The research proposed in this study aims at identifying molecules involved in the activation of store operated calcium entry through CRAC calcium channels in T cells. Our approach is to define the molecular causes for absent CRAC activation in T cells from human patients with a Severe Combined Immunodeficiency disease (SCID). Clinically these patients suffer from severe opportunistic infections due to impaired T cell activation, proliferation, cytokine production and overall gene expression. Ca 2+ signaling is almost completely absent in the SCID T cells as no increases in intracellular calcium concentrations in response to various T cell stimuli and no CRAC Ca 2+ currents (ICRAC) could be detected. CRAC is the predominant Ca 2+ channel in lymphocytes but neither its molecular identity nor its regulatory mechanisms are known. Candidate gene searches and conventional cloning methods used by many laboratories to identify CRAC have been unsuccessful in the past. In the SCID patients' T cells, none of the proposed CRAC candidate genes have been found mutated. Very likely, the protein mutated in this form of SCID involves a subunit of the CRAC channel or a component of the signaling pathway that leads to its activation. CRAC itself, like calcium channels in other tissues (e.g. the cardiovascular system) could be a promising target for therapeutical immune modulation. ? We are undertaking two major independent complementation cloning approaches to identify the gene product affected in the CRAC deficient T cells. (1) The first cloning approach aims at reconstituting CRAC function in the SCID T cells in vitro by transferring human chromosomes into the T cells by microcellmediated chromosome transfer (MMCT). We will try to establish this technique for use in lymphocytes and for complementation of highly regulated signaling proteins. (2) The second approach aims at developing a """"""""random retroviral insertional mutagenesis"""""""" method to target the healthy allele of the CRAC channel gene in heterozygous T cells from the patients' parents. The goal is to create a selectable Ca 2+ null phenotype (no ICRAC like) in the patients. The """"""""gene trapping"""""""" technique applied uses a mutagenizing retroviral expression vector, which randomly inserts into the genome of targeted cells. The viral integration site and thus the gene locus can be retrieved by RACE and the phenotype reversed by Cre-lox excision of the vector. ? ?
