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Traditionally prescribed for mood disorders, tricyclic antidepressants (TCAs) have shown promising therapeutic effects on chronic neuralgia and irritable bowel syndrome. However, the mechanism by which these atypical effects manifest is unclear. Among the proposed mechanisms is the well-known pain-related inhibitory G-protein coupled receptor (GiPCR), namely, the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR and regulates the gating of TRPC4, a downstream signaling of the Gi-pathway. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/Gi-pathway, treatment with amitriptyline (AMI) showed a decrease in [cAMP]i similar to that of the μOR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of μOR. A conserved aspartate residue of ORs was predicted to participate in salt bridge interaction with the amine group of TCAs, and in aspartate-to-arginine mutation, AMI did not decrease the FRET-based binding efficiency between the ORs and Gαi2. As an alternative way to monitor the downstream signaling of Gi-pathway, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gαi. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gαi2 or its dominant-negative mutant. As expected, TCA-evoked activation of TRPC4 was not observed in the aspartate mutants of OR. Taken together, OR could be proclaimed as a promising target among numerous binding partners of TCA, and TCA-evoked TRPC4 activation may help to explain the nonopioid analgesic effect of TCA.

Background: Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca2+-permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and by what mechanism in colonic myocytes consequently causes constipation.Methods: To prove the clinical benefit in patients with diarrhea by TCA treatment, we performed mechanical tension recording using human colonic migrating motor complexes (CMMCs) and isolated muscle strips. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes.Results: All TCA compounds significantly inhibited the spontaneous contraction of CMMCs and muscle strips isolated from CMMCs by TRPC4 inhibition of colonic myocytes. In TRPC4-overexpressed HEK cells, we observed TCA-evoked direct inhibition of TRPC4 with IC50 values. Compared with TRPC4-knockout mice, we identified that muscarinic cationic current (mIcat) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Conclusions: We investigated the inhibitory effect of the TRPC4 current by TCAs on colonic motility in TCA-induced constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy.

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels function as pacemaker channels in spontaneously active cells. We studied the existence of HCN channels and their functional roles in the interstitial cells of Cajal (ICC) from the mouse colon using electrophysiological, immunohistochemical and molecular techniques. HCN1 and HCN3 channels were detected in anoctamin‐1 (Ca2+‐activated Cl− channel; ANO1)‐positive cells within the muscular and myenteric layers in colonic tissues. The mRNA transcripts of HCN1 and HCN3 channels were expressed in ANO1‐positive ICC. In the deletion of HCN1 and HCN3 channels in colonic ICC, the pacemaking potential frequency was reduced. Basal cellular adenylate cyclase activity was decreased by adenylate cyclase inhibitor in colonic ICC, whereas cAMP‐specific phosphodiesterase inhibitors increased it. 8‐Bromo‐cyclic AMP and rolipram increased spontaneous intracellular Ca2+ oscillations. In addition, Ca2+‐dependent adenylate cyclase 1 (AC1) mRNA was detected in colonic ICC. Sulprostone, a PGE2‐EP3 agonist, increased the pacemaking potential frequency, maximum rate of rise of resting membrane in pacemaker potentials and basal cellular adenylate cyclase activity in colonic ICC. These results indicate that HCN channels exist in colonic ICC and participate in generating pacemaking potentials. Thus, HCN channels may be therapeutic targets in disturbed colonic motility disorders.

Transient receptor potential canonical (TRPC) channels are calcium permeable, non-selective cation channels with wide tissue-specific distribution. Among 7 TRPC channels, TRPC 1/4/5 and TRPC3/6/7 are subdivided based on amino acid sequence homology. TRPC4 and TRPC5 channels exhibit cationic current with homotetrameric form, but they also form heterotetrameric channel such as TRPC1/4 or TRPC1/5 once TRPC1 is incorporated. The expression of TRPC1 is ubiquitous whereas the expressions of TRPC4 and TRPC5 are rather focused in nervous system. With the help of conditional knock-out of TPRC1, 4 and/or 5 genes, TRPC channels made of these constituents are reported to be involved in various pathophysiological functions such as seizure, anxiety-like behaviour, fear, Huntington's disease, Parkinson's disease and many others. In heterologous expression system, many issues such as activation mechanism, stoichiometry and relative cation permeabilites of homomeric or heteromeric channels have been addressed. In this review, we discussed the role of TRPC1 channel per se in plasma membrane, role of TRPC1 in heterotetrameric conformation (TRPC1/4 or TRPC1/5) and relationship between TRPC1/4/5 channels, calcium influx and voltage-gated calcium channels.

