Search

(1) Background: In addition to the major phytocannabinoids, trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), the cannabis plant (Cannabis sativa L.) synthesizes over 120 additional cannabinoids that are known as minor cannabinoids. These minor cannabinoids have been proposed to act as agonists and antagonists at numerous targets including cannabinoid type 1 (CB1) and type 2 (CB2) receptors, transient receptor potential (TRP) channels and others. The goal of the present study was to determine the agonist effects of the minor cannabinoids: cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabitriol (CBT) and cannabidivarin (CBDV) at the CB1 receptor. In addition, the CB1 receptor antagonist effects of Δ9-tetrahydrocannabivarin (Δ9-THCV) were compared with its isomer Δ8-tetrahydrocannabivarin (Δ8-THCV). (2) Methods: CB1 receptor activity was monitored by measuring cannabinoid activation of G protein-gated inward rectifier K+ (GIRK) channels in AtT20 pituitary cells using a membrane potential-sensitive fluorescent dye assay. (3) Results: When compared to the CB1 receptor full agonist WIN 55,212-2 and the partial agonist Δ9-THC, none of the minor cannabinoids caused a significant activation of Gi/GIRK channel signaling. However, Δ9-THCV and Δ8-THCV antagonized the effect of WIN 55,212-2 with half-maximal inhibitory concentrations (IC50s) of 434 nM and 757 nM, respectively. Δ9-THCV antagonism of the CB1 receptor was “ligand-dependent”; Δ9-THCV was more potent in inhibiting WIN 55,212-2 and 2-arachidonoylglycerol (2-AG) than Δ9-THC. (4) Conclusions: While none of the minor cannabinoids caused Gi/GIRK channel activation, Δ9-THCV antagonized the CB1 receptor in an isomer- and ligand-dependent manner.

A graphene-based ion sensitive field effect transistor (GISFET) has been developed and investigated in terms of its ion sensing performance. The GISFET sensor was found to demonstrate a high detection sensitivity enabling direct measurement of K+ ion efflux from live cells. The sensing performance of the GISFET was directly compared to that of a commercial Si ISFET and very similar detection results were obtained, highlighting the promise of the GISFET sensor for ion-sensing applications. Additionally, fabrication of a GISFET array containing 25 devices using a CMOS compatible photolithographic process was demonstrated, which resulted in good uniformity across the array and high ion sensing properties of the devices, underlining their application potential for simultaneous multi-well testing with small sample volume.

Recreational use of synthetic cannabinoids (SCs) is associated with desirable euphoric and relaxation effects as well as adverse effects including anxiety, agitation and psychosis. These SC-mediated actions represent a combination of potentiated cannabinoid receptor signaling and “off-target” receptor activity. The goal of this study was to compare the efficacy of various classes of SCs in stimulating CB(1) receptors and activating “off-target” transient receptor potential (TRP) channels. Cannabinoid-type 1 (CB(1)) receptor activity was determined by measuring SC activation of G protein-gated inward rectifier K(+) (GIRK) channels using a membrane potential-sensitive fluorescent dye assay. SC opening of vanilloid type-1 (TRPV1) channels was measured by recording intracellular Ca(2+) transients. All of the SCs tested activated the GIRK channel with an efficacy of 4-fluoro MDMB-BUTINACA > 5-fluoro MDMB-PICA > MDMB-4en-PINACA ≈ WIN 55,212-2 > AB-FUBINACA > AM1220 ≈ JWH-122 N-(5-chloropentyl) > AM1248 > JWH-018 ≈ XLR-11 ≈ UR-144. The potency of the SCs at the CB(1) receptor was 5-fluoro MDMB-PICA ≈ 4-fluoro MDMB-BUTINACA > AB-FUBINACA ≈ MDMB-4en-PINACA > JWH-018 > AM1220 > XLR-11 > JWH-122 N-(5-chloropentyl) > WIN 55,212-2 ≈ UR-144 > AM1248. In contrast, when tested at a SC concentration that produced a maximal effect on the G(i)/GIRK channel, only XLR-11, UR-144 and AM1220 caused a significant activation of the TRPV1 channels. The TRPV1 channel/Ca(2+) signal measured during application of 10 µM XLR-11 was similar to the signal induced by the endocannabinoid N-arachidonoylethanolamine (AEA). Thus, while various SCs share the ability to stimulate CB(1) receptor/G(i) signaling, they display limited efficacy in opening TRPV1 channels.

