The identification and cloning of the CRAC channel by us and others (CRACM1/Orai1) has defined the basic molecular components required for store-operated calcium influx. Mutational analysis demonstrated that CRACM1/Orai1 homopolymerizes and that it constitutes the calcium selective pore of the CRAC channel. Importantly, using gene-trap technology to generate the Cracm1-/- mouse, we have demonstrated that CRACM1 is essential for mast cell effector functions and allergic responses in vivo. Surprisingly, we found that T cell development and proliferation were relatively unaffected in these mice. In order to gain a comprehensive view of SOCE in T cells, we are now studying CRACM2 and CRACM3. Our preliminary data suggest that these homologs may have distinct functions in T cells. We will dissect their respective roles by generating and characterizing knockout mice for each of these genes (Aim 1). Voltage operated calcium channels (VOCCs) are expressed in T cells, impact NFAT translocation and regulate cytokine production. But their mechanism of action is still unclear. We have generated an inducible CaV1.2 T cell knockout mouse and have observed that CaV1.2 loss significantly inhibits cytokine production in T cells. Intriguingly, our most recent data show that C-terminal domain of CaV1.2 and CRACM2 associate in resting T cells. These new data reveal an unexpectedly direct connection between at least one VOCC subunit and CRAC and provide a new framework in which to study all VOCC functions in T cells. We will fully characterize this interaction, dissect the mechanism of CaV1.2 function, and further define its role in T cells in vivo (Aim 2). Finally, we have discovered that the related VOCC channel CaV1.3 is localized to the ER and forms a stable protein complex with STIM1. This novel observation suggests another fundamental intersection between VOCC channels and CRAC. We hypothesize that CaV1.3 acts as a calcium sensor for STIM1 in the ER. We propose experiments to test this idea and fully dissect CaV1.3 function in T cells (Aim 3).

Public Health Relevance

The identification and cloning of the CRAC channel has defined the basic molecular components required for store-operated calcium entry (SOCE). We have recently demonstrated that CRACM1 deficiency results in minor T cell abnormalities in CRACM1-deficient mice. We now propose to study CRACM2 and CRACM3 function and determine their in vivo roles in T cells using deletion mouse models (Aim 1). In addition we will study how VOCC channel CaV1.
2 (Aim 2) and CaV1.
3 (Aim 3) interact and modulate CRAC channels. These studies will yield new insights into the molecular regulation of SOCE within T cells, enabling the development new approaches to treat autoimmune diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI075129-04
Application #
8296625
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Mallia, Conrad M
Project Start
2009-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2012
Total Cost
$416,500
Indirect Cost
$171,500
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215