Thursday, April 3, 2025
4/3/2025
Molecular and cellular mechanisms of store-operated calcium channels - Store-operated Ca2+ entry (SOCE) generates Ca2+ signals that are critical for many physiological processes, from immune cell activation and differentiation to muscle activity, secretion, and motility. Store-operated Ca2+ channels (SOCs) are activated by receptors that deplete Ca2+ from the ER; the loss of Ca2+ is sensed by STIM1, which then accumulates at ER-plasma membrane (ER-PM) junctions where it binds, traps, and activates calcium-selective Orai channels diffusing in the PM. Gain-of-function and loss-of-function mutations in this pathway have both been connected to serious human diseases, underscoring the critical importance of precise regulation. The long-term goal of our laboratory is to understand the molecular basis of SOC properties and regulation as well as their cellular roles. While the overall organization of the SOCE pathway is now known and many of the underlying proteins have been identified, major gaps still exist in our understanding of how they act to regulate SOCE location and amplitude. Over the next five years we aim to investigate three fundamental processes that regulate calcium influx through SOCs. (1) The dynamics of ER-PM junctions. These junctions where the ER closely approaches the PM are the only sites in the cell where STIM can bind and activate Orai, such that their size, abundance and location determine both the amplitude and location of Ca2+ entry. While a host of tethering proteins at junctional sites is known, their specific roles in junction initiation vs. turnover is unclear. By monitoring the appearance and removal of ER-PM junctions in living cells with fluorescent markers we will distinguish the different contributions of known tethering proteins to the initiation, lifetime and turnover rate of new junctions, as well as their ability to conduct SOCE. (2) The mechanism of STIM1 activation and its interaction with Orai1. The cytosolic domain of STIM1 undergoes a massive conformational change after ER Ca2+ depletion in order to unmask and extend the CRAC activation domain (CAD) to activate Orai in the plasma membrane. By studying STIM1 with single-molecule fluorescence and crosslinking techniques we aim to identify steps in the activation process and intermediate states that may help mitigate the energetic cost of unfolding and refolding STIM1. Similar approaches will be applied to determine basic features of the STIM-Orai interaction - the stoichiometry of the STIM-Orai complex, the conformation of CAD in the bound state, and the binding interface itself – which are currently not understood. (3) A molecular mechanism for Ca2+-dependent inactivation (CDI). Despite progress in identifying multiple residues and domains in STIM and Orai that are critical for CDI, an integrated mechanism is still lacking. We will use a pore accessibility assay to localize the position of the inactivation gate, and explore functional and physical interactions of CDI domains to understand how they cooperate to bring about CDI. Overall, the results of our studies will reveal fundamental cellular and molecular mechanisms that control the strength of store-operated calcium signals in diverse cells, and may suggest new strategies for regulating them to explore cellular functions and develop new treatments for human disease.
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