SUMMARY Store-operated Ca2+ channels activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) are a major Ca2+ entry pathway in non-excitable cells and are essential for T cell activation and adaptive immunity. Following store depletion, the ER Ca2+ sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N- and C-termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca2+ entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

Ca 2؉ -dependent inactivation (CDI) is a key regulator and hallmark of the Ca 2؉ release-activated Ca 2؉ (CRAC) channel, a prototypic store-operated Ca 2؉ channel. Although the roles of the endoplasmic reticulum Ca 2؉ sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CADinduced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470 -491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68 -91) that binds CaM in a Ca 2؉ -dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.calcium ͉ ion channel gating ͉ store-operated calcium entry ͉ patch-clamp ͉ calcium-binding proteins S tore-operated Ca 2ϩ channels provide a major route for receptor-stimulated Ca 2ϩ entry in nonexcitable cells (1). The Ca 2ϩ -release activated Ca 2ϩ (CRAC) channel, the bestcharacterized store-operated channel, is essential for generating Ca 2ϩ signals that drive the activation of T lymphocytes, mast cells, and platelets (2-4). CRAC channel activity is shaped by the combination of store-dependent activation and Ca 2ϩ -dependent inactivation (CDI) processes. The mechanisms linking depletion of Ca 2ϩ from the endoplasmic reticulum (ER) to activation of the CRAC channel are becoming well understood: reduction of ER luminal Ca 2ϩ causes the ER Ca 2ϩ sensor STIM1 (5, 6) to oligomerize (7), enabling its accumulation at ER-plasma membrane junctions (3,8,9), where it binds directly to the CRAC channel subunit to open the channel (13,14).Compared with activation, much less is known about the mechanisms underlying CDI, one of the hallmark characteristics of the CRAC current (I CRAC ) in mammalian cells. Initial studies in mast cells and T cells revealed that Ca 2ϩ influx at hyperpolarized potentials drives rapid inactivation on a time scale of tens of milliseconds through the binding of Ca 2ϩ to sites located several nanometers from the intracellular mouth of the pore (15, 16). Subsequent studies have suggested that calmodulin (CaM) and STIM1 both may contribute to this process. In a rat liver cell l...

Researchers have reevaluated the role of calmodulin and previously identified calmodulin binding sites in the mechanism by which Ca2+-release activated Ca2+ channels can be inactivated as Ca2+ ions enter cells.

Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.