Background/Aims: Anoctamin1 (Ca2+-activated Cl- channel, ANO1) is a specific marker of the interstitial cells of Cajal (ICC) in the gastrointestinal tract, and are candidate proteins that can function as pacemaker channels. Recently, novel selective ANO1 inhibitors were discovered and used to study Ca2+-activated Cl- channels. Therefore, to investigate whether ANO1 channels function as pacemaker channels, selective ANO1 inhibitors were tested with respect to the pacemaker potentials in ICC. Methods: Whole-cell patch-clamp recording, RT-PCR, and intracellular Ca2+ ([Ca2+]i) imaging were performed in cultured ICC obtained from mice. Results: Though CaCCinh-A01 (5 µM), T16Ainh-A01 (5 µM), and MONNA (5 µM) (selective ANO1 inhibitors) blocked the generation of pacemaker potentials in colonic ICC, they did not do so in small intestinal ICC. Though nifulmic acid (10 µM) and DIDS (10 µM) (classical Ca2+-activated Cl- channel inhibitors) also had no effect in small intestinal ICC, they suppressed the generation of pacemaker potentials in colonic ICC. In addition, knockdown of ANO1 reduced the pacemaker potential frequency in colonic ICC alone. Though ANO1 inhibitors suppressed [Ca2+]i oscillations in colonic ICC, they did not do so in small intestinal ICC. T-type Ca2+ channels were expressed in the both the small intestinal and colonic ICC, but mibefradil (5 µM) and NiCl2 (30 µM) (T-type Ca2+ channel inhibitors) inhibited the generation of pacemaker potentials in colonic ICC alone. Conclusion: These results indicate that though ANO1 and T-type Ca2+ channels participate in generating pacemaker potentials in colonic ICC, they do not do so in small intestinal ICC. Therefore, the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

Background The discovery of receptor activator of nuclear factor‐ĸB ligand (RANKL) as the final effector in the pathogenesis of osteoporosis has led to a better understanding of bone remodeling. When RANKL binds to its receptor (RANK), osteoclastic differentiation and activation are initiated. Herein, we propose a strategy using a novel RANKL variant as a competitive inhibitor for RANKL. The RANKL variant activates LGR4 signaling, which competitively regulates RANK and acts as an immunogen that induces anti‐RANKL antibody production. Methods We modified the RANK‐binding site on RANKL using minimal amino acid changes in the RANKL complex and its counterpart receptor RANK and tried to evaluate the inhibitory effects on osteoclastogenesis. Results The novel RANKL variant did not bind RANK in osteoclast progenitor cells, but activated LGR4 through the GSK3‐β signaling pathway, thereby suppressing activated T cell cytoplasmic nuclear factor calcineurin‐dependent 1 (NFATc1) expression and activity during osteoclastogenesis. Our RANKL variant generated high levels of RANKL‐specific antibodies, blocked osteoclastogenesis, and inhibited osteoporosis in ovariectomized mouse models. Generated anti‐RANKL antibodies showed a high inhibitory effect on osteoclastogenesis in vivo and in vitro. Conclusions We observed that the novel RANKL indeed blocks RANKL via LGR4 signaling and generates anti‐RANKL antibodies, demonstrating an innovative strategy in the development of general immunotherapy., Schematic diagram of mRANKL‐MT3 in dual inhibitory effect against RANKL during osteoclastogenesis. The first effect of mRANKL‐MT3 is induced to RANKL‐LGR4 modulation of the RANKL–NFATc1 signaling cascade by a negative‐feedback mechanism, which controls osteoclast activity. The second effect is induced to anti‐RANKL generation by mutant RANKL, which inhibits osteoclastogenesis and bone erosion.

Oxidative stress is a key mediator of neuronal death in acute brain injuries, such as epilepsy, trauma, and stroke. Although it is accompanied by diverse cellular changes, increases in levels of intracellular zinc ion (Zn2+) and calcium ion (Ca2+) may play a critical causative role in oxidative neuronal death. However, the mechanistic link between Zn2+ and Ca2+ dyshomeostasis in neurons during oxidative stress is not well-understood. Here, we show that the exposure of cortical neurons to H2O2 led to a zinc-triggered calcium influx, which resulted in neuronal death. The cyclin-dependent kinase inhibitor, NU6027, inhibited H2O2-induced Ca2+ increases and subsequent cell death in cortical neurons, without affecting the early increase in Zn2+. Therefore, we attempted to identify the zinc-regulated Ca2+ pathway that was inhibited by NU6027. The expression profile in cortical neurons identified transient receptor potential cation channel 5 (TRPC5) as a candidate that is known to involve in the generation of epileptiform burst firing and epileptic neuronal death (Phelan KD et al. 2012a; Phelan KD et al. 2013b). NU6027 inhibited basal and zinc-augmented TRPC5 currents in TRPC5-overexpressing HEK293 cells. Consistently, cortical neurons from TRPC5 knockout mice were highly resistant to H2O2-induced death. Moreover, NU6027 is neuroprotective in kainate-treated epileptic rats. Our results demonstrate that TRPC5 is a novel therapeutic target against oxidative neuronal injury in prolonged seizures and that NU6027 is a potent inhibitor of TRPC5.

