Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca2+) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca2+ stored within the ER lumen. As ER Ca2+ is released to generate primary Ca2+ signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca2+-selective Orai channels to mediate finely controlled Ca2+ signals and to homeostatically balance cellular Ca2+. Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC

The TRP family of ion channels transduce an extensive range of chemical and physical signals. TRPC6 is a receptor-activated nonselective cation channel expressed widely in vascular smooth muscle and other cell types. We report here that TRPC6 is also a sensor of mechanically and osmotically induced membrane stretch. Pressure-induced activation of TRPC6 was independent of phospholipase C. The stretch responses were blocked by the tarantula peptide, GsMTx-4, known to specifically inhibit mechanosensitive channels by modifying the external lipid-channel boundary. The GsMTx-4 peptide also blocked the activation of TRPC6 channels by either receptor-induced PLC activation or by direct application of diacylglycerol. The effects of the peptide on both stretch-and diacylglycerol-mediated TRPC6 activation indicate that the mechanical and chemical lipid sensing by the channel has a common molecular mechanism that may involve lateral-lipid tension. The mechanosensing properties of TRPC6 channels highly expressed in smooth muscle cells are likely to play a key role in regulating myogenic tone in vascular tissue.GsMTx-4 peptide ͉ mechanosensitivity ͉ tarantula venom ͉ myogenic tone ͉ calcium signals T he superfamily of TRP cation channels transduce a remarkable spectrum of signals ranging from small secondmessenger molecules to physical parameters including temperature, osmolarity, and touch (1, 2). Among the several subfamilies of TRP channels, the TRPC nonselective cation channels activated in response to PLC-coupled receptors are widely expressed among tissues (2, 3). The closely related subgroup comprising TRPC3, TRPC6, and TRPC7 channels are directly activated by diacylglycerol through a PKC-independent mechanism (3, 4). TRPC6 channels are highly expressed in a number of different tissues including vascular smooth muscle cells (5-8). Despite their abundance, the exact physiological role of TRPC6 channels has not been elucidated. Recently it has been suggested that TRPC6 channels are involved in hemodynamic regulation (6, 9) and may play a role in generating myogenic tone in response to intravascular pressure in arteries (6, 9). This is a key mechanism to control blood flow in arteries and arterioles (10, 11) involving depolarization, activation of Ca 2ϩ entry, and the contraction of vascular smooth muscle cells (11). Increases in Ca 2ϩ in response to pressure not only activate smooth muscle cell contraction but also modify their growth and differentiation (12). However, the identity and gating mechanisms of the mechanical sensors that mediate the depolarizing response have not been identified.TRPC6 channels are expressed predominantly in cells responding to hydrostatic pressure changes including vascular smooth muscle and glomerular podocytes and have been implicated in mediating pressure-induced responses (6, 13). TRPC6 channels mediate receptor-induced depolarization in smooth muscle cells (7,9), and opening of the related TRPC1 channel has been shown to be activated by stretch (14). In addition to being implicated in ...

Receptor-induced Ca 2؉ signals are key to the function of all cells and involve release of Ca 2؉ from endoplasmic reticulum (ER) stores, triggering Ca 2؉ entry through plasma membrane (PM) ''storeoperated channels'' (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca 2؉ -binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca 2؉ sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca 2؉ sensor, STIM1 functions within the PM to control operation of the Ca 2؉ entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca 2؉ release-activated Ca 2؉ (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current (I CRAC) into a constitutively active, Ca 2؉ store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties. Last, Ab externally applied to the STIM1 N-terminal EF hand blocks both I CRAC in hematopoietic cells and SOC-mediated Ca 2؉ entry in HEK293 cells, revealing that STIM1 has an important functional presence within the PM. The results reveal that, in addition to being an ER Ca 2؉ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca 2؉ -regulated processes including secretion, contraction, metabolism, cell division, and apoptosis.calcium signaling ͉ calcium channel ͉ patch-clamp ͉ mast cells ͉ T lymphocytes

Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (CaV1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with CaV1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both CaV1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed CaV1.2 and Orai channels has major implications for Ca2+ signal generation in excitable and nonexcitable cells.

The coupling mechanism between endoplasmic reticulum (ER) Ca(2+) stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1-3]. STIM1 was shown to play a crucial role in this coupling process [4-7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca(2+) entry signals.

STIM1 and CRACM1 (or Orai1) are essential molecular components mediating store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ (CRAC) currents. Although STIM1 acts as a luminal Ca2+ sensor in the endoplasmic reticulum (ER), the function of STIM2 remains unclear. Here we reveal that STIM2 has two distinct modes of activating CRAC channels: a store-operated mode that is activated through depletion of ER Ca2+ stores by inositol 1,4,5-trisphosphate (InsP3) and store-independent activation that is mediated by cell dialysis during whole-cell perfusion. Both modes are regulated by calmodulin (CaM). The store-operated mode is transient in intact cells, possibly reflecting recruitment of CaM, whereas loss of CaM in perfused cells accounts for the persistence of the store-independent mode. The inhibition by CaM can be reversed by 2-aminoethoxydiphenyl borate (2-APB), resulting in rapid, store-independent activation of CRAC channels. The aminoglycoside antibiotic G418 is a highly specific and potent inhibitor of STIM2-dependent CRAC channel activation. The results reveal a novel bimodal control of CRAC channels by STIM2, the store dependence and CaM regulation, which indicates that the STIM2/CRACM1 complex may be under the control of both luminal and cytoplasmic Ca2+ levels.