Channel-specific Inhibitor Research Articles

Time-resolved electromechanical and conductive behavior of nanostructured bilayers tethered to the surface of the electrode with incorporated channel proteins and peptides

The influence of incorporation of mitochondrial inner membrane potassium channel, and channel-forming peptide – Gramicidin on the ion transport and electromechanical properties of model lipid membranes tethered to gold electrode was electrochemically investigated by chronoamperometric and impedance spectroscopy techniques. In the case of the potassium channel the ion transport properties were modulated with channel-specific inhibitor – ATP-Mg2+ complex, whereas in the case of gramicidin peptide – by replacing potassium with sodium ions. The observed two exponential current–time responses of the systems studied were interpreted in terms of ion penetration and electrostriction of tethered lipid bilayer membrane, and conclusions supported with the experiments on alkanethiol self-assembled monolayers of different alkanethiol chain lengths deposited on gold.

Disease-modifying rdHSV-CA8* non-opioid analgesic gene therapy treats chronic osteoarthritis pain by activating Kv7 voltage-gated potassium channels.

Chronic pain is common in our population, and most of these patients are inadequately treated, making the development of safer analgesics a high priority. Knee osteoarthritis (OA) is a primary cause of chronic pain and disability worldwide, and lower extremity OA is a major contributor to loss of quality-adjusted life-years. In this study we tested the hypothesis that a novel JDNI8 replication-defective herpes simplex-1 viral vector (rdHSV) incorporating a modified carbonic anhydrase-8 transgene (CA8*) produces analgesia and treats monoiodoacetate-induced (MIA) chronic knee pain due to OA. We observed transduction of lumbar DRG sensory neurons with these viral constructs (vHCA8*) (~40% of advillin-positive cells and ~ 50% of TrkA-positive cells colocalized with V5-positive cells) using the intra-articular (IA) knee joint (KJ) route of administration. vHCA8* inhibited chronic mechanical OA knee pain induced by MIA was dose- and time-dependent. Mechanical thresholds returned to Baseline by D17 after IA KJ vHCA8* treatment, and exceeded Baseline (analgesia) through D65, whereas negative controls failed to reach Baseline responses. Weight-bearing and automated voluntary wheel running were improved by vHCA8*, but not negative controls. Kv7 voltage-gated potassium channel-specific inhibitor XE-991 reversed vHCA8*-induced analgesia. Using IHC, IA KJ of vHCA8* activated DRG Kv7 channels via dephosphorylation, but negative controls failed to impact Kv7 channels. XE-991 stimulated Kv7.2-7.5 and Kv7.3 phosphorylation using western blotting of differentiated SH-SY5Y cells, which was inhibited by vHCA8* but not by negative controls. The observed prolonged dose-dependent therapeutic effects of IA KJ administration of vHCA8* on MIA-induced chronic KJ pain due to OA is consistent with the specific activation of Kv7 channels in small DRG sensory neurons. Together, these data demonstrate for the first-time local IA KJ administration of vHCA8* produces opioid-independent analgesia in this MIA-induced OA chronic pain model, supporting further therapeutic development.

Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

Large-conductance and Ca2+-activated K+ (BK) channels are expressed in human hepatic stellate cells (HSCs), where they have roles in normal hepatic microcirculation, as well as in portal hypertension in liver cirrhosis through the regulation of contractility in activated HSCs. Nevertheless, whether BK channel activity exerts protective effects against aberrant HSC activation and hepatic fibrosis is unknown. Here, we report that BK channels are expressed in activated primary rat HSCs as well as in a human HSC line. Moreover, whole-cell K+ currents recorded from activated HSCs were markedly increased by exposure to rottlerin, a BK channel-specific activator, but were inhibited by treatment with the BK channel-specific inhibitor, paxilline, suggesting that BK channels are functional in activated HSCs. Overexpression but not downregulation of the BK channel pore-forming alpha subunit, KCNMA1, led to reduced migration and collagen expression in activated HSCs. Consistently, rottlerin treatment suppressed the fibrogenic cell function both in vitro and in CCl4-induced liver fibrosis in vivo. Microarray and pathway analysis, combined with a luciferase reporter assay and western blotting, further showed that rottlerin treatment led to a significant downregulation of the profibrotic TGFβ1/SMAD3 and JAK/STAT3 signaling pathways, both in vitro and in vivo. Our findings not only link BK channel function to profibrotic signaling pathways, but also provide evidence that BK channel activation represents a promising therapeutic strategy for the treatment of liver fibrosis.