Objective: Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. Methods: We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. Results: We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. Interpretation: GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated GABA signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. This article is protected by copyright. All rights reserved.

To investigate the role of ATP-sensitive K+(KATP) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca2+([Ca2+]i) imaging were performed in cultured colonic ICC. Pinacidil (a K+ channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a KATP channel blocker). RT-PCR showed that Kir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial KATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca2+([Ca2+]i) oscillations showed that glibenclamide increased the frequency of [Ca2+]i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and Kir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, KATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential.

EP receptor activation by PGE2 regulates gastrointestinal motility by modulating smooth muscle contractility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate smooth muscle activity. We aimed to determine effects of the EP3 receptor agonist sulprostone on pacemaker potentials in colonic ICCs. We performed a whole cell patch clamp, RT-PCR, and Ca2+ imaging in cultured ICCs from mouse colon. Sulprostone depolarized the membrane and increased pacemaker frequency. EP3 receptor antagonist blocked these sulprostone-induced effects. EP3 receptors were expressed in ANO1-positive ICCs. Phospholipase C inhibitor or Ca2+-ATPase inhibitor from the endoplasmic reticulum blocked the sulprostone-induced effects and sulprostone increased intracellular Ca2+ ([Ca2+]i) oscillations. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers also suppressed the sulprostone-induced effects. Sulprostone enhanced pacemaker activity through EP3 receptors by activating HCN channels via the [Ca2+]i release pathway. Therefore, EP3 receptor activation in ICCs may modulate colonic motility and could be a therapeutic target for enhancing colonic GI motility.

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16‑carbon fatty acid palmitate, is the most common acylation, and it enhances the membrane stability of ion channels. This post-translational modification (PTM) determines a functional mechanism of ion channel life cycle from maturation and membrane trafficking to localization. Especially, neurodevelopment is regulated by balancing the level of synaptic protein palmitoylation/depalmitoylation. Recently, we revealed the pathological role of the transient receptor potential canonical type 5 (TRPC5) channel in striatal neuronal loss during Huntington's disease (HD), which is abnormally activated by oxidative stress. Here, we report a mechanism of TRPC5 palmitoylation at a conserved cysteine residue, that is critical for intrinsic channel activity. Furthermore, we identified the therapeutic effect of TRPC5 depalmitoylation by enhancing the TRPC5 membrane instability on HD striatal cells in order to lower TRPC5 toxicity. Collectively, these findings suggest that controlling S-palmitoylation of the TRPC5 channel as a potential risk factor can modulate TRPC5 channel expression and activity, providing new insights into a therapeutic strategy for neurodegenerative diseases.

The study of the structure−function relationship of ion channels has been one of the most challenging goals in contemporary physiology. Revelation of the three-dimensional (3D) structure of ion channels has facilitated our understanding of many of the submolecular mechanisms inside ion channels, such as selective permeability, voltage dependency, agonist binding, and inter-subunit multimerization. Identifying the structure−function relationship of the ion channels is clinically important as well since only such knowledge can imbue potential therapeutics with practical possibilities. In a sense, recent advances in the understanding of the structure−relationship of transient receptor potential canonical (TRPC) channels look promising since human TRPC channels are calcium-permeable, non-selective cation channels expressed in many tissues such as the gastrointestinal (GI) tract, kidney, heart, vasculature, and brain. TRPC channels are known to regulate GI contractility and motility, pulmonary hypertension, right ventricular hypertrophy, podocyte injury, seizure, fear, anxiety-like behavior, and many others. In this article, we tried to elaborate recent findings of Cryo-EM (cryogenic-electron microscopy) based structural information of TRPC 4 and 5 channels and domain-specific functions of the channel, such as G-protein mediated activation mechanism, extracellular modification of the channel, homo/hetero-tetramerization, and pharmacological gating mechanisms.

The transient receptor potential canonical (TRPC) 1 channel is widely distributed in mammalian cells and is involved in many physiological processes. TRPC1 is primarily considered a regulatory subunit that forms heterotetrameric channels with either TRPC4 or TRPC5 subunits. Here, we suggest that the regulation of TRPC1/4 and TRPC1/5 heterotetrameric channels by the Gαq-PLCβ pathway is self-limited and dynamically mediated by Gαq and PI(4,5)P2. We provide evidence indicating that Gαq protein directly interacts with either TRPC4 or TRPC5 of the heterotetrameric channels to permit activation. Simultaneously, Gαq-coupled PLCβ activation leads to the breakdown of PI(4,5)P2, which inhibits activity of TRPC1/4 and 1/5 channels.

