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In the heart, TRPM4 is most abundantly distributed in the conduction system. Previously, a single mutation, ‘E7K’, was identified in its distal N-terminus to cause conduction disorder because of enhanced cell-surface expression. It remains, however, unclear how this expression increase leads to conduction failure rather than abnormally enhanced cardiac excitability. To address this issue theoretically, we mathematically formulated the gating kinetics of the E7K-mutant TRPM4 channel by a combined use of voltage jump analysis and ionomycin-perforated cell-attached recording technique and incorporated the resultant rate constants of opening and closing into a human Purkinje fiber single-cell action potential (AP) model (Trovato model) to perform 1D-cable simulations. The results from TRPM4 expressing HEK293 cells showed that as compared with the wild-type, the open state is much preferred in the E7K mutant with increased voltage-and Ca2+-sensitivities. These theoretical predictions were confirmed by power spectrum and single channel analyses of expressed wild-type and E7K-mutant TRPM4 channels. In our modified Trovato model, the facilitated opening of the E7K mutant channel markedly prolonged AP duration with concomitant depolarizing shifts of the resting membrane potential in a manner dependent on the channel density (or maximal activity). This was, however, little evident in the wild-type TRPM4 channel. Moreover, 1D-cable simulations with the modified Trovato model revealed that increasing the density of E7K (but not of wild-type) TRPM4 channels progressively reduced AP conduction velocity eventually culminating in complete conduction block. These results clearly suggest the brady-arrhythmogenicity of the E7K mutant channel which likely results from its pathologically enhanced activity.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABAA receptors (GABAARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABAARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABAARs. We here developed the first turn-on fluorescent imaging probe for GABAARs, which can be used to quantitatively evaluate ligand–receptor interactions under live cell conditions. Using noncovalent labeling of GABAARs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABAAR, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABAARs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods., Using a newly found turn-on fluorescent imaging probe for GABAARs, we developed a new imaging-based ligand assay system, which allows for the discovery of positive allosteric modulators for GABAARs.

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.

Mitochondria are an important source of reactive oxygen species (ROS). Although it has been reported that myocardial stretch increases cellular ROS production by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), referred to as X-ROS signalling, the involvement of mitochondria in X-ROS is not clear. Mitochondria are organelles that generate adenosine triphosphate (ATP) for cellular energy needs, which are mechanical-load-dependent. Therefore, it would not be surprising if these organelles had mechano-sensitive functions associated with stretch-induced ROS production. In the present study, we investigated the relation between X-ROS and mitochondrial stretch-sensitive responses in isolated mouse cardiac myocytes. The cells were subjected to 10% axial stretch using computer-controlled, piezo-manipulated carbon fibres attached to both cell ends. Cellular ROS production and mitochondrial membrane potential (Δψm) were assessed optically by confocal microscopy. The axial stretch increased ROS production and hyperpolarised Δψm. Treatment with a mitochondrial metabolic uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), at 0.5 μM did not suppress stretch-induced ROS production, whereas treatment with a respiratory Complex III inhibitor, antimycin A (5 μM), blunted the response. Although NOX inhibition by apocynin abrogated the stretch-induced ROS production, it did not suppress stretch-induced hyperpolarisation of Δψm. These results suggest that stretch causes activation of the respiratory chain to hyperpolarise Δψm, followed by NOX activation, which increases ROS production.

A series of ferrocene–porphyrin–fullerene linked triads (TC1, TC2, and TC4) possessing different numbers of cationic moieties were designed and prepared to achieve a high photoinduced charge-separation (CS) yield in a biological environment. In a solution, TC1, TC2, and TC4 demonstrated the formation of their nanoaggregates. Among the new triads, TC4 possessing the four cationic moieties exhibited the formation of a long-lived charge-separated state with the highest CS yield (86%) ever reported in cell membrane-like lipid bilayers, which is consistent with the largest change in the cell membrane potential of PC12 cells via the photoinduced CS under green light illumination. The highest CS yield in the biological environment can be rationalized by the well-tailored balance in hydrophobicity and hydrophilicity of TC4. This finding provides a strategy to improve greatly the photoinduced charge-separation yield of donor–acceptor linked molecules in the biological environment and also will be informative for e...

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 μM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca(2+)-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca(2+)-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca(2+) elevations and a disorganized cytoskeleton. CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC’s coiled-coil assembly which is essential for CaM binding. The resulting excess Ca(2+) may contribute to structural damage in the podocytes.

A Ca2+-activated monovalent cation-selective TRPM4 channel is abundantly expressed in the heart. Recently, a single gain-of-function mutation identified in the distal N-terminus of the human TRPM4 channel (Glu5 to Lys5, E7K) was found to be arrhythmogenic because of enhanced cell membrane expression. In this study, we conducted detailed analyses of this mutant channel from more functional aspects, in comparison with its wild type (WT). In an expression system, intracellular application of a short soluble PIP2 (diC8PIP2) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (Kd) of diC8PIP2 for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 μM). FRET-based PIP2 measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP2 level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent Kd values estimated from DrVSP-mediated PIP2 depletion: 0.97 and 1.06 μM, respectively), being associated with the differential PIP2-mediated modulation of voltage dependence. Moreover, intracellular perfusion of short N-terminal polypeptides containing either the ‘WT’ or ‘E7K’ sequences respectively attenuated the TRPM4 channel activation at whole-cell and single-channel levels, but in both configurations, the E7K polypeptide exerted greater inhibitory effects. These results collectively suggest that N-terminal interaction with endogenous PIP2 is essential for the TRPM4 channel to function, the extent of which may be abnormally strengthened by the E7K mutation through modulating voltage-dependent activation. The altered PIP2 interaction may account for the arrhythmogenic potential of this mutation.