Synthetic cannabinoids (SCs) are a class of new psychoactive substances (NPSs) that exhibit high affinity binding to the cannabinoid CB1 and CB2 receptors and display a pharmacological profile similar to the phytocannabinoid (-)-trans-Δ9-tetrahydrocannabinol (THC). SCs are marketed under brand names such as K2 and Spice and are popular drugs of abuse among male teenagers and young adults. Since their introduction in the early 2000s, SCs have grown in number and evolved in structural diversity to evade forensic detection and drug scheduling. In addition to their desirable euphoric and antinociceptive effects, SCs can cause severe toxicity including seizures, respiratory depression, cardiac arrhythmias, stroke and psychosis. Binding of SCs to the CB1 receptor, expressed in the central and peripheral nervous systems, stimulates pertussis toxin-sensitive G proteins (Gi/Go) resulting in the inhibition of adenylyl cyclase, a decreased opening of N-type Ca2+ channels and the activation of G protein-gated inward rectifier (GIRK) channels. This combination of signaling effects dampens neuronal activity in both CNS excitatory and inhibitory pathways by decreasing action potential formation and neurotransmitter release. Despite this knowledge, the relationship between the chemical structure of the SCs and their CB1 receptor-mediated molecular actions is not well understood. In addition, the potency and efficacy of newer SC structural groups has not been determined. To address these limitations, various cell-based assay technologies are being utilized to develop structure versus activity relationships (SAR) for the SCs and to explore the effects of these compounds on noncannabinoid receptor targets. This review focuses on describing and evaluating these assays and summarizes our current knowledge of SC molecular pharmacology.

A graphene-based ion sensitive field effect transistor (GISFET) has been developed and investigated in terms of its ion sensing performance. The GISFET sensor was found to demonstrate a high detection sensitivity enabling direct measurement of K

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

Graphene-based ion sensitive field effect transistors (GISFETs) with high sensitivity and selectivity for K+ ion detection have been demonstrated utilizing valinomycin based ion selective membrane. The performance of the GISFETs for K+ ion detection was studied in various media over a concentration range of 1 μM–2 mM. The sensitivity of the sensor was found to be >60 mV/decade, which is comparable to the best Si-based commercial ISFETs, with negligible interference found from Na+ and Ca2+ ions in high concentration. The sensor performance did not change significantly in Tris–HCl solution or with repeated testing over a period of two months highlighting its reliability and effectiveness for physiological monitoring. The performance of the sensor also remained unchanged when fabricated on biocompatible polyethylene terephthalate (PET) substrate, showing significant potential for developing flexible bio-implantable graphene-based ISFETs.

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K+ (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus. In whole-cell patch clamp experiments application of the cannabinoid agonist WIN 55,212-2 to AtT20 cells expressing the CB1 receptor (AtT20/CB1) activated GIRK currents that were blocked by BaCl2. WIN 55,212-2 activation of the GIRK channels was associated with a time- and concentration-dependent (EC50 = 309 nM) hyperpolarization of the membrane potential in the AtT20/CB1 cells when monitored using a fluorescent membrane potential-sensitive dye. The WIN 55,212-2-induced fluorescent signal was inhibited by pretreatment of the cells with either the GIRK channel blocker tertiapin-Q or the CB1 receptor antagonist SR141716. The cannabinoids displayed a response of WIN 55,212-2 ≈ anandamide (AEA) > CP 55,940 > Δ9-tetrahydrocannabinol (THC) when maximal concentrations of the four ligands were tested in the assay. Thus, the AtT20/CB1 cell fluorescent assay will provide a straightforward and efficient methodology for examining cannabinoid-stimulated Gi signaling.