White mulberry fruit polysaccharides enhance endothelial nitric oxide production to relax arteries in vitro and reduce blood pressure in vivo

Mulberry fruit polysaccharides have demonstrated excellent anti-inflammatory, antioxidant, hypolipidemic, and hypoglycemic properties. This study tested the effect of white mulberry fruit polysaccharides (WMFPs) on blood pressure. WMFPs induced endothelium-dependent relaxation in rat mesenteric arteries and NO production in endothelial cells, both of which were reversed by the NO synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride, a phosphoinositide 3-kinase inhibitor LY294002, a cell-permeable Ca2+ chelator (1,2-bis (o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid (acetoxymethyl ester)), and inhibitors of molecules downstream of NO, including the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, the potassium channel inhibitor tetraethylammonium chloride, the large conductance calcium-activated potassium channel-specific inhibitor iberiotoxin, and the KATP channel inhibitor glibenclamide. Intravenous injection of WMFPs reduced mean arterial blood pressure in both normotensive Sprague-Dawley and spontaneously hypertensive rats through enhanced endothelial NO production. This study demonstrated that WMFPs induce endothelium-dependent relaxation in rat mesenteric arteries to regulate blood pressure, suggesting that development of WMFPs as a novel antihypertensive agent is warranted.

NBCe1 is a novel regulator of pancreatic β‐cell function in diabetes

Dysregulation of glucose homeostasis in Type 2 diabetes mellitus (T2DM) is attributed to pancreatic β‐cell failure in maintaining adequate glucose‐stimulated insulin secretion (GSIS) to compensate for diminished insulin action. Recent single cell transcriptomics studies of human pancreatic islets have identified Slc4a4 (encoding an electrogenic Na+‐nHCO3− cotransporter, NBCe1) as a gene repressed in healthy non‐diabetic β‐cells, but expressed in human T2DM β‐cells. However the role of Slc4a4/NBCe1 in pancreatic β‐cell function remains unknown. Thus, in the current study we set out to test the hypothesis that mis‐expression of Slc4a4 in β‐cells contributes to loss of GSIS and impaired glucose homeostasis in T2DM. Subsequently, we first assessed Slc4a4 mRNA expression in isolated human islets (Control vs. T2DM) by qPCR and quantified NBCe1 protein expression by immunofluorescence in autopsy‐derived human pancreas tissue (Control vs. T2DM). We observed a 4‐fold induction in Slc4a4 mRNA expression in T2DM islets compared to non‐diabetic controls (p=0.004 vs. Control) and significantly increased NBCe1 co‐localization with insulin‐positive β‐cells of T2DM patients compared to controls (p<0.001 vs. Control). In addition, we also demonstrated that chronic inhibition of NBCe1 activity using the channel specific inhibitor (S0859) significantly enhanced GSIS in islets isolated from T2DM patients (~1.5‐fold increase vs. untreated, p=0.006). To delineate the role NBCe1 plays on β‐cell function in vivo, we generated β‐cell specific knockout (β‐Slc4a4−/−) mice by crossing Slc4a4fl/fl (G. Shull, UC) and Ins2‐Cre mouse models. We confirmed >50% reduction in Slc4a4 mRNA expression in β‐Slc4a4−/− islets and complete ablation of NBCe1 protein expression. In order to model the environmental stress that leads to metabolic defects common to T2DM, we exposed β‐Slc4a4−/− mice and control littermates to either standard chow diet or 60% high fat diet (HFD) for 8 weeks ad libitum. Under chow‐fed conditions, β‐Slc4a4−/− mice displayed normal glucose tolerance (p>0.05 vs. β‐Slc4a4+/+ Chow). Interestingly, β‐Slc4a4−/− mice exposed to HFD exhibited enhanced glucose tolerance and GSIS (p<0.05 vs. β‐Slc4a4+/+ HFD) and thus were protected from development of T2DM phenotype characteristics of HFD treatment. Finally, to understand the mechanism by which Slc4a4 deletion enhanced β‐cell function, we performed RNA‐sequencing on islets isolated from β‐Slc4a4−/− and β‐Slc4a4+/+ mice exposed to chow diet or HFD. Notably, gene‐set enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) defined biological pathways revealed that Slc4a4 deletion enriched genes associated with the cell cycle (p<0.001 vs. β‐Slc4a4+/+ Chow; p<0.001 vs. β‐Slc4a4+/+ HFD), while suppressing genes associated with apoptosis (p=0.002 vs. β‐Slc4a4+/+ Chow; p=0.004 vs. β‐Slc4a4+/+ HFD) in the presence of both chow diet and HFD. These result suggest that mis‐expression of Slc4a4 plays a novel role in the induction of β‐cell failure in T2DM. Future studies will delineate exact molecular mechanisms underlying NBCe1‐mediated regulation of β‐cell function in health and under conditions of metabolic stress.Support or Funding InformationMayo Clinic Center of Biomedical DiscoveryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

