Sustained calcium entry in mast cells plays a critical role in the initiation and maintenance of allergic responses associated with ligand binding to Fc epsilon RI. It is believed that the sustained calcium entry associated with engagement of Fc epsilon RI is mediated by the opening of calcium channels in the plasma membrane in response to depletion of a subset of calcium stores by the second messenger inositol-1,4,5-trisphosphate (IP3). Although the functional relationship between calcium store depletion and calcium entry is a well-documented phenomenon, there is little or no definitive data concerning the nature of the relevant calcium channels involved (referred to as Store Operated Channels or SOC), or the molecular mechanisms by which these channels are gated in response to calcium store depletion. Moreover, although recent data suggest that calcium entry may be regulated by pathways associated with the production of the second messengers sphingosine-1-phosphate, cyclic ADP-ribose or NAADP, the relationship between these putative pathways and calcium store depletion, and the generality of these pathways and the potential targets of these specific second messengers are either unknown or controversial. In summary, there is a significant lack of specific knowledge concerning the molecular mechanisms which regulate calcium entry into mast cells and other non-excitable cells. Because of the fundamental importance of calcium entry to mast cell function, the investigator's laboratory has embarked upon a series of experimental approaches to identify calcium entry regulatory proteins. In the preliminary data provided, experiments describe the identification and initial characterization of a novel family of putative calcium channels (CeCH proteins) which are widely expressed in non-excitable cells including mast cells. In the current application, experiments are proposed to analyze the function of these proteins in both mast cell and non-mast cell lines.
In specific aim 1, studies will be performed to analyze the assembly and transport of wild type CeCH proteins, to conduct structure/function analyses to identify structural features required for proper assembly and transport, and to isolate and characterize the role of CeCH-associated proteins in CeCH function.
In specific aim 2, experiments will analyze CeCH function in regulating calcium homeostasis and signaling through a combination of calcium imaging and electrophysiologic analysis of cultured cells expressing defined combinations of CeCH proteins under various types of stimulus conditions.
|Brixel, Lili R; Monteilh-Zoller, Mahealani K; Ingenbrandt, Claudia S et al. (2010) TRPM5 regulates glucose-stimulated insulin secretion. Pflugers Arch 460:69-76|
|Barbet, Gaetan; Demion, Marie; Moura, Ivan C et al. (2008) The calcium-activated nonselective cation channel TRPM4 is essential for the migration but not the maturation of dendritic cells. Nat Immunol 9:1148-56|
|Cheng, Henrique; Beck, Andreas; Launay, Pierre et al. (2007) TRPM4 controls insulin secretion in pancreatic beta-cells. Cell Calcium 41:51-61|
|Takezawa, Ryuichi; Cheng, Henrique; Beck, Andreas et al. (2006) A pyrazole derivative potently inhibits lymphocyte Ca2+ influx and cytokine production by facilitating transient receptor potential melastatin 4 channel activity. Mol Pharmacol 69:1413-20|
|Kraft, Stefan; Fleming, Tony; Billingsley, James M et al. (2005) Anti-CD63 antibodies suppress IgE-dependent allergic reactions in vitro and in vivo. J Exp Med 201:385-96|
|Launay, Pierre; Cheng, Henrique; Srivatsan, Subhashini et al. (2004) TRPM4 regulates calcium oscillations after T cell activation. Science 306:1374-7|
|Hermosura, Meredith C; Monteilh-Zoller, Mahealani K; Scharenberg, Andrew M et al. (2002) Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNuM. J Physiol 539:445-58|
|Launay, Pierre; Fleig, Andrea; Perraud, Anne Laure et al. (2002) TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 109:397-407|