The transient receptor potential canonical (TRPC) 1 channel is widely distributed in mammalian cells and is involved in many physiological processes. TRPC1 is primarily considered a regulatory subunit that forms heterotetrameric channels with either TRPC4 or TRPC5 subunits. Here, we suggest that the regulation of TRPC1/4 and TRPC1/5 heterotetrameric channels by the Gαq-PLCβ pathway is self-limited and dynamically mediated by Gαq and PI(4,5)P2. We provide evidence indicating that Gαq protein directly interacts with either TRPC4 or TRPC5 of the heterotetrameric channels to permit activation. Simultaneously, Gαq-coupled PLCβ activation leads to the breakdown of PI(4,5)P2, which inhibits activity of TRPC1/4 and 1/5 channels.

Oxidative stress is a key mediator of neuronal death in acute brain injuries, such as epilepsy, trauma, and stroke. Although it is accompanied by diverse cellular changes, increases in levels of intracellular zinc ion (Zn

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5'-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca2+ ([Ca2+]i) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na+-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca2+ or treatment with thapsigargin (inhibitor of Ca2+ uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca2+]i oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca2+ influx and [Ca2+]i release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.

To investigate the role of ATP-sensitive K

Crucial cysteine residues can be involved in the modulation of protein activity via the modification of thiol (-SH) groups. Among these reactions, disulfide bonds (S-S) play a key role in the folding, stability, and activity of membrane proteins. However, the regulation of extracellular cysteines in classical transient receptor potential (TRPC) channels remains controversial. Here, we examine the functional importance of the extracellular disulfide bond in TRPC5 in modulating channel gating and trafficking. Specifically, we investigated TRPC5 activity in transiently transfected HEK293 cells with wild-type (WT) or cysteine (C553 and C558) mutants in the pore loop. Using reducing agents, we determined that a disulfide linkage mediates the tetrameric formation of the TRPC5 channel. By measuring the TRPC5 current, we observed that C553S or C558S mutants completely lose channel activity induced by lanthanides or receptor stimulation. Co-expression of TRPC5 (WT) with mutants demonstrated a dominant-negative function in mutants, which inhibited the activity of TRPC5 (WT). We generated TRPC5-TRPC5 dimers and observed reduced activity of WT-mutant (C553S or C558S) dimers compared to WT-WT dimers. When pretreated with reducing agents for 12 h, the TRPC5 current decreased due to a reduction in membrane TRPC5 distribution. In addition, we identified a reduced expression of C553S mutant in plasma membrane. We analyzed a dimeric interaction of wild-type and mutant TRPC5 using co-immunoprecipitation and FRET method, indicating a weak interaction between dimeric partners. These results indicated that the disulfide bond between conserved extracellular cysteines, especially C553, is essential for functional TRPC5 activity by channel multimerization and trafficking.

Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear. NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB–TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6–NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB–TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

Background/Aims We investigated the role of representative endoplasmic reticulum proteins, stromal interaction molecule 1 (STIM1), and store-operated calcium entry-associated regulatory factor (SARAF) in pacemaker activity in cultured interstitial cells of Cajal (ICCs) isolated from mouse small intestine. Methods The whole-cell patch clamp technique applied for intracellular calcium ions ([Ca2+]i) analysis with STIM1 or SARAF overexpressed cultured ICCs from mouse small intestine. Results In the current-clamping mode, cultured ICCs displayed spontaneous pacemaker potentials. External carbachol exposure produced tonic membrane depolarization in the current-clamp mode, which recovered within a few seconds into normal pacemaker potentials. In STIM1-overexpressing cultured ICCs pacemaker potential frequency was increased, and in SARAF-overexpressing ICCs pacemaker potential frequency was strongly inhibited. The application of gadolinium (a non-selective cation channel inhibitor) or a Ca2+-free solution to understand Orai channel involvement abolished the generation of pacemaker potentials. When recording intracellular Ca2+ concentration with Fluo 3-AM, STIM1-overexpressing ICCs showed an increased number of spontaneous intracellular Ca2+ oscillations. However, SARAF-overexpressing ICCs showed fewer spontaneous intracellular Ca2+ oscillations. Conclusion Endoplasmic reticulum proteins modulated the frequency of pacemaker activity in ICCs, and levels of STIM1 and SARAF may determine slow wave patterns in the gastrointestinal tract.

