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 a 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. Specifically, we are intested in the roles of Stim1, which acts as a sensor for endoplasmic reticulum calcium, and Orai1, which is a subunit of the store-operated channels. 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 also interested in other calcium-permeable channels activated as a result of surface receptor activation, specifically the phospholipase C-regulated TRPC channels. There are 7 members of the TRPC family of ion channels identified in mammalian cells. In humans, there are 6, as TRPC2 is a pseudogene. These fall into three categories based on sequence similarity: TRPC1, TRPC3/6/7 and TRPC4/5. We are expressing these channel genes in cell types in which they are normally expressed and in cell types which do not express these channel proteins. We have found that one of these channels, TRPC3, can be regulated in two ways depending on its expression level: at low levels of expression it behaves as a capacitative calcium entry channel, while at higher levels of expression it behaves as a second messenger-regulated channel, responding to increases in diacylglycerols subsequent to activation of phospholipase C. In addition, we have found that TRPC7 can be regulated both by store depletion and by a phospholipase C-dependent mechanism in the same cells. We intend to examine the effects of interfering with, or deletion of these proteins by transfection of cells siRNAs, and cDNAs encoding for potentially dominant negative peptides. We have succeeded in knocking out a specific TRPC gene (TRPC7) 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.
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