Objectives Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. Methods Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. Results VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. Conclusions We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

A series of porphyrin–fullerene linked molecules has been synthesized to evaluate the effects of substituents and molecular structures on their charge-separation yield and the lifetime of a final charge-separated state in various hydrophilic environments. The selected high-performance molecule effectively achieved depolarization in a plasma cell membrane by visible light as well as two-photon excitation using a near-infrared light laser. Moreover, it was revealed that the depolarization can trigger neuronal firing in rat hippocampal neurons, demonstrating the potential and versatility for controlling cell functions using light.

Currently, cancer is one of major cause of death in patients with type 2 diabetes. We have previously reported the anti-prostate and anti-breast cancer effect of GLP-1R agonist Exendin-4 (Diabetes 2014, Endocrinology 2017). In the present study, we examined the anti-cancer effect of SGLT2 inhibitor ipragliflozin (Ipra) using a breast cancer model. In human breast cancer cell line, MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. 1-50nM Ipra significantly and dose-dependently suppressed the growth curve of MCF-7 cells. BrdU assay revealed that Ipra attenuates the proliferation rate of MCF-7 in a dose dependent manner. Further, apoptosis was also induced by Ipra in Tunel assay. Because the anti-breast cancer effect of Ipra was completely canceled by knocking down of SGLT2, this effect could be induced by SGLT2 inhibition by Ipra. We next measured membrane potential and whole cell current using the patch clamp technique. When we treated MCF-7 cell with Ipra or glucose free medium, membrane hyperpolarization was observed. In addition, the replacement of sodium with NMDG and knock-down of SGLT2 by siRNA suppressed glucose induced whole cell current of MCF-7 cell, suggesting that Ipra inhibits sodium and glucose incorporation through SGLT2. Further, mitochondrial membrane protein Bcl-2 was decreased and Bax was increased in western blotting, and JC-1 fluorescence was significantly increased, suggesting the change of mitochondrial membrane potential. These data suggest that SGLT2 inhibitor Ipra induces apoptosis in the breast cancer cell via membrane hyperpolarization and mitochondria dysfunction. Disclosure S. Komatsu: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.T. Numata: None.T. Kawanami: None.Y. Hamaguchi: None.T. Tanaka: None.R. Inoue: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.

To investigate the anti-fibrotic effects of the traditional oriental herbal medicine Daikenchuto (DKT) associated with transient receptor potential ankyrin 1 (TRPA1) channels in intestinal myofibroblasts.Inflammatory and fibrotic changes were detected in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) chronic colitis model of wild-type and TRPA1-knockout (TRPA1-KO) miceChronic treatment with TNBS caused more severe inflammation and fibrotic changes in TRPA1-KO than in wild-type mice. A one-week enema administration of DKT reduced fibrotic lesions in wild-type but not in TRPA1-KO mice. The active ingredients of DKT,The effects of DKT on the expression and activation of the TRPA1 channel could be advantageous for suppressing intestinal fibrosis, and benefit inflammatory bowel disease treatment.

Activation of transient receptor potential (TRP) canonical TRPC3/C6/C7 channels by diacylglycerol (DAG) upon stimulation of phospholipase C (PLC)-coupled receptors results in the breakdown of phosphoinositides (PIPs). The critical importance of PIPs to various ion-transporting molecules is well documented, but their function in relation to TRPC3/C6/C7 channels remains controversial. By using an ectopic voltage-sensing PIP phosphatase (DrVSP), we found that dephosphorylation of PIPs robustly inhibits currents induced by carbachol (CCh), 1-oleolyl-2-acetyl-sn-glycerol (OAG) or RHC80267 in TRPC3, TRPC6 and TRPC7 channels, though the strength of the DrVSP-mediated inhibition (VMI) varied among the channels with a rank order of C7>C6>C3. Pharmacological and molecular interventions suggest that depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is most likely the critical event for VMI in all three channels.When the PLC catalytic signal was vigorously activated through overexpression of the muscarinic type-I receptor (M1R), the inactivation of macroscopic TRPC currents was greatly accelerated in the same rank order as the VMI, and VMI of these currents was attenuated or lost. VMI was also rarely detected in vasopressin-induced TRPC6-like currents inA7r5 vascular smooth muscle cells, indicating that the inactivation by PI(4,5)P₂ depletion underlies the physiological condition. Simultaneous fluorescence resonance energy transfer (FRET)-based measurement of PI(4,5)P₂ levels and TRPC6 currents confirmed that VMI magnitude reflects the degree of PI(4,5)P₂ depletion. These results demonstrate that TRPC3/C6/C7 channels are differentially regulated by depletion of PI(4,5)P₂, and that the bimodal signal produced by PLC activation controls these channels in a self-limiting manner.

Objective— The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)–induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition. Methods and Results— Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II–induced contraction was largely dependent on Ca 2+ influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)–dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II–induced Ca 2+ influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II–induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6. Conclusion— PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.

