Calcium entry is required for fundamental processes in non-excitable cells. In human T cells (Jurkats), calcium influx is involved in activation and in murine erythroleukemia cells (MELC) in cellular differentiation. The objectives of the proposed research are: 1) to clone and express calcium channels from non-excitable cells (MELC and Jurkats); 2) to functionally characterize the cloned channels; and 3) to determine the role of such channels in the control of proliferation and induced differentiation in non-excitable cells. Preliminary studies in our laboratory indicate that MELC and Jurkats express calcium channels which are distinct from, but homologous to, the cardiac dihydropyridine receptor (DHPR). Our evidence for this includes: 1) the isolation of partial cDNAs encoding putative calcium channels expressed in MELC and Jurkats; 2) sequence analysis of these cDNAs demonstrating several additions, deletions and insertions compared to the DHPR, in particular the lack of the IIIS1 putative transmembrane region; 3) Northern blot analysis indicating that the putative calcium channel mRNAs are expressed at significant levels in MELC and Jurkats. We propose: 1) to clone and express complete cDNAs encoding calcium channels from MELC and Jurkat; 2) to functionally characterize the cloned MELC and Jurkat calcium channels in terms of blockers, activators, voltage-sensitivity, and ionic basis of channel function using Xenopus oocytes injected with in vitro transcribed RNA; 3) to examine the effects of agents which modulate calcium channel function on induced differentiation in MELC and Jurkats respectively; 4) to transfect MELC and Jurkats with sense and antisense cDNAs to establish lines which over and under express the calcium channels; 5) to characterize calcium influx in these cell lines during MELC differentiation and T cell activation. The significance of these studies will be the characterization of the structure and function of calcium channels expressed in non-excitable cells. These calcium channels may be involved in fundamental cellular functions, including erythroid cell differentiation and T cell activation.
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