TRPC4 and TRPC5: receptor-operated Ca-permeable nonselective cation channels

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca²⁺-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP₃) receptor activation or Ca²⁺ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca²⁺-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca²⁺ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4^−/− mice lack a highly Ca²⁺-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.     

Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production

Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1^?/? and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1^?/? mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1^?/? mice. Furthermore, contrary to expectations, Trpc1^?/? BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1^?/? BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1^?/? mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.     

Distribution of TRPC1 and TRPC5 in medial temporal lobe structures of mice

The transient receptor potential (TRP) superfamily comprises a group of non-selective cation channels that have been implicated in both receptor and store-operated channel functions. The family of the classical TRPs (TRPCs) consists of seven members (TRPC1-7). The presence of TRPC1 and TRPC5 mRNA in the brain has previously been demonstrated by real-time polymerase chain reaction. However, the distribution of these receptors within different brain areas of mice has not been investigated in detail. We have used antibodies directed against TRPC1 and TRPC5 to study the distribution and localization of these channels in murine medial temporal lobe structures. Both TRPC1 and TRPC5 channels are present in the various nuclei of the amygdala, in the hippocampus, and in the subiculum and the entorhinal cortex. We have found that TRPC1 channels are primarily expressed on cell somata and on dendrites, whereas TRPC5 channels are exclusively located on cell bodies. Moreover, TRPC1 channels are selectively expressed by neurons, whereas TRPC5 channels are mainly expressed by neurons, but also by non-neuronal cells. The expression of TRPC1 and TRPC5 channels in mammalian temporal lobe structures suggests their involvement in neuronal plasticity, learning and memory. This work was supported by the DFG (SFB 636/A5).     

TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats,but reduces severity of glomerular lesions

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in a variety of chronic kidney diseases including familial and acquired forms of focal and segmental glomerulosclerosis (FSGS) and renal fibrosis following ureteral obstruction. Here we have examined the role of TRPC6 in progression of inflammation and fibrosis in the nephrotoxic serum (NTS) model of crescentic glomerulonephritis. This was assessed in rats with non-functional TRPC6 channels due to genomic disruption of an essential domain in TRPC6 channels (Trpc6^del/del rats) and wild-type littermates (Trpc6^wt/wt rats). Administration of NTS evoked albuminuria and proteinuria observed 4 and 28 days later that was equally severe in Trpc6^wt/wt and Trpc6^del/del rats. By 28 days, there were dense deposits of complement and IgG within glomeruli in both genotypes, accompanied by severe inflammation and fibrosis readily observed by standard histological methods, and also by increases in renal cortical expression of multiple markers (α-smooth muscle actin, vimentin, NLRP3, and CD68). Tubulointerstitial fibrosis appeared equally severe in Trpc6^wt/wt and Trpc6^del/del rats. TRPC6 inactivation did not protect against the substantial declines in renal function (increases in blood urea nitrogen, serum creatinine and kidney:body weight ratio) in NTS-treated animals, and increases in a urine maker of proximal tubule pathology (β2-macroglobulin) were actually more severe in Trpc6^del/del animals. By contrast, glomerular pathology, blindly scored from histology, and from renal cortical expression of podocin suggested a partial but significant protective effect of TRPC6 inactivation within the glomerular compartment, at least during the autologous phase of the NTS model.     

Mouse transient receptor potential channel type 6 selectively regulates agonist-induced platelet function

While changes in intracellular calcium levels is a central step in platelet activation and thrombus formation, the contribution and mechanism of receptor-operated calcium entry (ROCE) via transient receptor potential channels (TRPCs) in platelets remains poorly defined. In previous studies, we have shown that TRPC6 regulates hemostasis and thrombosis, in mice. In the present studies, we employed a knockout mouse model system to characterize the role of TRPC6 in ROCE and platelet activation. It was observed that the TRPC6 deletion (Trpc6^?/?) platelets displayed impaired elevation of intracellular calcium, i.e., defective ROCE. Moreover, these platelets also exhibited defects in a host of functional responses, namely aggregation, granule secretion, and integrin αIIbβ3. Interestingly, the aforementioned defects were specific to the thromboxane receptor (TPR), as no impaired responses were observed in response to ADP or the thrombin receptor-activating peptide 4 (TRAP4). The defect in ROCE in the Trpc6^?/? was also observed with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Finally, our studies also revealed that TRPC6 regulates clot retraction. Taken together, our findings demonstrate that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Thus, TRPC6 may serve as a novel target for the therapeutic management of thrombotic diseases.     

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 Ca²⁺ influx. TRPCs are gated open by the endoplasmic reticulum Ca²⁺ 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 Ca²⁺ influx, and inhibition of Trpc3 had no further effect on Ca²⁺ 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.     

TRPC6 channel‐mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK,PI3K,and CAMKIV signaling

The non‐selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin‐induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient‐mediated changes in synaptic plasticity, ranging from calmodulin‐mediated Ras‐induced signaling cascades comprising the mitogen‐activated protein kinase, PI3K signal transduction pathways as well as Ca²⁺/calmodulin‐dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6‐mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen‐activated protein kinase/extracellular signal‐regulated kinases, phosphatidylinositide 3‐kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP‐response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract‐mediated antidepressant activity.