We investigated actions of uridine 5′-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X1 receptor, and a similar current was also induced by α,β-methylene adenosine 5′-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP–evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X1-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 μM) provoked a dose-dependent contraction of deendothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca2+ or bath-applied 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca2+]i were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X1 receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X1-like and P2Y receptors, but not TRPC3.[Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.10281FP] Keywords:: uridine 5′-triphosphate (UTP), P2X receptor, TRPC3, non-selective cation channel

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real‐time RT‐PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2‐APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (I spont) contributive to basal [Ca2+]i was identified by the nystatin‐perforated whole‐cell recording. I spont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA‐mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin‐induced γ‐globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

We investigated the inhibitory role of the nitric oxide (NO)–cGMP–protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by ∼70%) by a NO donor SNAP (100 μm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 μm) or after G-protein activation with intracellular perfusion of GTPγS (100 μm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 μm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of 32P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg8-vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO–cGMP–PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

Although Ca influx plays a pivotal role in neurohormonal and myogenic control of vascular tone and slow progressive vascular remodeling, the molecular identification of those Ca sources/pathways had long been elusive. The review will introduce recent discoveries that mammalian homologues of Drosophila transient receptor potential protein expressed in various regions of the vasculature exhibit appropriate features to elucidate these pathways, with associated dysfunctions.Recent investigations have revealed that expressed as well as native vascular transient receptor potentials behave as Ca-permeable cation channels that open in response to phospholipase C-coupled vasoconstrictors, mechanical forces and/or hypertrophic stimuli, thereby regulating the vascular resistance/tone and blood pressure/flow, and proliferative/apoptotic reorganization of vascular tissues. Notably, one vascular function relies on the coordinated interplay of multiple vascular transient receptor potential isoforms and vice versa. Imbalance of expression and altered activities of these vascular isoforms likely contribute to disorders such as hypertension, vasospasm, atherosclerosis and aneurysm.Such multifunctionality and multifaceted aspects of vascular transient receptor potentials not only suggest their unprecedented importance in regulating vascular functions, but may also offer the possibility of developing new drugs or 'calcium antagonists' that would work more selectively and elaborately for a wide range of vascular diseases based on entirely different strategies.

Background and purpose: Myosin light chain kinase (MLCK) plays a pivotal role in regulation of cellular functions, the evidence often relying on the effects of extracelluarly administered drugs such as ML-9. Here we report that this compound exerts non-specific inhibitory actions on the TRPC6 channel, a transient receptor potential (TRP) protein. Experimental approach: Macroscopic and single channel currents were recorded from transfected HEK293 cells by patch-clamp techniques. Key results: Cationic currents elicited by carbachol (CCh; 100 μM) in HEK293 cells overexpressing murine TRPC6 (ITRPC6) were dose-dependently inhibited by externally applied ML-9 (IC50=7.8 μM). This inhibition was voltage-dependent and occurred as fast as external Na+ removal. Another MLCK inhibitor, wortmannin (3 μM), and MLCK inhibitory peptides MLCK-IP11-19 (10 μM) and -IP480-501 (1 μM) showed little effects on ITRPC6 density and the inhibitory efficacy of ML-9. The extent of the inhibition also unchanged with co-expression of wild-type or a dominant negative mutant of MLCK. Inhibitory effects of ML-9 on ITRPC6 remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG (100 μM). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dose-dependently suppressed ITRPC6. In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished ∼35pS single TRPC6 channel activities. Contrarily, currents due to TRPC7 expression were rapidly enhanced by externally applied ML-9 and ML-7, which was not prevented by MLCK inhibitory peptides. Conclusion and implications: These results strongly suggest that ML compounds inhibit TRPC6 channels via a mechanism independent of inhibition of MLCK activity. British Journal of Pharmacology (2007) 152, 122–131; doi:10.1038/sj.bjp.0707368

A rationally designed donor–acceptor conjugate efficiently generates a photoinduced charge-separated state in a cellular environment, achieving photoinduction of neuronal firing., A series of porphyrin–fullerene linked molecules has been synthesized to evaluate the effects of substituents and molecular structures on their charge-separation yield and the lifetime of a final charge-separated state in various hydrophilic environments. The selected high-performance molecule effectively achieved depolarization in a plasma cell membrane by visible light as well as two-photon excitation using a near-infrared light laser. Moreover, it was revealed that the depolarization can trigger neuronal firing in rat hippocampal neurons, demonstrating the potential and versatility for controlling cell functions using light.

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4, 5-bisphosphate [PI(4, 5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4, 5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4, 5)P2 and the possible role of PI(4, 5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4, 5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4, 5)P2 using a model simulation that could explain the complicated effects of PI(4, 5)P2 on different ROCs.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

