T lymphocyte activation and proliferation underlies human adaptive immune response. It is well established that calcium signaling plays a crucial role in this process and its deficiencies result in immunodeficiency. Calcium signaling in lymphocytes is governed by periodic intracellular calcium ion elevations that participate in specific gene transcription. Two main sources of calcium are intracellular organelles such as endoplasmic reticulum and calcium influx pathways in the plasma membrane. In the last decade several of the main players involved in calcium influx have been identified at the molecular level. TRPM7 is a protein with dual ion channel and serine/threonine kinase function and is highly expressed in human T lymphocytes and other hematopoietic cells. A hallmark of this channel is its sensitivity to Mg2+ and pH in the cytoplasm. Understanding the mechanism of this regulation is important for the understanding of TRPM7 involvement in T cell calcium signaling. The functional kinase can autophosphorylate TRPM7 but its function in modulating the ion channel activity of this protein is not well understood. The kinase domain has also been proposed to mediate Mg2+ inhibition of this channel, but it is not known if the kinase participates in pH regulation. We propose to investigate the role of TRPM7 kinase and phosphoinositides in regulation pH in immune cells.
In Aim 1 we will compare TRPM7 channel properties in leukocytes isolated from wild type and kinase-dead mutant mice and determine the role of kinase in pH regulation of the channel.
In Aim 2 we will test if function of leukocyte such as phagocytosis and proliferation are influenced by inactivation of TRPM7 kinase.
In Aim 3 we will test the hypothesis that depletion of phosphoinositides in the plasma membranes of T cells affects pH sensitivity and activation of native TRPM7 channels. At the conclusion of these studies we will have elucidated the factors governing the regulation of TRPM7 channels in white blood cells and developed new tools to study the functions of the its kinase domain.
Cells of the human immune system require alkalinization and prolonged elevations of intracellular calcium ion concentration for fighting infections. Genetic disturbances of calcium handling result in immunodeficiencies. Conversely, immunosuppression is widely used in organ transplantation and autoimmune conditions. The current proposal aims to elucidate the cellular mechanisms of pH regulation and calcium handling in immune cells, to provide new information on therapeutic targets for immunosuppression and immunomodulation.
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