Cell-based screening assays are now widely used for identifying compounds that serve as ion channel modulators. However, instrumentation for the automated, real-time analysis of ion flux from clonal and primary cells is lacking. This study describes the initial development of an ion-sensitive field effect transistor (ISFET)-based screening assay for the acquisition of K(+) efflux data from cells cultured in multi-well plates. Silicon-based K(+)-sensitive ISFETs were tested for their electrical response to varying concentrations of KCl and were found to display a linear response relationship to KCl in the range of 10 µM-1 mM. The ISFETs, along with reference electrodes, were inserted into fast-flow chambers containing either human colonic T84 epithelial cells or U251-MG glioma cells. Application of the Ca(2+) ionophore A23187 (1 µM), to activate Ca(2+)-activated non-selective cation (NSC) channels (T84 cells) and large conductance Ca(2+)-activated K(+) (BK) channels (U251 cells), resulted in time-dependent increases in the extracellular K(+) concentration ([K(+)]o) as measured with the ISFETs. Treatment of the cells with blockers of either the NSC or BK channels, caused a strong inhibition of the A23187-induced increase in [K(+)]o. These results were consistent with ion current measurements obtained using the whole-cell arrangement of the patch clamp procedure. In addition, K(+) efflux data could be acquired in parallel from multiple cell chambers using the ISFET sensors. Given the non-invasive properties of the probes, the ISFET-based assay should be adaptable for screening ion channels in various cell types.

Tissue-transglutaminase (TG2), a dual function G-protein, plays key roles in cell differentiation and migration. In our previous studies we reported the mechanism of TG2-induced cell differentiation. In present study, we explored the mechanism of how TG2 may be involved in cell migration.To study the mechanism of TG2-mediated cell migration, we used neuroblastoma cells (SH-SY5Y) which do not express TG2, neuroblastoma cells expressing exogenous TG2 (SHYTG2), and pancreatic cancer cells which express high levels of endogenous TG2. Resveratrol, a natural compound previously shown to inhibit neuroblastoma and pancreatic cancer in the animal models, was utilized to investigate the role of TG2 in cancer cell migration. Immunofluorescence assays were employed to detect expression and intracellular localization of TG2, and calcium levels in the migrating cells. Native gel electrophoresis was performed to analyze resveratrol-induced cellular distribution and conformational states of TG2 in migrating cells. Data are presented as the mean and standard deviation of at least 3 independent experiments. Comparisons were made among groups using one-way ANOVA followed by Tukey-Kramer ad hoc test.TG2 containing cells (SHYTG2 and pancreatic cancer cells) exhibit increased cell migration and invasion in collagen-coated and matrigel-coated transwell plate assays, respectively. Resveratrol (1 μM-10 μM) prevented migration of TG2-expressing cells. During the course of migration, resveratrol increased the immunoreactivity of TG2 without affecting the total TG2 protein level in migrating cells. In these cells, resveratrol increased calcium levels, and depletion of intracellular calcium by a calcium chelator, BAPTA, attenuated resveratrol-enhanced TG2 immunoreactivity. In native-polyacrylamide gels, we detected an additional TG2 protein band with slower migration in total cell lysates of resveratrol treated cells. This TG2 form is non-phosphorylated, exclusively present in plasma membrane fractions and sensitive to intracellular Ca(2+) concentration suggesting a calcium requirement in TG2-regulated cell migration.Taken together, we conclude that resveratrol induces conformational changes in TG2, and that Ca(2+)-mediated TG2 association with the plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration.

The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF.

Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K+ (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K+ currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K+ current ( Ito) that activated at potentials positive to −40 mV and partially inactivated during depolarizing voltage steps. Ito was inhibited by the antiarrhythmic agent flecainide (100 μM) and BaCl2 (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K+ currents [ IK fast ( IKf) and IK slow ( IKs)] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited Ito but had no effect on IKf and IKs. Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 α-subunits but not Kv4.2 or Kv1.6 α-subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca2+ mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.

