Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging.

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100 µmol L-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and ß-actin. Neurones that did not respond during chloride imaging did not show KCC2 and ß-actin protein expression. The KCC2 antagonist (VU0240551,10 µmol L-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 µmol L-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 µmol L-1 ) and protein kinase inhibitor (K252a) (100 nmol L-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

Homer binds to Orai1 and TRPC channels in the neointima and regulates vascular smooth muscle cell migration and proliferation.

The molecular components of store-operated Ca2+ influx channels (SOCs) in proliferative and migratory vascular smooth muscle cells (VSMCs) are quite intricate with many channels contributing to SOCs. They include the Ca2+-selective Orai1 and members of the transient receptor potential canonical (TRPC) channels, which are activated by the endoplasmic reticulum Ca2+ sensor STIM1. The scaffolding protein Homer assembles SOC complexes, but its role in VSMCs is not well understood. Here, we asked whether these SOC components and Homer1 are present in the same complex in VSMCs and how Homer1 contributes to VSMC SOCs, proliferation, and migration leading to neointima formation. Homer1 expression levels are upregulated in balloon-injured vs. uninjured VSMCs. Coimmunoprecipitation assays revealed the presence and interaction of all SOC components in the injured VSMCs, where Homer1 interacts with Orai1 and various TRPC channels. Accordingly, knockdown of Homer1 in cultured VSMCs partially inhibited SOCs, VSMC migration, and VSMC proliferation. Neointimal area was reduced after treatment with an adeno-associated viral vector expressing a short hairpin RNA against Homer1 mRNA (AAV-shHomer1). These findings stress the role of multiple Ca2+ influx channels in VSMCs and are the first to show the role of Homer proteins in VSMCs and its importance in neointima formation.

Molecular determinants mediating gating of Transient Receptor Potential Canonical (TRPC) channels by stromal interaction molecule 1 (STIM1).

Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.

The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function.

Ca(2+) influx by store-operated Ca(2+) channels (SOCs) mediates all Ca(2+)-dependent cell functions, but excess Ca(2+) influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca(2+) sensor STIM1. Slow Ca(2+)-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448-530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448-490) and STIM1(490-530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448-490) lobe restricted, whereas the STIM1(490-530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.

Opposite regulatory effects of TRPC1 and TRPC5 on neurite outgrowth in PC12 cells.

The transient receptor potential (TRPC) family of Ca²âº permeable, non-selective cation channels is abundantly expressed in the brain, and can function as store-operated (SOC) and store-independent channels depending on their interaction with the ER Ca²âº sensor STIM1. TRPC1 and TRPC5 have critical roles in neurite outgrowth, however which of their functions regulate neurite outgrowth is unknown. In this study, we investigated the effects of TRPC channels and their STIM1-induced SOC activity on neurite outgrowth of PC12 cells. We report that PC12 cell differentiation down-regulates TRPC5 expression, whereas TRPC1 expression is retained. TRPC1 and TRPC5 interact with STIM1 through the STIM1 ERM domain. Transfection of TRPC1 and TRPC5 increased the receptor-activated Ca²âº influx that was markedly augmented by the co-expression of STIM1. Topical expression of TRPC1 in PC12 cells markedly increased neurite outgrowth while that of TRPC5 suppressed neurite outgrowth. Suppression of neurite outgrowth by TRPC5 requires the channel function of TRPC5. However, strikingly, multiple lines of evidence show that the TRPC1-induced neurite outgrowth was independent of TRPC1-mediated Ca²âº influx. Thus, a) TRPC1 and TRPC5 similarly increased Ca²âº influx but only TRPC1 induced neurite outgrowth, b) the constitutively STIM1(D76A) mutant that activates Ca²âº influx by TRPC and Orai channels did not increase neurite outgrowth, c) co-expression of TRPC5 with TRPC1 suppressed the effect of TRPC1 on neurite outgrowth, d) and most notable, channel-dead pore mutant of TRPC1 increased neurite outgrowth to the same extent as TRPC1(WT). Suppression of TRPC1-induced neurite outgrowth by TRPC5 was due to a marked reduction in the surface expression of TRPC1. We conclude that the regulation of neurite outgrowth by TRPC1 is independent of Ca²âº influx and TRPC1-promoted neurite outgrowth depends on the surface expression of TRPC1. It is likely that TRPC1 acts as a scaffold at the cell surface to assemble a signaling complex to stimulate neurite outgrowth.

STIM1-dependent and STIM1-independent function of transient receptor potential canonical (TRPC) channels tunes their store-operated mode.

Ca(2+) influx by store-operated Ca(2+) channels is a key component of the receptor-evoked Ca(2+) signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca(2+) influx. Recent studies have revealed how STIM1 activates TRPC1 in response to store depletion; however, the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. Here, we established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). Significantly, WT but not mutant TRPC channels were inhibited by scavenging STIM1 with Orai1(R91W), indicating the STIM1 dependence and independence of WT and mutant TRPC channels, respectively. Importantly, mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca(2+) influx.

Molecular determinants of fast Ca2+-dependent inactivation and gating of the Orai channels.