1. Pharmacological properties of nifedipine-insensitive, high voltage-activated Ca(2+) channels in rat mesenteric terminal arteries (NICCs) were investigated and compared with those of alpha1E and alpha1G heterologously expressed in BHK and HEK293 cells respectively, using the patch clamp technique. 2. With 10 mM Ba(2+) as the charge carrier, rat NICCs (unitary conductance: 11.5 pS with 110 mM Ba(2+)) are almost identical to those previously identified in a similar region of guinea-pig, such as in current-voltage relationship, voltage dependence of activation and inactivation, and divalent cation permeability. However, these properties are considerably different when compared with alpha1E and alpha1G. 3. SNX-482(200 nM and sFTX3.3 (1 micro M), in addition to omega-conotoxin GVIA (1 micro M) and omega-agatoxin IVA (100 nM), were totally ineffective for rat NICC currents, but significantly suppressed alpha1E (by 82% at 200 nM; IC(50)=11.1 nM) and alpha1G (by 20% at 1 micro M) channel currents, respectively. A non-specific T-type Ca(2+) channel blocker nimodipine (10 micro M) differentially suppressed these three currents (by 40, 3 and 85% for rat NICC, alpha1E and alpha1G currents, respectively). 4. Mibefradil, the widely used T-type channel blocker, almost equally inhibited rat NICC and alpha1G currents in a voltage-dependent fashion with similar IC(50) values (3.5 and 0.3 micro M and 2.4 and 0.14 micro M at -100 and -60 mV, respectively). Furthermore, other organic T-type channel blockers such as phenytoin, ethosuximide, an arylpiperidine derivative SUN N5030 (IC(50)=0.32 micro M at -60 mV for alpha1G) also exhibited comparable inhibitory efficacies for NICC currents (inhibited by 22% at 100 micro M; IC(50)=27.8 mM; IC(50)=0.53 micro M, respectively). 5. These results suggest that despite distinctive biophysical properties, the rat NICCs have indistinguishable pharmacological sensitivities to many organic blockers compared with T-type Ca(2+) channels.

We investigated the receptor-mediated regulation of nifedipine-insensitive, high voltage-activated Ca(2+) currents in guinea-pig terminal mesenteric arterioles (I(mVDCC)) using the whole-cell clamp technique. Screening of various vasoactive substances revealed that ATP, histamine and substance P exert modulatory effects on I(mVDCC). The effects of ATP on I(mVDCC) after complete P2X receptor desensitization exhibited a complex concentration dependence. With 5 mM Ba(2+), ATP potentiated I(mVDCC) at low concentrations (approximately 1-100 microM), but inhibited it at higher concentrations (100 microM). The potentiating effects of ATP were abolished by suramin (100 microM) and PPADS (10 microM) and by intracellular application of GDPbetaS (500 microM), whereas a substantial part of I(mVDCC) inhibition by milimolar concentrations of ATP remained unaffected; due probably to its divalent cation chelating actions. In divalent cation-free solution, I(mVDCC) was enlarged and underwent biphasic effects by ATPgammaS and ADP, while 2-methylthio ATP (2MeSATP) exerted only inhibition, and pyrimidines such as UTP and UDP were ineffective. ATP-induced I(mVDCC) potentiation was selectively inhibited by anti-Galpha(s) antibodies or protein kinase A (PKA) inhibitory peptides and mimicked by dibutyryl cAMP. In contrast, ATP-induced inhibition was selectively inhibited by Galpha(q/11) antibodies or protein kinase C (PKC) inhibitory peptides and mimicked by PDBu. Pretreatment with pertussis toxin was ineffective. The apparent efficacy for I(mVDCC) potentiation with PKC inhibitors was: ATPgammaSATP/=ADP and for inhibition with PKA inhibitors was: 2MeSATPATPgammaSATPADP. Neither I(mVDCC) potentiation nor inhibition showed voltage dependence. These results suggest that I(mVDCC) is multi-phasically regulated by external ATP via P2Y(11)-resembling receptor/G(s)/PKA pathway, P2Y(1)-like receptor/G(q/11)/PKC pathway, and metal chelation.

We investigated the effects of the novel gastroprokinetic agent Z-338 (N-(N-N'-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxybenzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate) on L-type voltage-dependent Ca2+ currents (ICa) in guinea pig gastric myocytes by using the whole-cell patch clamp technique. Bath-applied acetylcholine (ACh) produced biphasic effects on ICa, i.e., enhancement (1-100 nM) and inhibition (1-100 microM), both of which were abolished by pretreatment with atropine (10 microM) or intracellular perfusion of GDPbetaS (500 microM). Z-338 (> or = 1 nM, ED50: 120 nM) mimicked the enhancing effects of ACh, but did not inhibit ICa. The effects of Z-338 and ACh were non-additive and blocked by atropine and GDPbetaS, but not by pertussis toxin (PTX) pretreatment (500 ng/ml). ACh (> or = 1 microM) induced slow inward currents via activation of the muscarinic receptor/PTX-sensitive G-protein pathway, but Z-338 was devoid of these effects. Neither pirenzepine (1 microM), AF-DX116 (1 microM), nor oxybutynin (100 nM) could prevent Z-338 (1 microM) and ACh (10 nM) from enhancing ICa, whilst 4-DAMP (100 nM) blocked the effects of Z-338 and ACh. Bath-application of protein kinase C (PKC) activator PDBu (phorbol-12,13-dibutyrate) (250 nM) enhanced ICa, and conversely, pipette inclusion of PKC inhibitor peptide (150 microM) abolished the effects of ACh and Z-338 on ICa. These results collectively suggest that although contribution of the M3 receptor is not excluded, the major actions of Z-338 on gastric myocytes are potentiation of ICa through activation of M5-like receptor.

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C–insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.