The volume-sensitive chloride current ( ICl,swell) is found in the mammalian myocardium and is activated by osmotic swelling. The goal of this study was to examine the importance of the tyrosine kinases focal adhesion kinase (FAK) and Src kinase in cardiac ICl,swellregulation. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing β-galactosidase (AdLacZ), FAK, or FAK-related nonkinase. FAK-related nonkinase (FRNK) is an endogenous cardiac protein, which functions as an inhibitor of FAK. Whole cell patch-clamp recordings demonstrated that osmotic swelling was associated with the activation of an outward rectifying current in uninfected and AdLacZ-infected cells. Consistent with the properties of ICl,swell, this current displayed a reversal potential close to the equilibrium potential for Cl−; was inhibited by the Cl−channel blockers 4,4′-dinitrostilbene-2,2′-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and tamoxifen; and was eliminated in hypertonic solution. In addition to activating ICl,swell, hypotonic swelling enhanced the tyrosine phosphorylation of multiple cardiac proteins including those in the range of 68–70 and 120–130 kDa. Pretreatment of the cells with the drug 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of FAK and Src, diminished swelling-induced phosphorylation of these proteins but paradoxically increased ICl,swell. Furthermore, overexpression of FRNK but not FAK caused a twofold augmentation in ICl,swelland increased the rate of current activation. Thus the tyrosine kinases FAK and Src contribute to the regulation of ICl,swell.

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca2+ current ( ICa) is affected during transient increases in intracellular Ca2+ concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca2+ external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 μM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca2+ concentration was reduced to 0.5 mM or replaced with Ba2+. This Ca2+-dependent inhibition was also observed when the cells were treated with 1 μM isoproterenol, 100 μM 3-isobutyl-1-methylxanthine, or 500 μM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca2+/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca2+-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba2+. Thus Ca2+ entering the myocyte through the voltage-gated Ca2+ channel regulates the PKA responsiveness of ICa.

Objective: The goal of this study was to determine if the properties of the transient outward potassium ( I to), TTX-resistant sodium ( I Na) and L-type calcium ( I Ca) currents are altered during changes in cardiac cell shape. Methods: Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on either non-aligned or aligned collagen thin gels. In contrast to the flat, stellar-shaped myocytes obtained when the cells are plated on non-aligned collagen gels, myocytes plated on aligned gels display an elongated, rod-like shape. Ion channel expression was measured using the whole-cell arrangement of the patch clamp technique and Western blot analysis. Results: Peak values for I to, I Na and I Ca were 9±1, 71±13 and 7±1 pA/pF, respectively, in the flat cells, and increased to 21±2, 190±26 and 13±1 pA/pF, respectively, in the aligned cells. Application of forskolin (2 μM) and 3-isobutyl-1-methylxanthine (100 μM) resulted in a 101±18% increase in I Ca in the flat cells, but increased the current by only 43±9% in the aligned cells. Internal dialysis of the myocytes with cAMP strongly increased the peak I Ca in the flat cells, but caused no significant change in the aligned cells. While both basal and forskolin-stimulated levels of cAMP were the same in the two cell morphologies, the expression of the calcium channel α1C subunit was increased in the aligned cells. Conclusions: The expression and regulatory properties of voltage-gated calcium channels are modified during changes in neonatal rat myocyte shape.

The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding. Wild-type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild-type CFTR current by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) occurred in a voltage-dependent manner with preferential inhibition of the inward currents (Kd=166 μM at −90 mV). Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2-butylamino-5-nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd=243 μM), while addition of an extra phenyl ring at this position (5-nitro-2-(3,3-diphenylpropylamino)-benzoic acid) increased drug potency (Kd=58 μM). In contrast, removal of the benzoate ring (2-amino-4-phenylbutyric acid) or the 5-nitro group (2-(3-phenylpropylamino)-benzoic acid) of NPPB severely limited drug block of the wild-type channel. NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 μM, for the K335E and R347E mutants, respectively. NPPB block of the inward wild-type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN−. These studies present the first step in the development of high affinity probes to the CFTR channel. British Journal of Pharmacology (1999) 127, 369–376; doi:10.1038/sj.bjp.0702562