Scutellaria baicalensis Georgi extract (SBGE) is used in traditional herbal medicine and has also been used clinically to ameliorate the symptoms of various inflammatory diseases and cancer. In women, breast cancer is one of the most common diseases and numerous women succumb to it. The present study was undertaken to investigate the mechanism responsible for the SBGE‑induced apoptosis of MCF‑7 human breast cancer cells. SBGE was administered to cells at concentrations between 100 and 500 mg/ml, and cell viabilities were identified using an MTT assay. B‑cell lymphoma 2 (Bcl-2) and Bcl-2 X‑associated protein (Bax) family members were identified by western blotting, and the mRNA expression levels of the pro‑apoptosis genes Fas, Fas ligand (FasL) and tumor necrosis factor (TNF)‑α were assessed by reverse transcription‑polymerase chain reaction. It was identified that SBGE treatment for 24 h inhibited MCF‑7 proliferation and increased the sub‑G1 phase ratio. SBGE suppressed mitochondrial membrane potentials and SBGE‑induced apoptotic cell death was identified to be associated with downregulation of Bcl‑2, but upregulation of Bax. SBGE‑activated caspases 3 and 9, and increased reactive oxygen species generation. However, SBGE had no effect on the expression levels of Fas, FasL or TNF‑α. Furthermore, mitogen‑activated protein kinase and C‑Jun N‑terminal kinase inhibitors inhibited SBGE‑induced cell death. These results suggested that SBGE be considered as an agent for the treatment of breast cancer.

Transient receptor potential canonical (TRPC) 1, the first mammalian homologue of Drosophila trp gene, is distributed widely in mammalian cells and is involved in many physiological functions. TRPC1 is reported to be functional following heteromeric formation with other TRPC channels such as TRPC4 or TRPC5. It is known that the composition of this widely distributed TRPC1 is far from simple; functionality of such channels has been highly controversial. Furthermore, TRPC1 gene is known to have two splicing variants; one encodes long (TRPC1α) and the other encodes short (TRPC1β) TRPC1 isoforms, respectively. In this study, we examined the functionality of TRPC1/4 channels using various activation systems. Gq/11-coupled receptor (e.g., M1 or M3 receptors) stimulation significantly increased TRPC1α/4 currents but induced mild activation of TRPC1β/4. In addition, when expressed with TRPC4, TRPC1α acted as a pore-constituting subunit and not a β ancillary subunit. Multimerized with TRPC4, TRPC1α also generated strong pore field strength. We also found that Gi/o-coupled receptor (e.g., M2 receptor) stimulation was insufficient to activate TRPC1α/4 and TRPC1β/4 channels but selectively activated TRPC4 homomeric channels. These findings demonstrate that TRPC1/4 channel shows dynamic gating property depending on TRPC1 isoform subtypes and receptor stimulation system. Therefore, careful discrimination of the specificity of TRPC1 isoforms and upstream activation system is important in thorough understanding of TRPC1 and TRPC1/4 channels.

The transient receptor potential canonical 4 (TRPC4) channel is a Ca2+-permeable nonselective cation channel in mammalian cells and mediates a number of cellular functions. Many studies show that TRPC channels are activated by stimulation of Gαq-phospholipase C (PLC)-coupled receptors. However, our previous study showed that the TRPC4 current was inhibited by co-expression of a constitutively active form of Gαq (Gαq Q209L). A shortage of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in Gαq Q209L may be responsible for reduced TRPC4 activity. Here, we tested this hypothesis by using a rapamycin-inducible system that regulates PI(4,5)P2 acutely and specifically. Our results showed that the TRPC4β current was reduced by inducible Gαq Q209L, but not by the mutants with impaired binding ability to PLCβ. Depletion of PI(4,5)P2 by inducing the inositol polyphosphate 5-phosphatase to HEK293 cells that express TRPC4β led to an irreversible inhibition of TRPC4β currents. In contrast, inducing phosphatidylinositol 4-phosphate 5-kinase or intracellular PI(4,5)P2 application did not activate the TRPC4β current. Finally, we revealed that PI(4,5)P2 is important in delaying the desensitization of TRPC4β. Taken together, we suggest that PI(4,5)P2 is not the activator of TRPC4β activation, but it is still necessary for regulating TRPC4β activation.

The chloride channel (CLC) family of proteins consists of channels and transporters that share similarities in architecture and play essential roles in physiological functions. Among the CLC family, CLC-1 channels have the representative homodimeric double-barreled structure carrying two gating processes. One is protopore gating that acts on each pore independently by glutamate residue (Eext). The other is common gating that closes both pores simultaneously in association with large conformational changes across each subunit. In skeletal muscle, CLC-1 is associated with maintaining normal sarcolemmal excitability, and a number of myotonic mutants were reported to modify the channel gating of CLC-1. In this study, we characterized highly conserved helix O as a key determinant of structural stability in CLC-1. Supporting this hypothesis, myotonic mutant (G523D) at N-terminal of helix O showed the activation at hyperpolarizing membrane potentials with a reversed voltage dependency. However, introducing glutamate at serine residue (S537) at the C-terminal of the helix O on G523D restored WT-like voltage dependency of the common gate and showed proton insensitive voltage dependency. To further validate this significant site, site-specific mutagenesis experiments was performed on V292 that is highly conserved as glutamate in antiporter and closely located to S537 and showed that this area is essential for channel function. Taken together, the results of our study suggest the importance of helix O as the main contributor for stable structure of evolutionary conserved CLC proteins and its key role in voltage dependency of the CLC-1. Furthermore, the C-terminal of the helix O can offer a clue for possible proton involvement in CLC-1 channel.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.

TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.

Ginger extract is used as an analeptic in herbal medicine and has been reported to exert antioxidant effects. Transient receptor potential (TRP) canonical 5 (TRPC5), TRP cation channel, subfamily M, member 7 (TRPM7; melastatin 7), and TRP cation channel, subfamily A, member 1 (TRPA1; ankyrin 1) are non-selective cation channels that are modulated by reactive oxygen/nitrogen species (ROS/RNS) and subsequently control various cellular processes. The aim of this study was to evaluate whether ginger and its pungent constituents modulate these channels and exert antioxidant effects. It was found that TRPC5 and TRPA1 currents were modulated by ginger extract and by its pungent constituents, [6]-gingerol, zingerone and [6]-shogaol. In particular, [6]-shogaol markedly and dose-dependently inhibited TRPC5 currents with an IC50 of value of ~18.3 µM. Furthermore, the strong dose-dependent activation of TRPA1 currents by [6]-shogaol was abolished by A‑967079 (a selective TRPA1 inhibitor). However, ginger extract and its pungent constituents had no effect on TRPM7 currents. These results suggest the antioxidant effects of ginger extract and its pungent constituents are mediated through TRPC5 and TRPA1, and that [6]-shogaol is predominantly responsible for the regulation of TRPC5 and TRPA1 currents by ginger extract.

Canonical transient receptor potential (TRPC) channels are Ca2+-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gαs. Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gαs[Gαs(Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gαs(Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by Gs/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β2-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.

Aberrant glutathione or Ca(2+) homeostasis due to oxidative stress is associated with the pathogenesis of neurodegenerative disorders. The Ca(2+)-permeable transient receptor potential cation (TRPC) channel is predominantly expressed in the brain, which is sensitive to oxidative stress. However, the role of the TRPC channel in neurodegeneration is not known. Here, we report a mechanism of TRPC5 activation by oxidants and the effect of glutathionylated TRPC5 on striatal neurons in Huntington's disease. Intracellular oxidized glutathione leads to TRPC5 activation via TRPC5 S-glutathionylation at Cys176/Cys178 residues. The oxidized glutathione-activated TRPC5-like current results in a sustained increase in cytosolic Ca(2+), activated calmodulin-dependent protein kinase and the calpain-caspase pathway, ultimately inducing striatal neuronal cell death. We observed an abnormal glutathione pool indicative of an oxidized state in the striatum of Huntington's disease transgenic (YAC128) mice. Increased levels of endogenous TRPC5 S-glutathionylation were observed in the striatum in both transgenic mice and patients with Huntington's disease. Both knockdown and inhibition of TRPC5 significantly attenuated oxidation-induced striatal neuronal cell death. Moreover, a TRPC5 blocker improved rearing behaviour in Huntington's disease transgenic mice and motor behavioural symptoms in littermate control mice by increasing striatal neuron survival. Notably, low levels of TRPC1 increased the formation of TRPC5 homotetramer, a highly Ca(2+)-permeable channel, and stimulated Ca(2+)-dependent apoptosis in Huntington's disease cells (STHdh(Q111/111)). Taken together, these novel findings indicate that increased TRPC5 S-glutathionylation by oxidative stress and decreased TRPC1 expression contribute to neuronal damage in the striatum and may underlie neurodegeneration in Huntington's disease.

TPRC channels are Ca2+-permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). TRPC4 is commonly assumed to be activated by Gq/phospholipase C-coupled receptors. However, the other molecular mechanisms by which Gα proteins regulate TRPC4 remain unclear. Here, we found that Gαi2regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4β-CFP and the constitutively active mutant form of Gαi2was nearly 15% and was greater than that observed with wild-type Gαi2(nearly 5%). Gβγ and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4β-CFP and Gαi2(WT)-YFP from 4.7 ± 0.4% ( n = 7) to 12.6 ± 1.4% ( n = 7). We also found that the TRPC4 channel directly interacts with Gαi2, but not with Gαq, when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gαi2or Gαqusing the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gαi2-, and TRPC4-expressing cells showed a prolonged Ca2+influx compared with cells expressing only M2. Together, these data suggest that Gαi2activates the TRPC4 channel by direct binding, which then induces Ca2+entry.