     

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

The transient receptor potential (TRPC) family of Ca^2 + 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 Ca^2 + 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 Ca^2 + 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 Ca^2 + influx. Thus, a) TRPC1 and TRPC5 similarly increased Ca^2 + influx but only TRPC1 induced neurite outgrowth, b) the constitutively STIM1^D76A mutant that activates Ca^2 + 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 Ca^2 + 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.     

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells

Ca²⁺-permeable store-operated channels (SOCs) mediate Ca²⁺ entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1^?/? mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1^?/? VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1^?/? VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1^?/? VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.     

Activation of TRPC4β by Gαi subunit increases Ca selectivity and controls neurite morphogenesis in cultured hippocampal neuron

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca²⁺-permeable channels. TRPC channels are activated by stimulation of Gα_q-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gα_i. We studied the essential role of Gα_i subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gα_i2 subunit. Activation of TRPC4 by Gα_i2 increased Ca²⁺ permeability and Ca²⁺ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca²⁺ influx. Moreover, both TRPC4β and the TRPC4β-Gα_i2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gα_i2^Q205L-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gα_i proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.     

Group I metabotropic glutamate receptor-dependent TRPC channel trafficking in hippocampal neurons

The group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elicited two phases of synchronized neuronal (epileptiform) discharges in hippocampal slices: an initial phase of short duration discharges followed by a phase of prolonged discharges. We assessed the involvement of transient receptor potential canonical (TRPC) channels in these responses. Pre-treatment of hippocampal slices with TRPC channel blockers, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365) or 2-aminoethoxydiphenyl borate, did not affect the short epileptiform discharges but blocked the prolonged epileptiform discharges. SKF96365 suppressed ongoing DHPG-induced prolonged epileptiform discharges. Western blot analysis showed that the total TRPC4 or TRPC5 proteins in hippocampal slices were unchanged following DHPG. DHPG increased TRPC4 and TRPC5 in the cytoplasmic compartment and decreased these proteins in the plasma membrane. Translocation of TRPC4 and TRPC5 was suppressed when the epileptiform discharges were blocked by ionotropic glutamate receptor blockers. Translocation of TRPC4 and TRPC5 was also prevented in slices from phospholipase C (PLC) beta1 knockout mice, even when synchronized discharges were elicited by the convulsant 4-aminopyridine. The results suggest that TRPC channels are involved in generating DHPG-induced prolonged epileptiform discharges. This function of TRPC channels is associated with a neuronal activity- and PLCbeta1-dependent translocation of TRPC4 and TRPC5 proteins from the plasmalemma to the cytoplasmic compartment.     

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

TRPC3 ion channel subunit immunolocalization in the cochlea

Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca²⁺) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca²⁺ homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca²⁺ entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.     

A TRPC1 Protein-dependent Pathway Regulates Osteoclast Formation and Function

Ca²⁺ signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca²⁺ stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ϵ) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ϵ in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, mediating store-operated currents. TRPC1ϵ physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ϵ-Orai1 complex through TRPC1ϵ suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ϵ and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.     

Functional characteristics of TRPC4 channels expressed in HEK 293 cells

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by GTPγS was not desensitized. TPRC4 activation by GTPγS was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK _a of 7.3. Finally, TPRC4 activation by GTPγS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles. These authors contributed equally to this work.     

Distribution of TRPC4 in developing and adult murine brain

The transient receptor potential (TRP) superfamily comprises of a group of non-selective cation channels that have been implicated in both receptor and store-operated channel functions. The family of classical TRPs (TRPCs) consists of seven members (TRPC1-7), with TRPC4 possibly playing a role in neuronal signaling. We have examined the distribution pattern of TRPC4 mRNA and protein in the developing and postnatal murine brain by using in situ hybridization, Western blotting, and immunocytochemistry. Expression of TRPC4 mRNA starts at embryonic day 14.5 (E14.5) in the developing septal area and cerebellar anlagen. At E16.5, prominent expression is additionally seen in the hippocampal formation and cortical plate. High densities of cells expressing TRPC4 mRNA occur in the adult olfactory bulb and hippocampus, whereas the cortex and septum display lower densities of cells positive for TRPC4 mRNA. Analysis of the adult hippocampal formation has revealed TRPC4 immunoreactivity in hippocampal areas CA1 to CA3 and in the dentate gyrus. Functions consistent with this spatially restricted pattern of expression remain to be revealed.     

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

The Kv2.1 delayed rectifier potassium channel exhibits high‐level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity‐dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1^?/?) mice lacking this channel. Kv2.1^?/? mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1^?/? mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1^?/? mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1^?/? animals. Field recordings from hippocampal slices of Kv2.1^?/? mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1^?/? mice, long‐term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1^?/? mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1^?/? mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1^?/? mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function .     

TRPC6 and TRPC4 Heteromultimerization Mediates Store Depletion-Activated NCX1 Reversal in Proliferative Vascular Smooth Muscle Cells

Store depletion has been shown to induce Ca²⁺ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na⁺ was followed by a significant increase of cytosolic Ca²⁺ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca²⁺ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na⁺ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4–TRPC6 heteromultimerization linked Ca²⁺ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.