Abstract —Vascular endothelial cells are constantly exposed to mechanical forces resulting from blood flow and transmural pressure. The goal of this study was to determine whether mechanical stimulation alters the properties of endothelial voltage-gated K + channels. Cardiac microvascular endothelial cells (CMECs) were isolated from rat ventricular muscle and cultured on thin sheets of silastic membranes. Membrane currents were measured with the use of the whole-cell arrangement of the patch-clamp technique in endothelial cells subjected to static stretch for 24 hours and compared with measurements from control, nonstretched cells. Voltage steps positive to −30 mV resulted in the activation of a time-dependent, delayed rectifier K + current ( I K ) in the endothelial cells. Mechanically induced increases of 97%, 355%, and 106% at +30 mV were measured in the peak amplitude of I K in cells stretched for 24 hours by 5%, 10%, and 15%, respectively. In addition, the half-maximal voltage required for I K activation was shifted from +34 mV in the nonstretched cells to −5 mV in the stretched cells. Although I K in both groups of CMECs was blocked to a similar extent by tetraethylammonium, currents in the stretched endothelial cells displayed an enhanced sensitivity to inhibition by charybdotoxin. Preincubation of the CMECs with either pertussis toxin or phorbol 12-myristate 13-acetate during the 24 hours of cell stretch did not prevent the increase in I K . The application of phorbol 12-myristate 13-acetate and static stretch stimulated the proliferation of CMECs. Stretch-induced regulation of K + channels may be important to control the resting potential of the endothelium and may contribute to capillary growth during periods of mechanical perturbation.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate. GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3. Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

Cardiac ventricular myocytes from several species, including the guinea pig, possess a cAMP-dependent protein kinase A (PKA)-activated Cl- channel. In the present study, the properties of a protein kinase C (PKC)-activated Cl- current were studied in isolated guinea pig ventricular myocytes using the whole-cell arrangement of the patch-clamp technique. Intracellular dialysis of ventricular cells with PKC resulted in the activation of a large background current that displayed time-independent kinetics. In the presence of 146 mmol/L external Cl- and 71 mmol/L internal Cl-, the reversal potential (Erev) of the background current (-17 +/- 1 mV) was close to that of the Cl- equilibrium potential (-18 mV), and the current versus voltage relation for the current was outward rectifying in shape. When [Cl-]i or [Cl-]o was reduced by substitution of Cl- with aspartic acid, Erev for the background current shifted in a manner expected for a Cl(-)-selective channel. Based on Erev measurements, the permeability sequence for this PKC-activated Cl- channel was determined to be SCN- > I- > Br- congruent to Cl-. The PKC-activated Cl- current was not inhibited by the Cl- channel blocker 4,4'-dinitrostilbene-2,2'-disulfonic acid (100 mumol/L) but could be blocked by anthracene-9-carboxylic acid (1 mmol/L). Activation of the current was abolished in the presence of the PKC inhibitor staurosporine (2.5 mumol/L). Under conditions designed to cause a maximal activation of the Cl- channels by PKC, the addition of forskolin (1 mumol/L) to stimulate PKA caused only a slight further increase in the amplitude of the Cl- current. Thus, PKC activates a Cl- channel in guinea pig ventricular cells with properties similar but not identical to the PKA-activated channel.

G protein-coupled inward rectifier K+ (GIRK) channels represent novel targets for the development of new therapeutic agents. GIRK channels are activated by a large number of G protein-coupled receptors (GPCRs) and regulate the electrical activity of neurons, cardiac myocytes and β-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the patho-physiology of neuropathic pain, drug addiction, cardiac arrhythmias and other disorders. However, the pharmacology of these channels remains largely unexplored. In this paper we describe the development of a screening assay for identifying new modulators of neuronal and cardiac GIRK channels. Pituitary (AtT20) and cardiac (HL-1) cell lines expressing GIRK channels were cultured in 96-well plates, loaded with oxonol membrane potential-sensitive dyes and measured using a fluorescent imaging plate reader. Activation of the endogenous GPCRs in the cells caused a rapid, time-dependent decrease in the fluorescent signal; indicative of K+ efflux through the GIRK channels (GPCR stimulation versus control, Z’-factor = 0.5-0.7). As expected this signal was inhibited by addition of Ba2+ and the GIRK channel toxin tertiapin-Q. To test the utility of the assay for screening GIRK channel blockers, cells were incubated for 5 minutes with a compound library of Na+ and K+ channel modulators. Ion transporter inhibitors such as 5-(N,N-hexamethylene)-amiloride and SCH-28080 were identified as blockers of the GIRK channel at sub-micromolar concentrations. Thus, the screening assay will be useful for expanding the limited pharmacology of the GIRK channel and in developing new agents for the treatment of GIRK channelopathies.