T-type calcium channel enhancer SAK3 promotes dopamine and serotonin releases in the hippocampus in naive and amyloid precursor protein knock-in mice.

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer’s disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55–0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-β-erythroidine (100 μM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus.

Stim1 Deletion Synthetically Rescues Ezh2-Null Effector T Cells and Alloimmunity

Graft-versus-host disease (GVHD) remains a major barrier for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have identified the central role of the histone methyltransferase Ezh2 in regulating allogeneic T-cell expansion, differentiation and function. Conditional loss of Ezh2 in donor T cells inhibits GVHD in mice due to the inability of alloreactive T cells to persist. However, the molecular mechanism by which Ezh2 deficiency causes alloreactive T cell death remains unknown. Here we demonstrate that genetic deletion of Stromal Interaction Molecule (Stim) 1, a dynamic endoplasmic reticulum Ca2+ sensor and regulator of Ca2+ signaling, rescues antigen-activated Ezh2-null (Ezh2-/-) T cells, leading to restored persistence of alloreactive effector T cells in mice and severe GVHD. Using RNA-sequencing analysis, we found Ezh2-deficiency led to the upregulation of multiple genes (e.g., Ifng, Prf1, Ccl5, Ccl4, Upp1 and Spp1) known to be regulated by Ca2+ signals through calcineurin (CN), the primary target of the immunosuppressant cyclosporine A (CsA). This reverse correlation between Ezh2 inhibition and CsA-treatment for gene expression suggests that Ezh2 may antagonize Ca2+ signaling in activated T cells. Calcium signaling assays revealed higher cytosolic Ca2+ uptake and more frequent Ca2+ oscillations in Ezh2-/- T cells. Moreover, Ezh2-/- T cells exhibited significantly increased polarization of Stim1 and Orai1 in the cellular membrane. These data reveal an unexpected role of Ezh2 as a negative regulator of Ca2+ entry, thereby serving as a ‘brake’ for Ca2+ signaling. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we found significantly fewer Ezh2-/- or Stim1-/- IFN-g-secreting effector T cells compared to the WT counterparts on day 8 or 14 post-transplantation. In contrast, deleting Stim1 from Ezh2-/- donor T cells rescued the cells in the spleen and liver, producing even more donor T cells and IFN-g-secreting effector T cells compared to WT T cells and inducing severe GVHD. We further examined the cell autonomous effect of Stim1 deletion on the rescue of Ezh2-/- T cells by mixing WT T cells (B6/SJL, CD45.1) with Ezh2- and/or Stim1- conditional knockout T cells (i.e., Ezh2-/-, Stim1-/- or Ezh2-/- x Stim1-/- B6 T cells (CD45.2)) at a ratio of 1:1 before transferring into the Balb/c mice. While loss of either Ezh2 or Stim1 led to lower frequency of IFN-g+IL-2+ effector T cells, combined deletion of both genes restored the frequency and number of IFN-g+IL-2+ effector T cells to that of WT T cells. Thus, Stim1-mediated Ca2+ signals are crucial for mediating cell death in alloantigen-driven Ezh2-/- effector T cells. To further determine whether the inhibition of CN-NFAT contributes to the rescue, we treated T cell receptor (TCR)-activated Ezh2-/- T cells with CsA or the calcium release-activated channel specific inhibitor BTP2, respectively, in vitro. While BTP2 dramatically improved the survival of IFN-g-producing effector T cells, CsA did not, suggesting the involvement of CN-NFAT-independent pathways. Ca2+ overload is known to impair mitochondrial function and cause massive cell death. As compared to TCR-activated WT T cells, activated Ezh2-/- T cells displayed significantly less ATP, lower mitochondrial membrane potential, enlarged mitochondrial mass, and decreased capacity to upregulate oxidative phosphorylation. Stim1 deletion largely reversed the metabolic defect in Ezh2-/- T cells, indicating the critical role of mitochondrial metabolism in rescuing these T cells. Considered together, our findings identify the remarkable coordination between Ezh2- and Stim1-regulated effector T cell persistence. As such, these investigations may lead to new approaches to inhibit GVHD, with broad implications to defining fundamental mechanisms of T cell differentiation for control of adaptive immunity, such as tumor immunity and autoimmunity. DisclosuresReshef:Incyte: Consultancy; Takeda Pharmaceuticals: Consultancy; Pfizer: Consultancy; Kite Pharma: Consultancy; Atara Biotherapeutics: Consultancy; Bristol-Myers Squibb: Consultancy.

