The broad aim of this project is to understand at the cellular and molecular level the mechanisms involved in regulation of calcium entry across the plasma membrane in non-excitable cells. This is a process that is ubiquitous to non-excitable cells, occurs in many excitable cell types and which is known to play an important role in the control of a large variety of cell processes, including secretion, contraction, motility, growth, differentiation and apoptosis. This signaling process is vulnerable to environmental intervention by chemical and physical agents (such as EMF). Capacitative calcium entry is believed to involve an unknown signal generated in the endoplasmic reticulum when Ca2+ is released by IP3 and which then interacts with the plasma membrane to activate calcium channels. The nature of this signal and the nature of the channels involved are prime areas of investigation. We utilize as a model for studying this process the actions of a tumor-promoting plant product, thapsigargin. Thapsigargin acts by inhibiting endoplasmic reticulum Ca2+ pumps, thereby depleting intracellular Ca2+ stores in a passive manner. We are using molecular techniques of reverse transcriptase - PCR, cloning and heterologous expression to isolate and study candidate capacitative calcium entry channel molecules. To date, as many a 6 members of the TRP family of ion channels have been identified in mammalian cells. In humans, there are 5, as TRP2 is a pseudogene. These fall into three categories based on sequence similarity: TRP1, TRP3/6 and TRP4/5. We are expressing TRP1, TRP3 and TRP4 in cell types in which they are normally expressed and in cell types which do not express these channel proteins. In addition, we intend to examine the effects of interfering with, or deletion of this proteins by transfection of cells with cDNAs coding for antisense RNA, and for potentially dominant negative peptides. We plan to knock out specific TRP genes from chicken DT40 pre-B lymphocytes. We are hopeful that by achieving a better understanding of the molecular and cellular modes of regulation of this important signaling pathway, we can learn how capacitative calcium entry is altered by environmental factors and by disease states. - calcium, signal transduction, electrophysiology, calcium channels, inositol phosphates, epithelial cells, lymphocytes, fibroblasts, calcium indicators
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