Publications by authors named "Kristoffer Sahlholm"

36 Publications

Dopamine D Receptor Agonist Binding Kinetics-Role of a Conserved Serine Residue.

Int J Mol Sci 2021 Apr 15;22(8). Epub 2021 Apr 15.

Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden.

The forward (k) and reverse (k) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the ks and ks of four dopamine D receptor (DR) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S193 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar ks for the two 5-OH-DPAT enantiomers at wild-type (WT) DR, both being slower than the ks of DA and p-tyramine. Conversely, the k of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S193A mutation lowered the k of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Ks derived from the k and k estimates correlated well with EC values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing DR agonist candidate drugs.
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http://dx.doi.org/10.3390/ijms22084078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071183PMC
April 2021

Interaction of Ligands for PET with the Dopamine D3 Receptor: In Silico and In Vitro Methods.

Biomolecules 2021 Apr 2;11(4). Epub 2021 Apr 2.

Division of Nuclear Medicine and Clinical Molecular Imaging, Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

[F]Fallypride and [F]Fluortriopride (FTP) are two different PET radiotracers that bind with sub-nanomolar affinity to the dopamine D3 receptor (DR). In spite of their similar D affinities, the two PET ligands display very different properties for labeling the DR in vivo: [F]Fallypride is capable of binding to DR under "baseline" conditions, whereas [F]FTP requires the depletion of synaptic dopamine in order to image the receptor in vivo. These data suggest that [F]Fallypride is able to compete with synaptic dopamine for binding to the DR, whereas [F]FTP is not. The goal of this study was to conduct a series of docking and molecular dynamic simulation studies to identify differences in the ability of each molecule to interact with the DR that could explain these differences with respect to competition with synaptic dopamine. Competition studies measuring the ability of each ligand to compete with dopamine in the β-arrestin assay were also conducted. The results of the in silico studies indicate that FTP has a weaker interaction with the orthosteric binding site of the DR versus that of Fallypride. The results of the in silico studies were also consistent with the IC50 values of each compound in the dopamine β-arrestin competition assays. The results of this study indicate that in silico methods may be able to predict the ability of a small molecule to compete with synaptic dopamine for binding to the DR.
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http://dx.doi.org/10.3390/biom11040529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065765PMC
April 2021

V374A KCND3 Pathogenic Variant Associated With Paroxysmal Ataxia Exacerbations.

Neurol Genet 2021 Feb 6;7(1):e546. Epub 2021 Jan 6.

Department of Clinical Neuroscience (M.P., R.Å., T.L., Å.B., J.N., P.S.), Department of Molecular Medicine and Surgery (D.N.), Center for Molecular Medicine (D.N.), and Science for Life Laboratory (D.N.), Karolinska Institutet (S.L., I.S.), Stockholm; Department of Neurology (M.P., J.W., P.S.), Department of Clinical Genetics (D.N.), Department of Nuclear Medicine (I.S.), and Department of Neurophysiology (J.N.), Karolinska University Hospital (R.Å.), Stockholm; Department of Integrative Medical Biology (K.S.), Umeå University; and Department of Medical Sciences (J.N.), Örebro University, Sweden.

Objective: Ataxia channelopathies share common features such as slow motor progression and variable degrees of cognitive dysfunction. Mutations in potassium voltage-gated channel subfamily D member 3 (), encoding the K+ channel, Kv4.3, are associated with spinocerebellar ataxia (SCA) 19, allelic with SCA22. Mutations in potassium voltage-gated channel subfamily C member 3 (), encoding another K+ channel, Kv3.3, cause SCA13. First, a comprehensive phenotype assessment was carried out in a family with autosomal dominant ataxia harboring 2 genetic variants in and . To evaluate the physiological impact of these variants on channel currents, in vitro studies were performed.

Methods: Clinical and psychometric evaluations, neuroimaging, and genotyping of a family (mother and son) affected by ataxia were carried out. Heterozygous and homozygous Kv3.3 A671V and Kv4.3 V374A variants were evaluated in oocytes using 2-electrode voltage-clamp. The influence of Kv4 conductance on neuronal activity was investigated computationally using a Purkinje neuron model.

Results: The main clinical findings were consistent with adult-onset ataxia with cognitive dysfunction and acetazolamide-responsive paroxysmal motor exacerbations in the index case. Despite cognitive deficits, fluorodeoxyglucose (FDG)-PET displayed hypometabolism mainly in the severely atrophic cerebellum. Genetic analyses revealed the new variant c.1121T>C (V374A) in and c.2012T>C (A671V) in . In vitro electrophysiology experiments on oocytes demonstrated that the V374A mutant was nonfunctional when expressed on its own. Upon equal co-expression of wild-type (WT) and V374A channel subunits, Kv4.3 currents were significantly reduced in a dominant negative manner, without alterations of the gating properties of the channel. By contrast, Kv3.3 A671V, when expressed alone, exhibited moderately reduced currents compared with WT, with no effects on channel activation or inactivation. Immunohistochemistry demonstrated adequate cell membrane translocation of the Kv4.3 V374A variant, thus suggesting an impairment of channel function, rather than of expression. Computational modeling predicted an increased Purkinje neuron firing frequency upon reduced Kv4.3 conductance.