Pharmacological characterization of human beta-defensins 3 and 4 on potassium channels: Evidence of diversity in beta-defensin-potassium channel interactions

Recent reports have identified defensins as a new type of potassium channel inhibitors; differential binding mechanisms of human β-defensins hBD1 and hBD2 point to complex interactions between human β-defensins and potassium channels. We investigated the inhibitory effects of human defensins hBD3 and hBD4 on potassium channels. The data indicate that hBD3 is a voltage-gated channel subfamily A member 3 (Kv1.3) inhibitor with an IC50 value of 187.6 ± 25.7 nM; 1 μM hBD4 inhibited 34.0 ± 0.2% of Kv1.3 channel currents. Moreover, 1 μM hBD3 inhibited 50.6 ± 3.6% of Kv1.2 channel currents and had smaller effects on Kv1.1, SKCa3, and IKCa channel currents; these effects differed from the Kv1.3 channel-specific inhibitors hBD1 and hBD2. Similar to the pharmacological profiles of hBD1 and hBD2, hBD4 had lower inhibitory effects on Kv1.1, Kv1.2, SKCa3, and IKCa channels. Subsequent mutagenesis and channel activation experiments confirmed that hBD3 binds in a manner similar to that of hBD1, interacting with the outer pore region of the Kv1.3 channel without affecting Kv1.3 channel activation. Thus, the data indicate that the human β-defensin family is a novel group of potassium channel inhibitors with diverse types of human β-defensin-potassium channel interactions.