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). The TRPC4 channel is expressed in a punctate distribution in the membrane. To identify the regulating region of the channel trafficking to the membrane, we generated deletion mutants of the TRPC4 channel. We determined that when either region that was downstream of the 20 amino acids of the N terminus or the 700-730 amino acids was deleted, the mutants were retained in the endoplasmic reticulum. By coexpression of the wild-type TRPC4 with deletion mutants, we found that the 23-29 amino acids of the N terminus regulate a membrane trafficking. Additionally, by the fluorescence resonance energy transfer (FRET) method, we found that the regions downstream of the 99 amino acid region of the N terminus and upstream of the 730 amino acid region in the C terminus produce assembly of the TRPC4 tetramers. We inferred the candidate proteins that regulate or interact with the 23-29 domain of TRPC4.

ClC-1 is a member of a large family of voltage-gated chloride channels, abundantly expressed in human skeletal muscle. Mutations in ClC-1 are associated with myotonia congenita (MC) and result in loss of regulation of membrane excitability in skeletal muscle. We studied the electrophysiological characteristics of six mutants found among Korean MC patients, using patch clamp methods in HEK293 cells. Here, we found that the autosomal dominant mutants S189C and P480S displayed reduced chloride conductances compared to WT. Autosomal recessive mutant M128I did not show a typical rapid deactivation of Cl(-) currents. While sporadic mutant G523D displayed sustained activation of Cl(-) currents in the whole cell traces, the other sporadic mutants, M373L and M609K, demonstrated rapid deactivations. V1/2 of these mutants was shifted to more depolarizing potentials. In order to identify potential effects on gating processes, slow and fast gating was analyzed for each mutant. We show that slow gating of the mutants tends to be shifted toward more positive potentials in comparison to WT. Collectively, these six mutants found among Korean patients demonstrated modifications of channel gating behaviors and reduced chloride conductances that likely contribute to the physiologic changes of MC.

The transient receptor potential canonical 4 (TRPC4) channel is a Ca(2+)-permeable nonselective cation channel in mammalian cells and mediates a number of cellular functions. Many studies show that TRPC channels are activated by stimulation of Gαq-phospholipase C (PLC)-coupled receptors. However, our previous study showed that the TRPC4 current was inhibited by co-expression of a constitutively active form of Gαq (Gαq (Q209L)). A shortage of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in Gαq (Q209L) may be responsible for reduced TRPC4 activity. Here, we tested this hypothesis by using a rapamycin-inducible system that regulates PI(4,5)P2 acutely and specifically. Our results showed that the TRPC4β current was reduced by inducible Gαq (Q209L), but not by the mutants with impaired binding ability to PLCβ. Depletion of PI(4,5)P2 by inducing the inositol polyphosphate 5-phosphatase to HEK293 cells that express TRPC4β led to an irreversible inhibition of TRPC4β currents. In contrast, inducing phosphatidylinositol 4-phosphate 5-kinase or intracellular PI(4,5)P2 application did not activate the TRPC4β current. Finally, we revealed that PI(4,5)P2 is important in delaying the desensitization of TRPC4β. Taken together, we suggest that PI(4,5)P2 is not the activator of TRPC4β activation, but it is still necessary for regulating TRPC4β activation.

Canonical transient receptor potential (TRPC) channels are Ca 2+ -permeable, non-selective cation channels those are widely expressed in mammalian cells. Various molecules have been found to regulate TRPC both in vivo and in vitro , but it is unclear how heterotrimeric G proteins transmit external stimuli to regulate the activity of TRPC5. Here, we demonstrated that TRPC5 was potentiated by the Gα s regulatory pathway. Whole-cell TRPC5 current was significantly increased by β-adrenergic receptor agonist, isoproterenol (ISO, 246 ± 36%, n = 6), an activator of the adenylate cyclase, forskolin (FSK, 273 ± 6%, n = 5), or a membrane permeable cAMP analogue, 8-Br-cAMP (251 ± 63%, n = 7). In addition, robust Ca 2+ transient induced by isoproterenol was observed utilizing a Ca 2+ imaging technique. When intracellular [Ca 2+ ] i was buffered to 50 nM, cAMP-induced potentiation was attenuated. We also found that the Ca 2+ release is mediated by IP 3 since intracellular IP 3 infusion attenuated the potentiation of TRPC5 by Gα s cascade. Finally, we identified that the membrane localization of TRPC5 was significantly increased by ISO (155 ± 17%, n = 3), FSK (172 ± 39%, n = 3) or 8-Br-cAMP (216 ± 59%, n = 3). In conclusion, these results suggest that the Gα s –cAMP pathway potentiates the activity of TRPC5 via facilitating intracellular Ca 2+ dynamics and increasing channel trafficking to the plasma membrane.

