell line in the DT-40 B-cell system results in lethality. These results suggest that LTRPC7 function is linked to basic cellular processes required for cell survival. Experiments to address the unique structural features of LTRPC7 that could account for these functional effects and assessment of its ability to affect intracellular calcium are also proposed (Aim 3).
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