Endothelial Caveolae-TRPV4-SK Channel Interactions in Shear Stress-Mediated Coronary Arteriole Dilation

We have previously reported that shear stress-mediated dilation (SSD) of coronary arterioles is caveolae-dependent. We found that both TRPV4 and SK channels are targeted to the low-buoyant density caveolin-rich membrane fraction in freshly isolated bovine coronary endothelial cells (BCEC) and TRPV4 and SK3 channels are co-immunoprecipitated by anti-caveolin-1 antibody. Isoproterenol (1 µM) enhanced the whole-cell K+ currents in BCEC by 5-fold that are inhibited by apamin (200 nM), an SK channel-specific inhibitor, but not by iberiotoxin (100 nM) or Tram34 (200 nM). Exposure of the BCEC to the TRPV4 activator, GSK1016790A (150 nM) resulted in a 3.5-fold activation of SK currents. Acute exposure of BCEC seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in a 4-fold increase in TRPV4 currents that are inhibited by HC067047 (200 nM), a TRPV4-specific inhibitor, and a 3.5-fold increase in SK currents that are inhibited by apamin (200 nM). However, after incubation with HC067047, a TRPV4 specific inhibitor, SK currents could no longer be activated by SS, suggesting SK activation by SS was mediated through TRPV4. In addition, pre-incubation of isolated coronary arterioles with apamin (200 nM) resulted in a significant diminution of SSD while pre-incubation with HC067047 (150 nM) results in vasoconstriction by SS. Exposure of BECE to SS (10 dynes/cm2 12 h) enhanced the production of endothelium-derived vasodilators, NO and PGI2, and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated [Ca2+]i increase in BECE. These novel results indicate that there is a dynamic interaction in the vascular endothelium between caveolae, TRPV4 and SK channels in the modulation of SSD.

T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling.

Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca2+ influx, and highly selective T-type Ca2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca2+ channels, prevented enhanced axon growth. T-type Ca2+ channel-specific inhibitors eliminated Ca2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca2+ channels along sympathetic axons, which is required for enhanced axon growth.

Involvement of calcium/calmodulin-dependent protein kinase II in methamphetamine-induced neural damage.

Methamphetamine (METH), an illicit drug, is widely abused in many parts of the world. Mounting evidence shows that METH exposure contributes to neurotoxicity, particularly for the monoaminergic neurons. However, to date, only a few studies have tried to unravel the mechanisms involved in METH-induced non-monoaminergic neural damage. Therefore, in the present study, we tried to explore the mechanisms for METH-induced neural damage in cortical neurons. Our results showed that METH significantly increased intracellular [Ca(2) (+) ]i in Ca(2) (+) -containing solution rather than Ca(2) (+) -free solution. Moreover, METH also upregulated calmodulin (CaM) expression and activated CaM-dependent protein kinase II (CaMKII). Significantly, METH-induced neural damage can be partially retarded by CaM antagonist W7 as well as CaMKII blocker KN93. In addition, L-type Ca(2) (+) channel was also proved to be involved in METH-induced cell damage, as nifedipine, the L-type Ca(2) (+) channel-specific inhibitor, markedly attenuated METH-induced neural damage. Collectively, our results suggest that Ca(2) (+) -CaM-CaMKII is involved in METH-mediated neurotoxicity, and it might suggest a potential target for the development of therapeutic strategies for METH abuse. Copyright © 2016 John Wiley & Sons, Ltd.

Blockage of KCa3.1 and Kv1.3 channels of the B lymphocyte decreases the inflammatory monocyte chemotaxis

ObjectiveTo investigate the effects of Ca2+ activated potassium channel KCa3.1 and voltage-gated potassium channel Kv1.3 of B lymphocyte on inflammatory monocytes chemotaxis and the potential mechanisms. Materials and methodsThanswell test was used to detect the inflammatory monocyte (Ly-6Chi) chemotaxis caused by the B lymphocyte. Enzyme-linked immunosorbent assay (ELISA) was applied to detecting the C-C motif ligand 7 (CCL7) in cultured media. Cell counting kit-8 (CCK) was used to detect the proliferation of B lymphocytes after activation and blockage of both KCa3.1 and Kv1.3 channels. Western blot was used to detect the expression of phosphorylated extracellular signal-regulated kinase (P-ERK) of the B lymphocytes. ResultsWhen activated, B lymphocytes significantly proliferated. After application of KCa3.1 channel-specific inhibitor TRAM-34 and potent Kv1.3 channel inhibitor ShK, both B lymphocytes proliferation and Ly-6Chi monocyte chemotaxis were significantly inhibited. The expression of chemotaxis related factor CCL7 decreased remarkably. ConclusionThe opening of KCa3.1 and Kv1.3 channels promote B lymphocyte activation, proliferation and Ly-6Chi monocyte chemotaxis. The increase of CCL7 secretion by B lymphocyte may explain the pro migration effects.

