The research proposed here aims at characterizing the mechanism of store-operated calcium entry (SOCE) in T cells by (i) identifying the genetic defect causing human Severe Combined Immunodeficiency (SCID) disease associated with a defect in SOCE and (ii) generating an animal model for abrogated SOCE by conditionally targeting the gene locus for stromal interaction proteins (STIM) 1 and 2, respectively. We have identified a novel form of SCID due to a severe defect in store-operated Ca2+ entry and undetectable CRAC channel currents. Using a genetic mapping approach we have determined a region on chromosome 12, which is linked to the SCID disease. In this region we identified a mutation in a novel transmembrane protein, which is very likely to be a component of the CRAC channel complex.
Aim 1 : We will validate the relevance of this mutation for the defect in SOCE and CRAC channel function in the SCID patients by both genetic and functional analysis. Since this is a novel protein, we will perform structure-function analysis of the protein with respect to its ability to promote SOCE and CRAC currents, evaluate its interaction with other proteins, especially STIM1 and STIM2, and establish an in vivo model of the role of this protein in T cell development and T cell-mediated immune responses by conditional gene targeting.
Aim 2 : We will conditionally target the genes of STIM1 and STIM2, respectively, in mouse T cells using the Cre-loxP system to establish an animal model for the role of store-operated Ca2+ entry in vivo. Exons 2 and 3 of STIM1 and STIM2, respectively, encoding for an EF hand motif will be flanked by loxP sites to allow for T cell specific deletion. We expect that T cells from STIM1-/- and STIM2-/- mice will be compromised in SOCE. We will evaluate the role of SOCE for T cell development, T cell homeostasis, CD4+ and CD8+ T cell-dependent immune responses in vivo and in the context of animal models of autoimmunity. ? ? ?
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