Tissue-transglutaminase (TG2) is a dual function G-protein with Ca2+-dependent transglutaminase activity and GTP-binding/hydrolyzing activity. TG2 is physiologically involved in cell differentiation, cell death, migration, invasion of cancer cells. In the extracellular matrix (ECM), TG2 mediates cell- ECM interactions through fibronectin and integrins and promotes cell attachment, migration and invasion. When present on the cytoplasmic side of the plasma membrane TG2 activates phospholipase C and RhoA-ROCK-2 signaling pathways that promote cell differentiation. While mechanism of TG2-induced cell differentiation is well characterized, the mechanism underlying TG2-mediated cell migration is not known. In present study, we explored the mechanism of how TG2 may be involved in cell migration by using wild type neuroblastoma cells (SH-SY5Y) which do not express TG2, neuroblastoma cells expressing exogenous TG2 (SHYTG2), and a transamidation-deficient TG2 containing SH-SY5Y cells (SHYmutant). Our cell migration assays (scratch assay and collagen-coated transwell assay) and cell invasion assay (matrigel-coated trasnwell assay) indicate that TG2 expressing cells exhibit higher migrating and invasive properties. To further investigate the role of TG2 in cell migration, we utilized a natural compound, resveratrol. Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic and phytoalexin compound present in grapes and blueberries and has shown promising results in the treatment of neuroblastoma. In our studies, resveratrol (1 μM -10 μM) significantly prevented migration and invasion of TG2-expressing cells. In migrating cells, resveratrol increases the immunoreactivity of TG2 without affecting the total TG2 protein level. In these cells, resveratrol increases calcium levels, and depletion of intracellular calcium by a calcium chelator, BAPTA, attenuate resveratrol-enhanced TG2 immunoreactivity. To further analyze resveratrol-induced cellular distribution and conformational states of TG2, we performed native gel electrophoresis and detected an additional TG2 protein band with slower migration in resveratrol-treated migrating cells. This TG2 form is non-phosphorylated, exclusively present in plasma membrane fractions and sensitive to intracellular Ca2+ concentrations suggesting a calcium requirement in TG2-regulated cell migration. The observed less mobility of transamidation-defective SH-SY5Y cells (SHYmutant) and the reduced migration of SHYTG2 cells in the presence of a TG2 transamidation inhibitor, monodensylcadevarine, in scratch assays further support the requirement of TG2 in cell migration. Our mechanistic studies suggest that resveratrol induces conformational changes in TG2 (compact to open structure form), and Ca2+-mediated TG2 association to plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration. Together, present study indicates that TG2 plays a key role in neuroblastoma cell migration. Although higher concentrations of resveratrol (∼100 μM) induces cell death in cancer cells, the current study suggest that lower physiologically relevant dose of resveratrol (∼1 μM) prevent the migration and invasion of cancer cells from the primary site. Since retinoic acid treatment increases TG2 protein level and resveratrol inhibits the migration of TG2-expressing cells, this study also proposes the use of resveratrol with retinoic acid for the treatment of neuroblatoma. Citation Format: Ambrish Kumar, Jianjun Hu, Holly A. LaVoie, Kenneth B. Walsh, Donald J. DiPette, Ugra S. Singh. Calcium-sensitized tissue transglutaminase translocates to the plasma membranes and inhibits neuroblastoma cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B52.