Canonical type of TRP channels have been reckoned as a molecular candidate for Ca2+-permeable, nonselective cation channels which are ubiquitously expressed in mammalian cells. While there are various already-reported regulators both in vivo and in vitro, little is known, however, how heterotrimeric G-protein mediates external stimuli into TRPC5 function. We previously reported that Gs negatively regulates TRPC5 via direct phosphorylation by PKA and here, we demonstrate novel regulatory pathway regarding TRPC5 of which Gs downstream molecules govern through IP3-mediated Ca2+ release. We performed various drug administrations during whole-cell patch clamp recording in the hTRPC5-transiently expressing HEK293 cells. Confocal Laser Scanning Microscopy was aided in addition to patch-clamping to measure membrane portion of TRPC5. We found that TRPC5 whole-cell current was increased by β-adrenergic receptor specific agonist, isoproterenol (246 ± 36%), adenylyl cyclase activator, forskolin (273 ± 6%), and membrane permeable analogue of cAMP, 8-Br-cAMP (232 ± 14%), whereas translocation onto plasma membrane was not the case. To assert the possibility of mediating Ca2+-release, we infused inositol triphosphate (IP3) in pipette solution which diminished potentiating effect of the drugs. In addition, population Ca2+ imaging showed robust Ca2+ release after drug treatment. Altogether, we conclude that Gs pathway regulates TRPC5 via not only inhibitory PKA phosphorylation but also activating Ca2+ release.∗This research was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MEST) (2008-2005948 and 2010-0019472).

The voltage-gated K(+) channel Kv2.1 is expressed as a highly phosphorylated protein in most central neurons, where it plays a key role in regulating neuronal membrane excitability. Previous studies have shown that Kv2.1 channel activity is upregulated by Src-mediated phosphorylation through an unknown mechanism. However, a systematic analysis of the molecular mechanism of Kv2.1 channel phosphorylation by Src is lacking. Here, we show that tyrosine phosphorylation by Src plays a fundamental role in regulating Kv2.1-mediated K(+) current enhancement. We found that the level of expression of the Kv2.1 protein is increased by Src kinase. Using mass spectrometric proteomic techniques, we identified two novel phosphotyrosine sites, Y686 and Y810, in the cytoplasmic domains of Kv2.1. We found that Src-dependent phosphorylation at these sites affects Kv2.1 through distinct regulatory mechanisms. Whereas phosphorylation at Y686 regulates Kv2.1 activity similarly to the known site Y124, phosphorylation at Y810 plays a significant role in regulating the intracellular trafficking of Kv2.1 channels. Our results show that these two novel tyrosine phosphorylation sites of Kv2.1 are crucial to regulating diverse aspects of Kv2.1 channel function and provide novel insights into molecular mechanisms for the regulation of Src-dependent modulation of Kv2.1 channels.

We investigated the molecular identity and functional activity of STIM1 and ORAI in human cavernous smooth muscle. We also determined whether transferring dominant negative mutants of the STIM1 or ORAI gene would correct diabetes related erectile dysfunction in a rat model.Reverse transcriptase-polymerase chain reaction was done to identify ORAI and STIM in human cavernous smooth muscle. For the in vivo study intracavernous pressure, blood pressure and their ratio were assessed after cavernous nerve stimulation to diabetic rats transfected with pcDNA encoding the ORAI1(DN) or the STIM1(DN) gene.ORAI (1, 2 and 3) and STIM (1 and 2) were identified in human cavernous smooth muscle cells. After [Ca(2+)] depletion by thapsigargin and cyclopiazonic acid we recorded store operated Ca(2+) entry in human cavernous smooth muscle cells. Entry was decreased by the store operated Ca(2+) channel blockers La(3+) and SKF96365. Mean ± SE intracavernous pressure/blood pressure in rats with ORAI1(DN) or STIM1(DN) gene transfer was 78.8% ± 2.2% and 77.1% ± 1.2% in 11 and 10, respectively. This result was significantly higher than that in 10 diabetic controls (51.0% ± 3.7%) and similar to that in 9 normal controls (85.8% ± 2.6%). Using reverse transcriptase-polymerase chain reaction we confirmed transgene expression in rat cavernous tissue.Transfer of ORAI(DN) or STIM1(DN) genes restored erectile function in diabetic rats. It might be applicable to develop new therapy for erectile dysfunction.

In this paper two variants of an autoregressive, leading indicator (ARLI) model are employed to forecast turning points in the growth rates of 18 countries' annual real output for the years 1974–1986. Bayesian predictive densities have been employed to compute probabilities of downturns and upturns, and they have been employed in a Bayesian decision-theoretic framework to obtain optimal turning point forecasts. Various versions of the ARLI model, with and without pooling of data across countries, produced about 70 to 80 percent correct forecasts for 158 turning points. Time-varying parameter models performed slightly better than others. The performance of the ARLI models in forecasting turning points is shown to be considerably better than that of four naive methods of forecasting turning points.