The regulation of calcium entry in B-cells and other types of immune system cells is poorly understood. We and others have identified a new family of calcium channels provisionally designated the C. elegans Channel Homologue (CeCH) family, which are expressed in many types of non-excitable cells including immune system cells. We have cloned a novel member of this family, provisionally designated CeCH2, which is expressed in all types of immune tissues and cells. Preliminary data from gene targeting experiments in the DT-40 B-cell system demonstrate that CeCH2 is required for B-cell viability, while overexpression of CeCH2 in the HEK293 cell system is toxic, suggesting that CeCH2 is likely to be closely linked to basic cellular processes required for cell survival. Furthermore, we have identified a novel domain in CeCH2, which is highly homologous to the catalytic domain of the myosin heavy chain kinase (MHCK)/eukaryotic elongation factor-2 kinase (EEF2 kinase) family and our preliminary data implicate this domain in the gating mechanism of CeCH2. We propose to further explore CeCH2 function through the following lines of investigation:
Specific Aim 1 : Characterization of the role of CeCH2 in cell viability and calcium signaling, in this aim we will characterize the effect of CeCH2 overexpression in the HEK293 system and the CeCH2-deficient phenotype in the DT-40 system by analyzing cellular growth characteristics and calcium homeostasis/signaling in wild type or CeCH2-altered conditions.
Specific Aim 2 : Characterization of the MHCK-homology domain of CeCH2. In this aim we will determine whether the novel MHCK-homology domain of CeCH2 is required for CeCH2 function, and analyze whether its potential protein kinase activity might play a role in CeCH2 function. We will also analyze the role of this domain in CeCH2 function in terms of its serving as a protein or small molecule interaction domain.
Specific Aim 3 : Analysis of CeCH2 structure/function relationships. In this aim we will perform a detailed structure/function analysis of CeCH2 to attempt to understand how CeCH2 structure relates to its requirement for cell viability in the DT-40 system and its toxicity in the HEK293 system. These analyses will include testing whether other CeCH family channels, chimeric CeCH channels or CeCH2 channels with mutations in several highly conserved regions are toxic when expressed in HEK293 cells and/or are able to complement CeCH2 function in the DT-40 system. Together these analyses will provide significant insight into CeCH2 function and its role in B-cell calcium homeostasis and signaling.
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