9-Phenanthrol inhibits recombinant and arterial myocyte TMEM16A channels.

In arterial smooth muscle cells (myocytes), intravascular pressure stimulates membrane depolarization and vasoconstriction (the myogenic response). Ion channels proposed to mediate pressure-induced depolarization include several transient receptor potential (TRP) channels, including TRPM4, and transmembrane protein 16A (TMEM16A), a Ca(2+) -activated Cl(-) channel (CaCC). 9-Phenanthrol, a putative selective TRPM4 channel inhibitor, abolishes myogenic tone in cerebral arteries, suggesting that either TRPM4 is essential for pressure-induced depolarization, upstream of activation of other ion channels or that 9-phenanthrol is non-selective. Here, we tested the hypothesis that 9-phenanthrol is also a TMEM16A channel blocker, an ion channel for which few inhibitors have been identified. Patch clamp electrophysiology was used to measure rat cerebral artery myocyte and human recombinant TMEM16A (rTMEM16A) currents or currents generated by recombinant bestrophin-1, another Ca(2+) -activated Cl(-) channel, expressed in HEK293 cells. 9-Phenanthrol blocked myocyte TMEM16A currents activated by either intracellular Ca(2+) or Eact , a TMEM16A channel activator. In contrast, 9-phenanthrol did not alter recombinant bestrophin-1 currents. 9-Phenanthrol reduced arterial myocyte TMEM16A currents with an IC50 of ∼12 μM. Cell-attached patch recordings indicated that 9-phenanthrol reduced single rTMEM16A channel open probability and mean open time, and increased mean closed time without affecting the amplitude. These data identify 9-phenanthrol as a novel TMEM16A channel blocker and provide an explanation for the previous observation that 9-phenanthrol abolishes myogenic tone when both TRPM4 and TMEM16A channels contribute to this response. 9-Phenanthrol may be a promising candidate from which to develop TMEM16A channel-specific inhibitors.

Tumor-associated macrophage-derived IL-6 and IL-8 enhance invasive activity of LoVo cells induced by PRL-3 in a KCNN4 channel-dependent manner.

BackgroundTumor-associated macrophages (TAMs) are known to promote cancer progression and metastasis through the release of a variety of cytokines. Phosphatase of regenerating liver (PRL-3) has been considered as a marker of colorectal cancer (CRC) liver metastasis. Our previous research suggests that PRL-3 can enhance the metastasis of CRC through the up-regulation of intermediate-conductance Ca2+-activated K+ (KCNN4) channel, which is dependent on the autocrine secretion of tumor necrosis factor-alpha (TNF-α). However, whether TAMs participate in the progression and metastasis of CRC induced by PRL-3 remains unknown.MethodsWe used flow cytometry, coculture, western blotting, invasion assays, real-time quantitative PCR, chromatin immunoprecipitation, luciferase reporter assays, and immunofluorescence staining to determine the effect of TAMs on the ability of PRL-3 to promote invasiveness of CRC cells.ResultsIn this study, we found that TAMs facilitated the metastasis of CRC induced by PRL-3. When TAMs were cocultured with CRC cells, the expression of KCNN4 was increased in TAMs and the invasion of CRC cells was enhanced. Furthermore, cytokines that were secreted by TAMs, such as IL-6 and IL-8, were also significantly increased. This response was attenuated by treating TAMs with the KCNN4 channel-specific inhibitor, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), which suggested that KCNN4 channels may be involved in inducing the secretion of IL-6 and IL-8 by TAMs and improving CRC cell invasiveness. Moreover, the expression of KCNN4 channels in TAMs was regulated through the NF-κB signal pathway, which is activated by TNF-α from CRC cells. Immunofluorescence analysis of colorectal specimens indicated that IL-6 and IL-8 double positive cells in the stroma showed positive staining for the TAM marker CD68, suggesting that TAMs produce IL-6 and IL-8. Increased numbers of these cells correlated with higher clinical stage.ConclusionsOur findings suggested that TAMs participate in the metastasis of CRC induced by PRL-3 through the TNF-α mediated secretion of IL-6 and IL-8 in a paracrine manner.

