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

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.

The coupling between receptor-mediated Ca 2؉ store release and the activation of "store-operated" Ca 2؉ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca 2؉ entry within 10 min with an IC 50 of 0.1-0.3 M. Store-operated Ca 2؉ entry induced by Ca 2؉ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr 2؉ entry activated by muscarinic receptors was also blocked by BTP2 with an IC 50 of <0.3 M. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC 50 ϳ 0.3 M). BTP2 still blocked TRPC3 in medium with Nmethyl-D-glucamine-chloride replacing Na ؉ , indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 M BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca 2؉ -selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.Cytosolic Ca 2ϩ signals control a wide array of cellular functions ranging from short-term responses such as contraction and secretion to longer-term regulation of cell growth and proliferation (1). Ca 2ϩ signals generated in response to receptors are complex, involving two closely coupled components: rapid, transient release of Ca 2ϩ stored in the endoplasmic reticulum (ER), 1 followed by slowly developing extracellular Ca 2ϩ entry (1-4). Receptors coupled to activation of either PLC-␤ or PLC-␥ generate the two messengers, InsP 3 and DAG. InsP 3 diffuses rapidly within the cytosol to interact with InsP 3 receptors in the ER that serve as Ca 2ϩ channels to release luminal stored Ca 2ϩ and generate the initial Ca 2ϩ signal phase (1). The resulting depletion of Ca 2ϩ stored within the ER lumen serves as the primary trigger for a message that is returned to the plasma membrane resulting in the activation of storeoperated channels (SOCs) that...

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca 2؉ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-snglycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca 2؉ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca 2؉ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca 2؉ entry was suppressed by more than 50%. In contrast, OAG-induced Ca 2؉ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca through plasma membrane channels (1-3). Although identification of the latter has proven elusive, members of the canonical transient receptor potential (TRPC) 3 channel family have been leading contenders (2-5). The TRPC channels all appear to be activated in response to phospholipase C (PLC)-coupled receptors (2-8). Within the TRPC family, there are two structurally divided subgroups: TRPC3, TRPC6, and TRPC7 channels (TRPC3/6/7) and TRPC1, TRPC4, and TRPC5 (TRPC1/4/5). One functional characteristic distinguishing these two subgroups is the ability of diacylglycerol (DAG) to activate TRPC3/6/7 channels but not TRPC1/4/5 channels (2, 6 -11). DAG also activates TRPC2 channels; however, this channel is not expressed in higher mammals and is restricted mostly to the vomeronasal organ (12). As a product of receptor-induced PLC activation, DAG is an obvious mediator of TRPC channel activation. However, its role in the activation of endogenously expressed TRPC3/6/7 channels is uncertain (7,8,(13)(14)(15). The majority of studies revealing the action of DAG on TRPC channels have been undertaken using overexpression systems (2,7,8,14,15). Such expression may differ from endogenous TRPC expression. For example, considering that TRPC channels probably function as tetramers (4, 5), overexpression may result in a predominance of homotetrameric structures, whereas endogenous expression may reflect heteromers between TRPC channel subtypes, resulting in quite different properties (2, 4, 5). In addition, since TRPC channels may function within ...

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zδ values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zδ), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.

We have employed both in vitro patch clamp recordings of hair cell synaptic vesicle fusion and in vivo single unit recording of cochlear nerve activity to study, at the same synapse, the time course, control, and physiological significance of readily releasable pool dynamics. Exocytosis of the readily releasable pool was fast, saturating in less than 50 ms, and recovery was also rapid, regaining 95% of its initial amplitude following a 200-ms period of repolarization. Longer depolarizations (greater than 250 ms) yielded a second, slower kinetic component of exocytosis. Both the second component of exocytosis and recovery of the readily releasable pool were blocked by the slow calcium buffer, EGTA. Sound-evoked afferent synaptic activity adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. Comparison of readily releasable pool amplitude, capture distances of calcium buffers, and number of vesicles tethered to the synaptic ribbon suggested that readily releasable pool dynamics reflect the depletion of release-ready vesicles tethered to the synaptic ribbon and the reloading of the ribbon with vesicles from the cytoplasm. Thus, we submit that rapid recovery of the cochlear hair cell afferent fiber synapse from short-term adaptation depends on the timely replenishment of the synaptic ribbon with vesicles from a cytoplasmic pool. This apparent rapid reloading of the synaptic ribbon with vesicles underscores important functional differences between synaptic ribbons in the auditory and visual systems.