Structural Determinants of CRACM and STIM Interaction

The prototypical store-operated calcium-influx pathway I CRAC (for "calcium-release activated calcium current") originally has been identified in Prof. Penner`s laboratory in 1992. Since then this lab, and others have acquired substantial information about I CRAC `s physiological and clinical importance. Changes in intracellular free Ca2+ concentration ([Ca 2+]i) probably represent the most wide-spread and most important signaling event in cellular physiology, since transient elevations of [Ca 2+]i directly or indirectly control and regulate a plethora of cellular responses such as enzyme activation, gene transcription, cell proliferation, and apoptotic cell death. Ca2+ release from stores and Ca2+ influx are intimately linked through a process termed store- operated Ca2+ (SOC) entry. The relevance of SOC for immune cells is highlighted by the fact that lymphocytes with defective SOC are unable to mount an immune response and patients develop severe combined immunodeficiency (SCID). I CRAC represents the best characterized SOC current. However, the molecular composition of the CRAC channels has still remained elusive. Only recently a significant finding has established that a protein called stromal interaction molecule (STIM1), acting as the sensor for store Ca2+ content, is required for functional store-operated Ca2+ influx. This exciting finding has been followed by the identification of another essential protein for store-operated calcium entry called CRAC Modulator 1 (CRACM1) by Dr. Penner`s group and Orai1 by an independent study from the Rao and Lewis laboratories. Suppression of this protein by RNAi abolishes CRAC channel activity, as does a point mutation of CRACM1 in lymphocytes of some patients suffering from SCID. The combined overexpression of STIM1 and CRACM1 greatly amplifies store- operated currents and these currents possess the most defining characteristics of I CRAC . Work from three laboratories (Cahalan, Rao, and Penner) has identified amino acids in transmembrane and loop domains of CRACM1 that affect ion selectivity, demonstrating that CRACM1 represents a pore-forming subunit of the CRAC channel. Further data show that the CRACM1 homologs CRACM2 and CRACM3 also form store-operated channels with distinct properties. My proposal focuses on STIM1 and CRACM homologs, as a group of proteins that mediate store-operated Ca2+ entry with distinct functional properties. I propose biophysical, molecular, and functional approaches to investigate the structural and mechanistic aspects of these proteins and their roles in store-operated Ca2+ entry.