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.

Effects of propofol on responses of rat isolated renal arteriole to vasoactive agents

Rat renal arterial rings were suspended in organ chambers for isometric tension recording. The effects of propofol on the resting tone, KCl-, norepinephrine (NE)-, serotonin- and thromboxane A 2 analog U46619-induced contractions were observed. The relaxation responses to propofol on KCl-, NE- and U46619-induced contractions were assessed in the absence or presence of cyclooxygenase inhibitor, nitric oxide synthetase inhibitor or specific K + channel inhibitors. Propofol did not significantly affect the resting tone, but inhibited the contractions induced by KCl-, NE-, serotonin- and U46619. Propofol (1–100 µM) concentration-dependently relaxed 60 mM KCl-, 10 µM NE-, and 1 µM U46619-induced contractions with the values of RC 50 (concentration to decline the precontraction by 50%) being 18.9 µM, 70.6 µM and 12.7 µM, respectively. Propofol-induced relaxation was attenuated by indomethacin, but not by either N G nitro- l-arginine methyl ester (L-NAME) or any K + channel specific inhibitors used. The vasorelaxations induced by acetylcholine, sodium nitroprusside and amrinone were not affected by the presence of propofol. The present results indicate that propofol antagonizes provoked contractions of the arteriole and suggest that inhibition of extracellular Ca 2+ influx and synthesis of vasodilator prostanoid may be involved in propofol-induced relaxation of the arteriole.

Expression and function of calcium‐activated potassium channels in human glioma cells

Ca(2+)-activated K(+) (K(Ca)) channels are a unique family of ion channels because they are capable of directly communicating calcium signals to changes in cell membrane potential required for cellular processes including but not limited to cellular proliferation and migration. It is now possible to distinguish three families of K(Ca) channels based on differences in their biophysical and pharmacological properties as well as genomic sequence. Using a combination of biochemical, molecular, and biophysical approaches, we show that human tumor cells of astrocytic origin, i.e. glioma cells, express transcripts for all three family members of K(Ca) channels including BK, IK, and all three SK channel types (SK1, SK2, and SK3). The use of selective pharmacological inhibitors shows prominent expression of currents that are inhibited by the BK channel specific inhibitors iberiotoxin and paxilline. However, despite the presence of transcripts for IK and SK, neither clotrimazole, an inhibitor of IK channels, nor apamin, known to block most SK channels inhibited any current. The exclusive expression of functional BK channels was further substantiated by shRNA knockdown experiments, which selectively reduced iberiotoxin sensitive currents. Western blotting of patient biopsies with antibodies specific for all three KCa channel types further substantiated the exclusive expression of BK type KCa channels in vivo. This finding is in sharp contrast to other cancers that express primarily IK channels.

Characterization of the Functional Coupling of Bovine Brain Vacuolar-type H+-translocating ATPase: EFFECT OF DIVALENT CATIONS, PHOSPHOLIPIDS, AND SUBUNIT H (SFD)