Ca(2+) influx by store-operated Ca(2+) influx channels (SOCs) mediates many cellular functions regulated by Ca(2+), and excessive SOC-mediated Ca(2+) influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca(2+)-dependent inactivation, which limits Ca(2+) influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca(2+)-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca(2+)-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca(2+)-dependent inactivation. Fast Ca(2+)-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca(2+)-dependent inactivation that has implications for Ca(2+) influx by SOC in physiological and pathological states.

TRPC channels as STIM1-regulated SOCs.

Store-operated Ca(2+) channels (SOCs) are Ca(2+) influx channels at the plasma membrane whose opening is determined by the level of Ca(2+) stored in the endoplasmic reticulum lumen. SOCs are activated in response to receptor-mediated or passive depletion of ER Ca(2+) to regulate many Ca(2+)-dependent cellular functions. Early work implicated the TRPC channels as SOCs. More recently, it was found that the Orai channels mediate the CRAC current and that the Ca(2+) binding protein STIM1 functions as the ER Ca(2+) sensor that mediates activation of the SOCs in response to depletion of ER Ca(2+). Key questions are whether both TRPC and Orai channels are opened by STIM1 and the molecular mechanism by which STIM1 opens the SOCs. Ample biochemical and functional evidence indicate interaction of the TRPC channels with STIM1. Furthermore, it was found that STIM1 gates TRPC channels by electrostatic interaction of STIM1(K684,K685) in the polybasic domain of STIM1 with two negative charges (aspartates or glutamates) that are conserved in all TRPC channels. Charge mutants of STIM1(K684,K685) and TRPC1(D639,D640) and TRPC3(D697,D698) were used to develop further direct evidence for the function of TRPC channels as SOCs. The evidence in favor of TRPC channels as SOCs are discussed.

SOAR and the polybasic STIM1 domains gate and regulate Orai channels.

Influx of Ca(2+) through store-operated Ca(2+) channels (SOCs) is a central component of receptor-evoked Ca(2+) signals. Orai channels are SOCs that are gated by STIM1, a Ca(2+) sensor located in the ER but how it gates and regulates the Orai channels is unknown. Here, we report the molecular basis for gating of Orais by STIM1. All Orai channels are fully activated by the conserved STIM1 amino acid fragment 344-442, which we termed SOAR (the STIM1 Orai activating region). SOAR acts in combination with STIM1 (450-485) to regulate the strength of interaction with Orai1. Activation of Orai1 by SOAR recapitulates all the kinetic properties of Orai1 activation by STIM1. However, mutations of STIM1 within SOAR prevent activation of Orai1 but not co-clustering of STIM1 and Orai1 in response to Ca(2+) store depletion, indicating that STIM1-Orai1 co-clustering is not sufficient for Orai1 activation. An intact carboxy terminus alpha-helicial region of Orai is required for activation by SOAR. Deleting most of the Orai1 amino terminus impaired Orai1 activation by STIM1, but Orai1(Delta1-73) interacted with and was fully activated by SOAR. Accordingly, the characteristic inward rectification of Orai is mediated by an interaction between the polybasic STIM1 (672-685) and a Pro-rich region in the N terminus of Orai1. Hence, the essential properties of Orai1 function can be rationalized by interactions with discrete regions of STIM1.

STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction.

The receptor-evoked Ca(2+) signal includes activation of the store-operated channels (SOCs) TRPCs and the Orais. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1((639)DD(640)) with the positively charged STIM1((684)KK(685)) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1((684)EE(685))+TRPC1((639)RR(640)), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observed with TRPC3((697)DD(698)). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.

TRPC channels as STIM1-regulated store-operated channels.

Receptor-activated Ca(2+) influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca(2+) influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca(2+) influx channels. The findings that STIM1 is an ER resident Ca(2+) binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca(2+) stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels demonstrate their regulation by STIM1 and indicate that all TRPC channels, except TRPC7, function as SOCs.

STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels.

Stromal interacting molecule 1 (STIM1) is a Ca(2+) sensor that conveys the Ca(2+) load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La(3+), suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.

STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

XTRPC1-dependent chemotropic guidance of neuronal growth cones.

Calcium arising through release from intracellular stores and from influx across the plasma membrane is essential for signalling by specific guidance cues and by factors that inhibit axon regeneration. The mediators of calcium influx in these cases are largely unknown. Transient receptor potential channels (TRPCs) belong to a superfamily of Ca2+-permeable, receptor-operated channels that have important roles in sensing and responding to changes in the local environment. Here we report that XTRPC1, a Xenopus homolog of mammalian TRPC1, is required for proper growth cone turning responses of Xenopus spinal neurons to microscopic gradients of netrin-1, brain-derived neurotrophic factor and myelin-associated glycoprotein, but not to semaphorin 3A. Furthermore, XTRPC1 is required for midline guidance of axons of commissural interneurons in the developing Xenopus spinal cord. Thus, members of the TRPC family may serve as a key mediator for the Ca2+ influx that regulates axon guidance during development and inhibits axon regeneration in adulthood.