Research
In various cell types including the immune cells, the depletion of intracellular Ca2+ stores by receptor stimulation triggers Ca2+ entry through the store-operated Ca2+ (SOC) channels. The increased Ca2+ levels activate a plethora of signalling pathways for proliferation, cytokine production, and cell death. The specific type of SOC channels in immune cells (e.g. T or B cells) is defined as Ca2+ release-activated Ca2+ (CRAC) channels due to their unique electrophysiological properties including high selectivity for Ca2+ ions, low unitary conductance, and inward rectification. Although the CRAC current was biophysically characterized earlier, the molecular identity of the CRAC channel stayed elusive until 2006 when we and two other groups discovered the gene encoding a critical subunit of the CRAC channels. In collaboration with Dr. Norbert Perrimon's group using genome-wide RNAi screen in Drosophila cells, the long-sought pore component of CRAC channels was identified as olf-186F. The gene was re-named by the PI as Drosophila Orai (dOrai) and its mammalian homologues as Orai1, Orai2, and Orai3. In addition, it was found that a point mutation in the ORAI1 gene in human patients leads to lethal, severe combined immune deficiency (SCID). These results indicate a pivotal role of Orai1 in maintaining a functional immune system. The identification of Orai provides us a molecular handle to study the macromolecular structure and mechanism of regulation of CRAC channels. In addition to Orai1, another important signalling molecule STIM1 that mediates store depletion and CRAC channel activation was identified by two labs (Dr. Michael Cahalan and Tobias Meyer) a year earlier. STIM1 detects the Ca2+stores in the endoplasmic reticulum (ER) by its EF-hand motif. Upon ER store-depletion, STIM1 aggregates and translocates into the junctional regions near the plasma membrane to physically interact with Orai1 and mediate Ca2+ entry. Presently, the major focus of our lab is:
i) Identification of novel regulators for Orai1 and STIM1. Using integrative information from the genome-wide RNAi screen, expression pattern analysis, and proteomics approaches, we haveidentified several novel molecules that play an important role in the function of Orai1 and STIM1. One of those candidates named as CRACR2A (CRAC Regulator 2A) has been recently published by our group (Srikanth et al, Nature Cell Biology, 2010). In the follow up studies, we have generated knockout animal models to elucidate the in vivo function of this novel regulator in modulating CRAC channel activity in immune cells. We are also focusing on other regulators identified from the screens.
ii) Structure/function analysis of Orai1. We have generated various point mutants of Orai1 and examined SOCE after reconstitution in Orai1-deficient cells using single cell ratiometric Ca2+ imaging and whole-cell patch clamp electrophysiological methods. This study aims to understand the functional domains of Orai1 important for its activity and binding to various regulators.
iii) Elucidation of physiological roles of store-operated Ca2+ entry in the immune system using animal models. We have previously generated Orai1-deficient (Orai1-/-) mice and observed that the mice show immunodeficiency phenotype, similar to human patients expressing non-functional Orai1. In the follow up studies, we are examining the role of Orai1 in death of immune cells. Immune cells upon activation undergo robust proliferation to combat the pathogen. After the pathogen clearance, a majority of these activated cells need to die to maintain homeostasis. Intriguingly, Ca2+ has been shown to play a role in both, the activation phase as well as death of activated cells. We are analyzing this cell death phenomenon to examine the role of Orai1 in stimulation-induced death of T cells.
In the long term, we are also interested in examining the role of Orai1 in other cell types where Ca2+/Calcineurin/NFAT signaling is known to play an important role.