Cellular stimulation from the surrounding extracellular environment via receptors and other pathways evoke activation of Ca2+-permeable cation channels that form essential signaling pathways in controlling biological responses. An important clue to understand the molecular mechanisms underlying these cation channels (tentatively termed as receptor-mediated cation channels (RMCC)) was first provided through molecular studies of the transient receptor potential (trp) protein (TRP), which controls light-induced depolarization in Drosophila photoreceptor cells. Use of the genetic information and recombinant expression technique lead to the discovery of numerous mammalian TRP homologues revealing novel RMCCs. In this review, we focus on the dramatic progress in the molecular investigation of RMCC in mammalian systems. The recent findings should provide powerful tools for the development of novel pharmaceutical targets.

Abstract —We have found nifedipine-insensitive (NI), rapidly inactivating, voltage-dependent Ca 2+ channels (current, NI- I Ca ) with unique biophysical and pharmacological properties in the terminal branches of guinea pig mesenteric artery, by using a whole-cell mode of the patch-clamp technique. The fraction of NI- I Ca appeared to increase dramatically along the lower branches of mesenteric artery, amounting to almost 100% of global I Ca in its periphery. With 5 mmol/L Ba 2+ as the charge carrier, NI- I Ca was activated with a threshold of −50 mV, peaked at −10 mV, and was half-activated and inactivated at −11 and −52 mV, respectively, generating a potential range of constant activation near the resting membrane potential. The NI- I Ca was rundown resistant, was not subject to Ca 2+ -dependent inactivation, and exhibited the pore properties typical for high voltage–activated Ca 2+ channels; Ba 2+ is ≈2-fold more permeable than Ca 2+ , and Cd 2+ is a better blocker than Ni 2+ (IC 50 , 6 and 68 μmol/L, respectively). Relatively specific blockers for N- and P/Q-type Ca 2+ channels such as ω-conotoxins GVIA and MVIIC (each 1 μmol/L) and ω-agatoxin IVA (1 μmol/L) were ineffective at inhibiting NI- I Ca , whereas nimodipine partially (10 μmol/L; ≈40%) and amiloride potently (≈75% with 1 mmol/L; IC 50 ; 107 μmol/L) blocked the current. Although these properties are reminiscent of R-type Ca 2+ channels, expression of the α 1E mRNA was not detected using reverse transcriptase–polymerase chain reaction. These results strongly suggest the predominant presence of NI, high voltage–activated Ca 2+ channels with novel properties, which may be abundantly expressed in peripheral small arterioles and contribute to their tone regulation.

1. The effects of NO donors on Ca2+-dependent Cl- currents (ICl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole-cell patch clamp technique. 2. With nystatin-perforated whole-cell recording, carbachol (CCh, >/= 1 microM) induced a transient inward current (ICCh) with a reversal potential of about -20 mV. Activation of ICCh probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) greatly inhibited this current, while 11-(2-(diethylamino)methyl)-1-piperidinylacetyl)-5, 11-dihydro-6H-pyrido (2,3beta) (1,4)benzodiazepine-6-one (AF-DX 116) or pirenzepine at concentrations of up to 1 microM were almost ineffective. 3. Chloride channel/transporter blockers such as DIDS (100 microM), anthracene-9-carboxylic acid (9-AC, 100 microM) and niflumic acid (100 microM) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 microM), Zn2+ (500 microM) or Gd3+ (500 microM), were without effect. 4. Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 microM) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml-1) in the pipette abolished a substantial part of ICCh. These results suggest that ICCh is ICl(Ca), which is activated by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release. 5. The nitric oxide donor S-nitroso-N-acetyl penicillamine (SNAP) reduced the amplitude of ICCh dose dependently (IC50, approximately 10 microM). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (+/-)-E-methyl-2-(E-hydroxyimitol)-5-nitro-6-methoxy-3- hexeneamide (NO-R). 6. SNAP-induced ICCh inhibition was effectively antagonized by Methylene Blue (1-100 nM), and mimicked by dibutyryl cGMP (db-cGMP) (0.5-1 mM), whereas two structurally distinct types of cGMP-dependent (G)-kinase inhibitor, N-(2-aminoethyl)-5-isoquinilinesulphonamide (H-8, 2.5 microM) and KT5823 (1 microM), failed to counteract the inhibitory effects of SNAP or db-cGMP. Another G-kinase-specific inhibitor Rp-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS; 1 microM) itself caused a marked reduction in ICCh. 7. SNAP (100 microM) or db-cGMP (100 microM) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current. 8. These results suggest that inhibition of ICCh by NO donors involves a cGMP-dependent but G-kinase-independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.

Nonselective cation channels (NSCCs) are defined as the channel exhibiting better selectivity for cations over anions. This nomenclature is however confusing, because the channels which fall into this category show a high degree of heterogeneity in their biophysics, physiology and pharmacology. Since little molecular information is yet available for most types of NSCC (except for the fast extracellular ligand-gated NSCC), one useful criterion to sub-classify them is the mode of activation. Based on this, more than 10 subclasses of NSCC, including the G-protein-coupled NSCC as a newly emerging member, can be distinguished (Table 1). Furthermore, the biophysical and pharmacological profile of these subclasses of NSCC suggest that varying extents of structural similarities may exist. Such a duality, i.e. heterogeneity and similarity of NSCC is on one hand, the obstacle to exploiting selective drugs for NSCCs, but on the other hand, may account for a broad repertoire of functions that NSCCs subserve under various patho-physiological conditions. This short paper briefly overviews, from such an aspect, the physiology and pharmacology of NSCCs of the plasma membrane, which have rather poorly been elucidated so far.