Objectives: The aim of this study was to determine the effects of Cl− channel blockers on the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl− current ( I Cl) and the protein kinase A-regulated L-type calcium current (PKA- I Ca). Methods: Whole-cell I Cl and Z Ca were recorded from isolated guinea pig ventricular myocytes using the patch clamp technique during stimulation of PKA by forskolin (1 or 2 μM). Results: The inhibitory effects of clofibric acid, p -chlorophenoxy propionic acid, gemfibrozil, diphenylamine-2-carboxylate (DPC), anthracene-9-carboxylate, 4,4′dinitrostilbene-2,2′-disulfonic acid and indanyloxyacetic acid 94 were examined on the two currents. Clofibric acid (1 mM), p -chlorophenoxy propionic acid (1 mM) and gemfibrozil (250 μM) produced an approximate 50% decrease in I Cl, but had no effect on the PKA- I Ca. Surprisingly, application of DPC (500 μM and 1 mM) and anthracene-9-carboxylate (500 μM) strongly reduced both currents. However, inhibition of the Ca2+ and Cl− channels by DPC could be differentiated in two important ways. First, increasing the pH of the external solution from 7.4 to 10.0 prevented the block of I Cl by DPC, but did not attenuate the reduction in the PKA- I Ca. Second, DPC inhibited the PKA- I Ca in mouse atrial myocytes which lacked I Cl. Neither 4,4′dinitrostilbene-2,2′-disulfonic acid (100 μM) nor indanyloxyacetic acid 94 (50 μM) caused any change in either of the guinea pig ventricular currents. Conclusions: Drugs such as DPC and anthracene-9-carboxylate which block the cardiac CFTR Cl− channel also inhibit the regulation of the L-type I Ca. During β-adrenergic stimulation, changes produced by these drugs on the cardiac action potential duration will be attributable to inhibition of both the Cl− and Ca2+ currents. Analogues of clofibric acid may serve as selective blockers of the CFTR Cl− channel that can be used to determine the physiological function of I Cl in cardiac excitation.

Background Tissue-transglutaminase (TG2), a dual function G-protein, plays key roles in cell differentiation and migration. In our previous studies we reported the mechanism of TG2-induced cell differentiation. In present study, we explored the mechanism of how TG2 may be involved in cell migration. Methods To study the mechanism of TG2-mediated cell migration, we used neuroblastoma cells (SH-SY5Y) which do not express TG2, neuroblastoma cells expressing exogenous TG2 (SHYTG2), and pancreatic cancer cells which express high levels of endogenous TG2. Resveratrol, a natural compound previously shown to inhibit neuroblastoma and pancreatic cancer in the animal models, was utilized to investigate the role of TG2 in cancer cell migration. Immunofluorescence assays were employed to detect expression and intracellular localization of TG2, and calcium levels in the migrating cells. Native gel electrophoresis was performed to analyze resveratrol-induced cellular distribution and conformational states of TG2 in migrating cells. Data are presented as the mean and standard deviation of at least 3 independent experiments. Comparisons were made among groups using one-way ANOVA followed by Tukey-Kramer ad hoc test. Results TG2 containing cells (SHYTG2 and pancreatic cancer cells) exhibit increased cell migration and invasion in collagen-coated and matrigel-coated transwell plate assays, respectively. Resveratrol (1 μM-10 μM) prevented migration of TG2-expressing cells. During the course of migration, resveratrol increased the immunoreactivity of TG2 without affecting the total TG2 protein level in migrating cells. In these cells, resveratrol increased calcium levels, and depletion of intracellular calcium by a calcium chelator, BAPTA, attenuated resveratrol-enhanced TG2 immunoreactivity. In native-polyacrylamide gels, we detected an additional TG2 protein band with slower migration in total cell lysates of resveratrol treated cells. This TG2 form is non-phosphorylated, exclusively present in plasma membrane fractions and sensitive to intracellular Ca2+ concentration suggesting a calcium requirement in TG2-regulated cell migration. Conclusions Taken together, we conclude that resveratrol induces conformational changes in TG2, and that Ca2+-mediated TG2 association with the plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration.