86Rb Efflux Research Articles

Functional characterization of activating mutations in the sulfonylurea receptor 1 (ABCC8) causing neonatal diabetes mellitus in Asian Indian children.

Gain-of-function of ATP-sensitive K+ (KATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in KATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known KATP inhibitors in intact cells were determined by 86 Rb efflux assays. Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP4- inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the KATP channel inhibitors glibenclamide and carbamazepine. The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect KATP channel function to cause the disease. The reduced ATP4- sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium (K2P ) channel opener, reduces rat dorsal root ganglion neuron excitability.

Background and PurposeTREK two‐pore‐domain potassium (K2P) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI‐530159.Experimental ApproachThe effect of GI‐530159 on TREK channels was demonstrated using 86Rb efflux assays, whole‐cell and single‐channel patch‐clamp recordings from recombinant TREK channels. The expression of K2P2.1 (TREK1), K2P10.1 (TREK2) and K2P4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current‐clamp recordings from cultured rat DRG neurons were used to measure the effect of GI‐530159 on neuronal excitability.Key ResultsFor recombinant human TREK1 channels, GI‐530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current‐clamp recordings from cultured rat DRG neurones showed that application of GI‐530159 at 1 μM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential.Conclusions and ImplicationsThis study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI‐530159 could aid in the development of novel analgesic agents.Linked ArticlesThis article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc

Effect of piracetam on Ca2+-induced K+ efflux from sickle cells

We have therefore studied the in vitro effect of piracetam on Ca 2+ -activated K + ( 86 Rb) efflux from sickle cells. We have aslo tested the protective effect of piracetam on the deformability of sickle cells when entry of Ca 2+ was induced by repetitive cycles of oxygenation-deoxygenation for 15 hours

Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast.

Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established.

High Incidence of Heterozygous ABCC8 and HNF1A Mutations in Czech Patients With Congenital Hyperinsulinism.

Congenital hyperinsulinism of infancy (CHI) represents a group of heterogeneous disorders characterized by oversecretion of insulin from pancreatic β-cells causing severe hypoglycemia. We studied the distribution of genetic causes of CHI in a Czech population. Countrywide collection of patients with CHI included 40 subjects (12 females, median age of diagnosis, 1 wk [interquartile range, 1-612 wk]). We sequenced the ABCC8, KCNJ11, GLUD1, GCK, HADH, UCP2, SLC16A1, HNF4A, and HNF1A genes and investigated structural changes in the ABCC8 gene. We functionally tested novel variants in the ABCC8 gene by Rb(86+) efflux assay and novel variants in the HNF1A gene by transcriptional activation and DNA-binding tests. We found causal mutations in 20 subjects (50%): 19 carried a heterozygous mutation while one patient was homozygous for mutation in the ABCC8 gene. Specifically, we detected 11 mutations (seven novel) in ABCC8, one novel mutation in KCNJ11, five mutations (two novel) in HNF1A, two novel mutations in HNF4A, and one in GCK. We showed a decrease of activation by diazoxide in mutant KATP channels with novel ABCC8 variants by 41-91% (median, 82%) compared with wild-type (WT) channels and reduced transcriptional activity of mutant HNF1A proteins (2.9% for p.Asn62Lysfs93* and 22% for p.Leu254Gln) accompanied by no DNA-binding ability compared with WT HNF1A. We detected a higher proportion of heterozygous mutations causing CHI compared with other cohorts probably due to lack of consanguinity and inclusion of milder CHI forms. Interestingly, HNF1A gene mutations represented the second most frequent genetic cause of CHI in the Czech Republic. Based on our results we present a genetic testing strategy specific for similar populations.

Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

Methadone is a non-competitive antagonist at the α4β2 and α3* nicotinic acetylcholine receptors and an agonist at the α7 nicotinic acetylcholine receptor.

Nicotine-methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non-competitive antagonist at rat α3β4 nicotinic acetylcholine receptors (nAChR) and an agonist at human α7 nAChRs. In this study, we used cell lines expressing human α4β2, α7 and α3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [(3) H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and (86) Rb(+) efflux and changes in intracellular calcium [Ca(2+) ]i were used to assess changes in the functional activity of the receptors. Methadone displaced [(3) H]epibatidine from nicotinic agonist-binding sites in SH-EP1-hα7 and SH-SY5Y cells, but not in SH-EP1-hα4β2 cells. The Ki values for methadone were 6.3μM in SH-EP1-hα7 cells and 19.4μM and 1008μM in SH-SY5Y cells. Methadone increased [Ca(2+) ]i in all cell lines in a concentration-dependent manner, and in SH-EP1-hα7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH-EP1-hα4β2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre-treatment abolished the nicotine-induced response in [Ca(2+) ]i in all cell lines expressing nAChRs. In SH-EP1-hα4β2 and SH-SY5Y cells, methadone had no effect on the (86) Rb(+) efflux, but it antagonized the nicotine-induced (86) Rb(+) ion efflux in a non-competitive manner. These results suggest that methadone is an agonist at human α7 nAChRs and a non-competitive antagonist at human α4β2 and α3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co-administration in human beings and might be of interest to the field of drug discovery.

Intermediate Conductance Ca2+-Activated K+ Channels Modulate Human Placental Trophoblast Syncytialization

Regulation of human placental syncytiotrophoblast renewal by cytotrophoblast migration, aggregation/fusion and differentiation is essential for successful pregnancy. In several tissues, these events are regulated by intermediate conductance Ca2+-activated K+ channels (IKCa), in part through their ability to regulate cell volume. We used cytotrophoblasts in primary culture to test the hypotheses that IKCa participate in the formation of multinucleated syncytiotrophoblast and in syncytiotrophoblast volume homeostasis. Cytotrophoblasts were isolated from normal term placentas and cultured for 66 h. This preparation recreates syncytiotrophoblast formation in vivo, as mononucleate cells (15 h) fuse into multinucleate syncytia (66 h) concomitant with elevated secretion of human chorionic gonadotropin (hCG). Cells were treated with the IKCa inhibitor TRAM-34 (10 µM) or activator DCEBIO (100 µM). Culture medium was collected to measure hCG secretion and cells fixed for immunofluorescence with anti-IKCa and anti-desmoplakin antibodies to assess IKCa expression and multinucleation respectively. K+ channel activity was assessed by measuring 86Rb efflux at 66 h. IKCa immunostaining was evident in nucleus, cytoplasm and surface of mono- and multinucleate cells. DCEBIO increased 86Rb efflux 8.3-fold above control and this was inhibited by TRAM-34 (85%; p<0.0001). Cytotrophoblast multinucleation increased 12-fold (p<0.05) and hCG secretion 20-fold (p<0.05), between 15 and 66 h. Compared to controls, DCEBIO reduced multinucleation by 42% (p<0.05) and hCG secretion by 80% (p<0.05). TRAM-34 alone did not affect cytotrophoblast multinucleation or hCG secretion. Hyposmotic solution increased 86Rb efflux 3.8-fold (p<0.0001). This effect was dependent on extracellular Ca2+, inhibited by TRAM-34 and 100 nM charybdotoxin (85% (p<0.0001) and 43% respectively) but unaffected by 100 nM apamin. In conclusion, IKCa are expressed in cytotrophoblasts and their activation inhibits the formation of multinucleated cells in vitro. IKCa are stimulated by syncytiotrophoblast swelling implicating a role in syncytiotrophoblast volume homeostasis. Inappropriate activation of IKCa in pathophysiological conditions could compromise syncytiotrophoblast turnover and volume homeostasis in pregnancy disease.

Mice expressing the ADNFLE valine 287 leucine mutation of the Β2 nicotinic acetylcholine receptor subunit display increased sensitivity to acute nicotine administration and altered presynaptic nicotinic receptor function

Several mutations in α4 or β2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the β2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (β2VL). Previous studies indicated that the β2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of β2VL mice. No differences in β2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the β2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [3H]-dopamine release and 86Rb efflux were observed. Maximal nAChR-mediated [3H]-GABA release in the cortex was also decreased in the MT, but maximal [3H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic–clonic seizure (EEG recordable) 3min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.

ENU Mutagenesis in the Mouse for Identification of Genes Regulating Erythropoiesis: a Mouse Mutant with An Activating Mutation of the KCl Cotransporter, KCC1 Causing Dehydrated Red Cells

Abstract 684Genome-wide phenotype-driven studies have emerged as a powerful tool for the identification of novel genes in a biological process of interest, new functions for existing genes and the establishment of mouse models of human disease. Recent advances in exomic and whole genome sequencing has simplified mutation detection, making genetic screens in the mouse more facile. We have employed a large-scale mutagenesis screen in mice using the chemical mutagen N-ethyl-N-nitrosourea (ENU) to identify genes regulating erythropoiesis. In this dominant screen, G1 progeny were screened at 7 weeks of age by automated hematological analysis for isolated abnormalities of red cell indices (HCT, RCC and MCV) defined as greater than three standard deviations from the wild type population. Here, we describe the RBC10 mouse line, in which homozygotes had mild anemia with microcytosis, target cells, spherocytes, reticulocytosis and increased resistance to osmotic stress. A combination of SSLP and SNP mapping isolated the genetic mutation to a 3.65 Mb region on chromosome 8. Whole genome Next Generation Sequencing of the mapped interval identified an A to T nucleotide substitution within the coding region of the potassium chloride (KCl) co-transporter, Slc12a4 (KCC1), resulting in a non-conservative amino acid substitution from methionine to lysine at position 935 within the C-terminal cytoplasmic domain. KCC1 and KCC3 function are the major erythroid KCl co-transporters that export KCl in response to hypotonic swelling. We hypothesized that the M935K was an activating mutation of KCC1, based upon the microcytosis and resistance to osmotic stress, which contrasts with the macrocytic phenotype of red cells lacking both KCC1 and KCC3. 86Rb efflux assay confirmed increased KCC1 activity of RBC mutant red cells. We propose that the M935K mutation increases activity of KCC1 by preventing phosphorylation of the nearby threonine reside, a critical regulatory target of the serine-threonine kinase, WNK1. Disclosures:No relevant conflicts of interest to declare.

Hyperinsulinaemic hypoglycaemia and diabetes mellitus due to dominant ABCC8/KCNJ11 mutations

Aims/hypothesisDominantly acting loss-of-function mutations in the ABCC8/KCNJ11 genes can cause mild medically responsive hyperinsulinaemic hypoglycaemia (HH). As controversy exists over whether these mutations predispose to diabetes in adulthood we investigated the prevalence of diabetes in families with dominantly inherited ATP-sensitive potassium (KATP) channel mutations causing HH in the proband.MethodsWe studied the phenotype of 30 mutation carriers (14 children and 16 adults) from nine families with dominant ABCC8/KCNJ11 mutations. Functional consequences of six novel missense mutations were examined by reconstituting the KATP channel in human embryonic kidney 293 (HEK293) cells and evaluating the effect of drugs and metabolic poisoning on the channels using the 86Rb flux assay.ResultsThe mutant channels all showed a lack of 86Rb efflux on exposure to the channel agonist diazoxide or metabolic inhibition. In the families, dominant ABCC8/KCNJ11 mutations were associated with increased birthweight (median + 1.56 SD score [SDS]). Fourteen children had HH and five adults were reported with HH or hypoglycaemic episodes (63%). Progression from hypoglycaemia to diabetes mellitus occurred in two individuals. Eight adults had a history of gestational diabetes in multiple pregnancies or were diabetic (diagnosed at a median age of 31 years). Within these families, none of the 19 adults who were not carriers of the ABCC8/KCNJ11 mutation was known to be diabetic.Conclusions/interpretationThe phenotype associated with dominant ABCC8/KCNJ11 mutations ranges from asymptomatic macrosomia to persistent HH in childhood. In adults, it may also be an important cause of dominantly inherited early-onset diabetes mellitus.

Down‐regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

Gain of function of Kir4.1 channel increases cell resistance to changes of potassium fluxes and cell volume evoked by ammonia and hypoosmotic stress

The Kir4.1 channel is an inward rectifying potassium channel involved in the control of potassium and water movement in mammalian cells. To evaluate independently the role of Kir4.1 alone and without interaction with other cellular effectors, we compared 86Rb fluxes and cell volume in Kir4.1 transfected cells (Kir4.1+) with cells transfected with an empty vector (Kir4.1–). Transfection with Kir4.1 neither increased 86Rb uptake nor 86Rb efflux from cells in isotonic medium. Pretreatment with ammonia (5mM ammonium chloride) in isotonic medium produced a pronounced increase of 86Rb uptake and a moderate decrease of cell volume in Kir4.1– but not in Kir4.1+ cells. However, pretreatment evoked no change in 86Rb efflux in either cell type. Hypotonic treatment (HT) markedly increased 86Rb efflux in Kir4.1– cells and increased cell volume in both cell types. Although pretreatment with ammonia did not alter the effect of HT on 86Rb efflux in either Kir4.1+ or Kir4.1– cells, it potentiated the effect of hypotonic treatment in increasing cell volume in Kir4.1– cells. The results demonstrate that the presence of Kir4.1in cells increases their resistance to alterations of potassium fluxes and/or cell volume imposed by ammonia and hypotonicity.