Vacuolar-type H+-translocating ATPases (V-ATPases or V-pumps) are complex proteins containing multiple subunits and are organized into two functional domains: a peripheral catalytic sector V1 and a membranous proton channel V0. The functional coupling of ATP hydrolysis activity to proton transport in V-pumps requires a regulatory component known as subunit H (SFD) as has been shown both in vivo and in vitro (Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993) J. Biol. Chem. 268, 18286-18292; Xie, X. S., Crider, B. P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Ca2+ is thought to uncouple V-pumps because it is found to support ATP hydrolysis but not proton transport, while Mg2+ supports both activities. The direct effect of phospholipids on the coupling of V-ATPases has not been reported, likely due to the fact that phospholipids are constituents of biological membranes. We now report that Ca2+-induced uncoupling of the bovine brain V-ATPase can be reversed by imposition of a favorable membrane potential. Furthermore we report a simple "membrane-free" assay system using the V0 proton channel-specific inhibitor bafilomycin as a probe to detect the coupling of V-ATPase under certain conditions. With this system, we have characterized the functional effect of subunit H, divalent cations, and phospholipids on bovine brain V-ATPase and have found that each of these three factors plays a critical role in the functional coupling of the V-pump.

Activation of Calcium Signaling in Isolated Rat Hepatocytes Is Accompanied by Shape Changes of Microvilli

Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca2+signaling pathway, the ATP-dependent Ca2+store and the store-coupled Ca2+influx pathway, are both located in microvilli covering the surface of these cells. Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors. These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca2+, ATP, and IP3. The entry of Ca2+into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body. Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP3-sensitive Ca2+store that can be emptied by profilin-induced depolymerization or reorganization [K. Lange and U. Brandt (1996)FEBS Lett.395, 137–142]. Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca2+signaling system in isolated rat hepatocytes. Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the “diffusion barrier” concept of Ca2+signaling: Irrespective of the type of the applied stimulus, activation of the Ca2+influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function. We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type. Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed. Under these conditions the cells displayed the well known rather high basal [Ca2+]iof 200–250 nMas repeatedly demonstrated for freshly isolated hepatocytes. However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca2+level to about 100 nM.Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca2+signaling pathway by vasopressin, as well as by the IP3-independent acting Ca2+store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca2+]iinduced by the effectors. Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca2+/Fura-2 compartment which is rapidly depleted from Ca2+by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca2+influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca2+via the microvillar tip region in the cytoplasm.

Nifedipine-sensitive intramembrane charge movement in Purkinje cells from mouse cerebellum.

1. The intramembrane charge movement was recorded in freshly dissociated Purkinje cells from 14- to 18-day-old mouse cerebellum using the whole-cell voltage clamp technique. 2. After pharmacological elimination of all ionic currents, a depolarizing pulse from a holding potential of -80 mV revealed a transient capacitive outward current at the onset and a transient inward current at the end of the pulse. The amount of charge transferred at the onset (Qon) was equivalent to that moved at the end of the pulse (Qoff). The decay time course of Qon can be fitted by a single exponential curve with a maximum time constant of 1.89 +/- 0.35 ms at 20 mV (n = 11). 3. The charge movement had an S-shaped dependence on test membrane potential, according to a two-state Boltzmann function. The maximum amount (Qmax) of Qon that could be moved was 17.46 +/- 0.83 nC muF-1; the membrane potential at which half the charge movement occurred (V) was 13.48 +/- 2.20 mV and the slope factor (k) was 16.83 +/- 0.84 mV (n = 27). 4. Phenylglyoxal (2 mM), an arginine-specific modifying reagent, reduced Qmax to 60% of control after 20 min treatment. 5. The charge movement was partially immobilized by nifedipine in a dose-dependent manner with an IC50 of 70 nM. The fraction of the nifedipine-sensitive component was 39% of the total charge movement. The potential dependence of the nifedipine-sensitive charge movement could be expressed by a Boltzmann function with values of 7.00 +/- 0.53 nC muF-1 for Qmax, 31.44 +/- 4.23 mV for V and 21.53 +/- 3.18 mV for k (n = 8). 6. The P-type calcium channel specific inhibitor, omega-Aga IVA (250 nM), had no effect on intramembrane charge movement. 7. The above results show that part of the intramembrane charge movement in Purkinje cells may be related to a conformational change of DHP receptors upon membrane depolarization.