Potentiating actions of external lanthanum (La3+) on muscarinic receptor-activated nonselective cation current (Icat) were investigated in myocytes dissociated from the longitudinal muscle layer of guinea-pig ileum, with a whole-cell variant of the patch clamp technique. Icat was dissected from other membrane currents by loading Cs-aspartate into the cell. Application of submilimolar concentrations of La3+ following 300 microM ACh into the bath caused a dose-dependent increase in the amplitude of Icat. The apparent Kd value for this increase was 190 microM, with a cooperativity factor of 1.7. La(3+)-induced increase in Icat amplitude was not associated with either changes in the reversal potential of Icat or altered sensitivity of muscarinic receptor to ACh, and paralleled by the conductance increase of Icat, the maximum of which (Gmax) occurred at about 1 mM La3+. Voltage-jump experiments revealed that the rate of current relaxation at hyperpolarizing potentials was greatly reduced in the presence of La3+, and correspondingly the steady state activation curve shifted toward more negative potentials. Divalent cations such as Cd2+ or Ni2+, which have been known to block Icat, antagonized the augmentative effect of La3+ on Icat in a competitive fashion, suggesting that the site of their actions might be similar. Furthermore, single Icat activities induced by internal perfusion of GTP gamma S (100 microM) was also greatly enhanced by external addition of 1 mM La3+. Under current clamp conditions, 1 mM La3+ blocked spontaneous Ca2+ spike activities, but was almost without effect on the membrane depolarization induced by ACh. In contrast, milimolar concentrations of Cd2+ and Ni2+ abolished both Ca2+ spike activities and ACh-induced depolarization. Potential importance of La3+ as a tool to investigate the external Ca(2+)-dependence of Icat has been discussed.

The effects of hydrogen peroxide H2O2 (10−6 and 10−3 M) on membrane potential, membrane currents, intracellular calcium concentration, resting muscle tone and contractions elicited by electrical field stimulation (EFS) and carbachol were examined in cat tracheal strips and isolated smooth muscle cells. H2O2 (10−4 and 10−5 M) enhanced the amplitude of contractions and excitatory junction potentials (e.j.p.) evoked by EFS without changing muscle tone and resting membrane potential of the tracheal smooth muscle, and enhanced the contraction induced by carbachol (10−8 M). At an increased concentration (10−3 M), H2O2 elevated resting muscle tone and marginally hyperpolarized the membrane in the majority of the cells. In 51 out of 56 cells examined, H2O2 (10−6–10−3 M) elicited an outward current at a holding potential of −40 mV and enhanced the frequency of the spontaneous transient outward current (STOC). In 20 cells the outward current was preceded by a small inward current. In the other cells, H2O2 elicited only an inward current or did not affect the background current. In Ca2+ free solution the action of H2O2 on the resting muscle tone, STOCs, background current and on the current induced by ramp depolarization was significantly reduced. H2O2 (10−4 M) increased the intracellular ionized calcium concentration both in the absence and presence of external Ca2+. However, the effect developed faster and was of a higher amplitude in the presence of external Ca2+. These results suggest that H2O2 increases intracellular Ca2+, with a subsequent augmentation of stimulation-evoked contractions, and enhances Ca2+ and voltage-sensitive potassium conductance.

Stimulation of excitatory receptors in smooth muscle often leads to the opening of ROCC. These channels exhibit considerable permeability to Ca2+, and they have been regarded as the most probable candidate for the "receptor-operated Ca2+ entry" pathway. The muscarinic receptor ROCC in guinea pig ileum (mROCC) have a unitary conductance of -25pS and are activated through a pertussis toxin-sensitive G protein. mROCC permeate Ca2+ and Ba2+ several fold more preferably than monovalent cations, and they are inhibited by various types of K channel blockers, diphenylamine-2-carboxylate derivatives and even by nicardipine and D-600 at high concentrations. mROCC are efficiently regulated by various physiological factors including the membrane potential, intracellular Ca2+ concentration, external pH and osmolarity. The effective ranges of these factors span their dynamic ranges under physiological conditions. In addition to these properties, mROCC have several sites sensitive to external polyvalent cations. The alpha 1-adrenergic receptor ROCC in rabbit portal vein resemble mROCC in many respects, e.g., the unitary conductance, ionic selectivity, activation kinetics, sensitivity to polyvalent cations and voltage-dependence. These complex characteristics of ROCC suggest that they play other roles in addition to being just a passive cation permeable pore in agonist-mediated Ca2+ mobilization in smooth muscle.

The effects of external H+ on the acetylcholine-induced inward current (nonselective cationic current; InsACh) in guinea pig ileal smooth muscle were investigated using the conventional whole cell patch-clamp technique. When the external pH (pHo) was lowered, the amplitude of InsACh was increased, with no significant change in the reversal potential or no detectable induction of other ionic permeabilities. The dose-response curve for this effect was best described by a Hill-type equation with an apparent pKa value of 7.4 and a Hill coefficient of approximately 1. The effect of pHo was associated with a shift of the steady-state activation curve for InsACh; the half-maximum activation potential became more negative on lowering pHo. Similar results were obtained when InsACh was activated by intracellularly applied guanosine 5'-O-(3-thiotriphosphate). These results indicate that the external H+ activity is an efficient regulator of InsACh channel, and this may have a physiological importance for controlling the muscarinic receptor-mediated contractions in this muscle.