Congenital Hyperinsulinism and Glucose Hypersensitivity in Homozygous and Heterozygous Carriers of Kir6.2 (KCNJ11) Mutation V290M Mutation

OBJECTIVEThe ATP-sensitive K+ channel (KATP) controls insulin secretion from the islet. Gain- or loss-of-function mutations in channel subunits underlie human neonatal diabetes and congenital hyperinsulinism (HI), respectively. In this study, we sought to identify the mechanistic basis of KATP-induced HI in two probands and to characterize the clinical course.RESEARCH DESIGN AND METHODSWe analyzed HI in two probands and characterized the course of clinical treatment in each, as well as properties of mutant KATP channels expressed in COSm6 cells using Rb efflux and patch-clamp methods.RESULTSWe identified mutation V290M in the pore-forming Kir6.2 subunit in each proband. In vitro expression in COSm6 cells supports the mutation resulting in an inactivating phenotype, which leads to significantly reduced activity in intact cells when expressed homomerically, and to a lesser extent when expressed heteromerically with wild-type subunits. In one heterozygous proband, a fluoro-DOPA scan revealed a causal focal lesion, indicating uniparental disomy with loss of heterozygosity. In a second family, the proband, homozygous for the mutation, was diagnosed with severe diazoxide–unresponsive hypersinsulinism at 2 weeks of age. The patient continues to be treated successfully with octreotide and amlodipine. The parents and a male sibling are heterozygous carriers without overt clinical HI. Interestingly, both the mother and the sibling exhibit evidence of abnormally enhanced glucose tolerance.CONCLUSIONSV290M results in inactivating KATP channels that underlie HI. Homozygous individuals may be managed medically, without pancreatectomy. Heterozygous carriers also show evidence of enhanced glucose sensitivity, consistent with incomplete loss of KATP channel activity.

Rb+ Efflux Assay for Assessment of Non-Selective Cation Channel Activities

ABSTRACT Transient receptor potential (TRP) channels have been found to play important roles in cellular physiology and hold promise as therapeutic targets. These channels activate in response to a variety of chemical or physical stimuli and conduct non-selective cation currents (NSCC). Due to their unique activation properties, application of automated electrophysiology to measure the channel activity has been difficult. Using HEK293 cells stably expressing human TRP channels, hTRPC6 and hTRPA1, we developed and validated a high-throughput Rb(+) efflux assay for NSCC channels. The assay was performed in cell-based 96-well format. A significant increase in Rb(+) efflux can be detected upon channel activation by specific agonists, confirming that both TRPC6 and TRPA1 channels are permeable to Rb(+) ions. The agonists induced Rb(+) efflux can be blocked by known channel blockers and selected compounds from our high-throughput screening (HTS) hits. The assay is suitable for HTS with Z' factors of 0.53 and above. We also tested the Ca(2+) effect on channel activities in this assay. Both TRPC6 and TRPA1 channels were found to be inhibited by increasing the concentration of Ca(2+) in the assay buffer. However, Ca(2+) significantly reduced the potency of allyl isothiocyanate (AITC) on TRPA1 but did not affect the potency of carbochol on TRPC6. Using this assay for secondary confirmation screen, we successfully identified and confirmed the positive hits as TRPC6 inhibitors.

Role of α5 Nicotinic Acetylcholine Receptors in Pharmacological and Behavioral Effects of Nicotine in Mice

Incorporation of the alpha5 nicotinic acetylcholine receptor (nAChR) subunit can greatly influence nAChR function without altering receptor number. Although few animal studies have assessed the role of the alpha5 nAChR in nicotine-mediated behaviors, recent evidence suggests an association between polymorphisms in the alpha5 nAChR gene and nicotine dependence phenotypes in humans. Thus, additional studies are imperative to elucidate the role and function of the alpha5 nAChR subunit in nicotine dependence. Using alpha5(-/-) mice, the current study aimed to examine the role of alpha5 nAChRs in the initial pharmacological effects of nicotine, nicotine reward using the conditioned place preference model, and the discriminative effects of nicotine using a two-lever drug discrimination model. (86)Rb(+) efflux and (125)I-epibatidine binding assays were conducted to examine the effect of alpha5 nAChR subunit deletion on expression and activity of functional nAChRs. Results show that alpha5(-/-) mice are less sensitive to the initial effects of nicotine in antinociception, locomotor activity, and hypothermia measures and that the alpha5 nAChR is involved in nicotine reward. Alternatively, alpha5(-/-) mice did not differ from wild-type littermates in sensitivity to the discriminative stimulus effects of nicotine. Furthermore, deletion of the alpha5 nAChR subunit resulted in a statistically significant decrease in function in the thalamus and hindbrain, but the decreases noted in spinal cord were not statistically significant. Receptor number was unaltered in all areas tested. Taken together, results of the study suggest that alpha5 nAChRs are involved in nicotine-mediated behaviors relevant to development of nicotine dependence.

Two haplotypes of the P2X7receptor containing the Ala‐348 to Thr polymorphism exhibit a gain‐of‐function effect and enhanced interleukin‐1β secretion

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

Effects of fusicoccin on ion fluxes in guard cells

The pharmacology has been further investigated of the two transport systems mediating potassium (rubidium) (K(+)(Rb(+))) release from the guard cell vacuole, responsible, respectively, for the resting efflux and abscisic acid (ABA)-induced transient stimulation of efflux, and for the transient stimulation induced by hypotonic treatment. Here, the effects of fusicoccin and of butyrate-induced cytoplasmic acidification on (86)Rb efflux were measured in isolated guard cells of Commelina communis. Fusicoccin (10 microM) inhibited the resting efflux at the tonoplast and the ABA-induced transient, but had no effect on the hypotonic transient. All three processes were inhibited by cytoplasmic acidification. Fusicoccin did not inhibit efflux at the plasmalemma. As the hypotonic response is inhibited by cytoplasmic acidification but not by fusicoccin, the effect of fusicoccin on the resting efflux and ABA response must be direct, and not the result of fusicoccin-induced cytoplasmic acidification. The collected tonoplast efflux properties resemble those of TPC1 (two-pore channel) rather than TPK1 (two-pore K channel). The flux and TPC1 are both activated by Ca(2+), but inhibited by phenylarsine oxide and by cytoplasmic acidification. The flux is inhibited by fusicoccin. TPC1 is inhibited by 14-3-3 proteins and has the C-terminal sequence STSDT, a type III binding site for 14-3-3 proteins, of the kind involved in fusicoccin binding.

Functional Characterization of Mutations in Kir4.1 (KCNJ10) Associated with the SeSAME Syndrome

Kir4.1 channels are expressed in many brain cells, particularly astrocytes, and may be responsible for the K+ buffering action of the glia (J Biol Chem 270: 16339-46, 1995). In addition, Kir4.1 channels are found in the basolateral membrane of distal convoluted tubule cells, where they contribute to renal electrolyte homeostasis. Mutations in KCNJ10, the gene encoding Kir4.1, have been associated to the newly described SeSAME syndrome (Proc Natl Acad Sci USA 106: 5842-47, 2009), a unique set of symptoms that include sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. To determine the functional significance of these mutations, we performed radiotracer efflux assays and inside-out membrane patch clamping in COSm6 cells expressing wild-type (WT) or mutant (R65P, C140R, T164I, A167V, R199Stop, and R297C) channels. All mutations lead to varying degrees of loss of Kir4.1 channel function. In untransfected cells, the 86Rb efflux rate constant was 0.008 min−1 ± 0.001 (n=3), and in cells transfected with WT, the rate of Kir4.1-mediated 86Rb efflux (proportional to K+ conductance) was 0.018 min−1 ± 0.001 (n=3). The mutant Kir4.1-mediated rate constants were 60% (A167V), 21% (R297C), 20% (R65P), 15% (C140R), 12% (T164I), and 1% (R199Stop), relative to WT. No measurable currents were recorded from cells expressing C140R, T164I, R199Stop or R297C. Some of these mutations (R297C, R199Stop) are away from the channel pore, and ongoing studies are examining the potential for altered trafficking. In R65P and A167V, on-cell inward rectification, as well as sensitivity to block by spermine and barium were normal. However, while the current amplitude was similar to WT immediately upon patch excision, it decreased 50-80% within the first 2 min, suggesting that these mutations, located in the potential PIP2 binding site or at the PIP2-dependent gate, reduce open state stability.

Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at alpha4beta2 * nicotinic acetylcholine receptors, has moderate affinity (K(i)=5.46microM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2-20 of lobeline were synthesized and evaluated for interaction with alpha4beta2 * and alpha7 * neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at alpha7 * nAChRs. Similar to lobeline (K(i)=4nM), sulfonic acid esters had high affinity at alpha4beta2 * (K(i)=5-17nM). Aromatic carboxylic acid ester analogs of lobeline (2-4) were 100-1000-fold less potent than lobeline at alpha4beta2 * nAChRs, whereas aliphatic carboxylic acid ester analogs were 10-100-fold less potent than lobeline at alpha4beta2 *. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the (36)Rb(+) efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC(50) values of 0.85microM and 1.60microM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13-45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K(i)=1.98-10.8microM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at alpha4beta2 * nAChRs (K(i)=19.3microM) and was equipotent with lobeline, at VMAT2 (K(i)=2.98microM), exhibiting a 6.5-fold selectivity for VMAT2 over alpha4beta2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.