The transient receptor potential (TRP) proteins form a large Ca2+-permeable nonselective cation channel superfamily activated by physicochemical stimuli, and participate in a wide array of biological functions including sensory signal transduction. Recent investigations have disclosed that many of TRP channels expressed in the cardiovascular system (CVS) are activated by mechanical stresses operating therein such as membrane stretch, hypoosmolarity and shear stress. Although mechanisms proposed for mechanical signal transduction are diverse, accumulating evidence suggests that lipid mediators derived from phospholipase C (PLC)- and phospholipase A2 (PLA2)-dependent pathways may play central roles in the activation and regulation of these TRP channels. In this review, we focus on the lipid-mediated regulation of two TRP channels abundantly expressed in the CVS, i.e. TRPC6 and TRPV4, with particular interest in the synergistic interaction between receptor-mediated and mechanical stimulations, and discuss about their complex functional antagonism in vascular tone and blood pressure regulation.

1. The effects of isoprenaline, a forskolin derivative NKH-477, and dibutyryl cyclic AMP (db cyclic AMP) on the membrane potential, conductance and cell volume of the dog non-pigmented ciliary epithelium (NPE) were investigated by intracellular potential recording, nystatin-perforated patch clamp technique and videomicroscopic cytometry. 2. The resting membrane potential of NPE was about -70 mV in physiological saline and was depolarized by isoprenaline in a dose-dependent manner with an ED50 of about 3 nM. This depolarization was competitively antagonized by the beta-adrenoceptor antagonist, timolol (pA2 = ca. 9) and almost completely blocked by the Cl transport blocker, DIDS. 3. In single dissociated NPE cells, 10 microM isoprenaline induced an inward current and caused a concomitant decrease in cell volume. The reversal potential measurement indicated that this inward current was carried mainly by Cl ion. DIDS (10 microM) abolished both the current and cell volume decrease. 4. NKH-477 (10 microM) or db cyclic AMP (1 mM) also induced an inward current together with a cell volume decrease, the properties of which were similar to those caused by isoprenaline. 5. These results suggest that beta-adrenoceptor stimulation in NPE leads to an increased rate of aqueous humour production by increasing Cl- efflux via an elevation of cyclic AMP and this effect is efficiently blocked by timolol.

There is growing body of evidence that nitric oxide (NO)-cGMP-PKG signaling plays a central role in negative regulation of cardiovascular (CV) responses and its disorders through suppressed Ca(2+) dynamics. Other lines of evidence also reveal the stimulatory effects of this signaling on some CV functions. Recently, transient receptor potential (TRP) channels have received much attention as non-voltage-gated Ca(2+) channels involved in CV physiology and pathophysiology. Available information suggests that these channels undergo both inhibition and activation by NO via PKG-mediated phosphorylation and S-nitrosylation, respectively, and also act as upstream regulators to promote endothelial NO production. This review summarizes the roles of NO-cGMP-PKG signaling pathway, particularly in regulating TRP channel functions with their associated physiology and pathophysiology.

TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E2(PGE2) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca2+. In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca2+-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca2+influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd3+-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca2+influx by TNF-α, whereas potentiated TNF-α induced PGE2production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca2+influx, and COX-2-dependent PGE2production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE2production without affecting TRPC1 expression and the Ca2+influx. Finally, the suppression of store-dependent Ca2+influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE2production, respectively, at the downstream of TRPC1-associated Ca2+influx.

1. The excitatory actions of phenylephrine (Phe) and acetylcholine (ACh) on the smooth muscle of rabbit portal vein were investigated and compared by using whole-cell and single channel configurations of the patch clamp technique, in combination with a modified concentration jump method. 2. At negative holding potentials with KCl (0.1 mM EGTA) electrodes, rapid applications of Phe (> 1 microM) and ACh (> 10 microM) both resulted in biphasic responses consisting of fast outward or inward currents and a long-lasting inward current with an increased noise level. 3. The slow inward current was still recorded when the cell was dialysed with caesium aspartate solution complemented with 10 mM EGTA, in order to eliminate contributions of calcium-dependent conductances (K+ and Cl- currents). Phe was more potent at activating the current than ACh. Ion replacement experiments revealed that both the Phe- and ACh-induced slow inward currents are cation-selective conductances (CS currents). 4. The I-V relationships of the Phe- or ACh-induced CS currents were similar both for the instantaneous peak and the steady state. The tail current analysis over a wide range of membrane potentials (-150 to +100 mV) showed that depolarizations to very positive potentials (> +50 mV) from near the resting membrane potential (-40 mV) can produce a several-fold increase in the steady-state activation of the CS currents, but the maximal activations were in most cases not observed even at +100 mV. 5. Externally applied Cd2+ produced a quick and reversible inhibition of both the Phe- and ACh-induced CS currents. This inhibition seemed almost voltage independent and the concentrations of half-inhibition were 100 and 129 microM for Phe and ACh, respectively. 6. Single channel activities were recorded in the presence of Phe or ACh using the outside-out membrane patches. The unitary conductances and reversal potentials of the Phe- and ACh-activated channels were 23 pS, +5.2 mV and 25 pS, +4.1 mV, respectively, and the open lifetimes evaluated for 50 microM Phe and 500 microM ACh were of similar order (the longer open times at -60 mV for Phe and ACh were 4.3 and 4.4 ms, respectively). In addition, the relative open probability (Po,rel) was no more than 0.2 in the voltage range of -100 to -30 mV for both 50 microM Phe and 500 microM ACh, suggesting low open probabilities of the Phe- and ACh-activated CS channels near the resting membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of pinacidil were investigated on the resting membrane potential and excitatory neuro-effector transmission in the smooth muscle tissue of dog trachea using tension recording, microelectrode and double sucrose gap methods. Pinacidil (>5×10 -7 M) increased the ionic conductance and hyperpolarized smooth muscle cell membranes in dog trachea, in a dose-dependent manner. This pinacidil-induced hyperpolarization was antagonized by glibenclamide (2×10 -6 M)

TRPC6 is a non–voltage-gated Ca 2+ entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible interactions of receptor and mechanical stimulations in activating this channel using the patch clamp technique. In HEK293 cells expressing TRPC6, application of mechanical stimuli (hypotonicity, shear, 2,4,6-trinitrophenol) caused, albeit not effective by themselves, a prominent potentiation of cationic currents ( I TRPC6 ) induced by a muscarinic receptor agonist carbachol. This effect was insensitive to a tarantula toxin GsMTx-4 (5 μmol/L). A similar extent of mechanical potentiation was observed after activation of I TRPC6 by GTPγS or a diacylglycerol analog 1-oleoyl-2-acetyl- sn -glycerol (OAG). Single TRPC6 channel activity evoked by carbachol was also enhanced by a negative pressure added in the patch pipette. Mechanical potentiation of carbachol- or OAG-induced I TRPC6 was abolished by small interfering RNA knockdown of cytosolic phospholipase A 2 or pharmacological inhibition of ω-hydroxylation of arachidonic acid into 20-HETE (20-hydroxyeicosatetraenoic acid). Conversely, direct application of 20-HETE enhanced both OAG-induced macroscopic and single channel TRPC6 currents. Essentially the same results were obtained for TRPC6-like cation channel in A7r5 myocytes, where its activation by noradrenaline or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation of receptor and mechanical stimulations may synergistically amplify transmembrane Ca 2+ mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A 2 /ω-hydroxylase/20-HETE pathways.

Transient receptor potential (TRP) proteins constitute a large non-voltage-gated cation channel superfamily, activated polymodally by various physicochemical stimuli, and are implicated in a variety of cellular functions. Known activators for TRP include not only chemical stimuli such as receptor stimulation, increased acidity and pungent/cooling agents, but temperature change and various forms of mechanical stimuli such as osmotic stress, membrane stretch, and shear force. Recent investigations have revealed that at least ten mammalian TRPs exhibit mechanosensitivity (TRPC1, 5, 6; TRPV1, 2, 4; TRPM3, 7; TRPA1; TRPP2), but the mechanisms underlying it appear considerably divergent and complex. The proposed mechanisms are associated with lipid bilayer mechanics, specialized force-transducing structures, biochemical reactions, membrane trafficking and transcriptional regulation. Many of mechanosensitive (MS)-TRP channel likely undergo multiple regulations via these mechanisms. In the cardiovascular system in which hemodynamic forces constantly operate, the impact of mechanical stress may be particularly significant. Extensive morphological and functional studies have indicated that several MS-TRP channels are expressed in cardiac muscle, vascular smooth muscle, endothelium and vasosensory neurons, each differentially contributing to cardiovascular (CV) functions. To further complexity, the recent evidence suggests that mechanical stress may synergize with neurohormonal mechanisms thereby amplifying otherwise marginal responses. Furthermore, the currently available data suggest that MS-TRP channels may be involved in CV pathophysiology such as cardiac arrhythmia, cardiac hypertrophy/myopathy, hypertension and aneurysms. This review will overview currently known mechanisms for mechanical activation/modulation of TRPs and possible connections of MS-TRP channels to CV disorders.

Recent investigations have revealed that mammalian homologues of transient receptor potential (TRP) protein (TRP1-7) are promising candidates for Ca2+ entry mechanisms (or channels) associated with various metabotropic G protein-coupled receptors (GPCRs) in smooth muscle, stimulation of which generates lipid second messengers and depletes internal stores. RT-PCR and immunocytochemical experiments have demonstrated that although the level of expression varies depending on tissues, the major TRP isoforms expressed in smooth muscle are TRP4, 6 and 7. In some vascular preparations, the significant expression of TRP1 mRNA and protein is also detected. Consistent with these findings, recent functional studies using TRP6- and TRP1-specific antisense oligonucleotides and antibodies have suggested that TRP6 is the essential component of alpha1-adrenoceptor activated, store depletion-independent Ca2+ entry channels, while TRP1 is partly involved in Ca2+ entry associated with store depletion or capacitative Ca2+ entry. In addition, coexpression of different TRP isoforms results in the appearance of cation channels showing novel properties reminiscent of some native GPCR-activated Ca2+-permeable non-selective cation channels. Thus, at present, TRP proteins may be the most important clues for elucidating the molecular entities of receptor- and store-operated Ca2+ entry mechanisms in smooth muscle and their roles in smooth muscle functions.