Conclusions: Our findings suggest that Kv4.3 V374A is likely pathogenic and associated with paroxysmal ataxia exacerbations, a new trait for SCA19/22. The present FDG PET findings contrast with a previous study demonstrating widespread brain hypometabolism in SCA19/22.
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http://dx.doi.org/10.1212/NXG.0000000000000546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862093PMC
February 2021

Inhibition of Tryptophan Hydroxylases and Monoamine Oxidase-A by the Proton Pump Inhibitor, Omeprazole- and Investigations.

Front Pharmacol 2020 26;11:593416. Epub 2020 Nov 26.

Department of Biomedicine, University of Bergen, Bergen, Norway.

Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A , we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.
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http://dx.doi.org/10.3389/fphar.2020.593416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726444PMC
November 2020

Voltage-Dependent Dopamine Potency at D-Like Dopamine Receptors.

Front Pharmacol 2020 7;11:581151. Epub 2020 Oct 7.

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

In recent years, transmembrane voltage has been found to modify agonist potencies at several G protein-coupled receptors (GPCRs). Whereas the voltage sensitivities of the Gα-coupled dopamine D-like receptors (DR, DR, DR) have previously been investigated, the putative impact of transmembrane voltage on agonist potency at the mainly Gα-coupled dopamine D-like receptors (DR, DR) has hitherto not been reported. Here, we assayed the potency of dopamine in activating G protein-coupled inward rectifier potassium (GIRK) channels co-expressed with DR and DR in oocytes, at -80 mV and at 0 mV. Furthermore, GIRK response deactivation rates upon dopamine washout were measured to estimate dopamine dissociation rate (k) constants. Depolarization from -80 to 0 mV was found to reduce dopamine potency by about 7-fold at both DR and DR. This potency reduction was accompanied by an increase in estimated dopamine ks at both receptors. While the GIRK response elicited via DR was insensitive to pertussis toxin (PTX), the response evoked via DR was reduced by 64% (-80 mV) and 71% (0 mV) in the presence of PTX. Injection of oocytes with Gα antisense oligonucleotide inhibited the DR-mediated response by 62% (-80 mV) and 76% (0 mV) and abolished the DR response when combined with PTX. Our results suggest that depolarization decreases dopamine affinity at DR and DR. The voltage-dependent affinities of dopamine at DR and DR may be relevant to the functions of these receptors in learning and memory.
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http://dx.doi.org/10.3389/fphar.2020.581151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577048PMC
October 2020

Decreased striatal adenosine A-dopamine D receptor heteromerization in schizophrenia.

Neuropsychopharmacology 2021 02 3;46(3):665-672. Epub 2020 Oct 3.

Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, UB, L'Hospitalet de Llobregat, Barcelona, Spain.

According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A receptors (ARs) or their degree of functional heteromerization with dopamine D receptors (DR). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native AR-DR heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal DR without changes in AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal AR-DR heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of AR-DR heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both DR and AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal AR-DR heteromers. The degree of AR-DR heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.
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http://dx.doi.org/10.1038/s41386-020-00872-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027896PMC
February 2021

Ligand with Two Modes of Interaction with the Dopamine D Receptor-An Induced-Fit Mechanism of Insurmountable Antagonism.

ACS Chem Neurosci 2020 10 15;11(19):3130-3143. Epub 2020 Sep 15.

Department of Neuroscience, Karolinska Institutet, Stockholm 171 77, Sweden.

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D receptor (DR). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in oocytes to measure the kinetics of DR antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of DR decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by modeling, whereby SV-III-130 initially binds reversibly to the DR, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.
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http://dx.doi.org/10.1021/acschemneuro.0c00477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553383PMC
October 2020

Discovery and biological characterization of a novel scaffold for potent inhibitors of peripheral serotonin synthesis.

Future Med Chem 2020 08 5;12(16):1461-1474. Epub 2020 Aug 5.

Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Postboks 7804, 5020 Bergen, Norway.

Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Screening using a thermal shift assay for TPH1 binders yielded Compound (2-(4-methylphenyl)-1,2-benzisothiazol-3(2)-one), which showed high potency (50% inhibition at 98 ± 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure-activity relationships studies revealed several analogs of showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of , with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.
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http://dx.doi.org/10.4155/fmc-2020-0127DOI Listing
August 2020

A Neanderthal Sodium Channel Increases Pain Sensitivity in Present-Day Humans.

Curr Biol 2020 Sep 23;30(17):3465-3469.e4. Epub 2020 Jul 23.

Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany; Okinawa Institute of Science and Technology, Onna-son, Okinawa 904-0495, Japan. Electronic address:

The sodium channel Nav1.7 is crucial for impulse generation and conduction in peripheral pain pathways [1]. In Neanderthals, the Nav1.7 protein carried three amino acid substitutions (M932L, V991L, and D1908G) relative to modern humans. We expressed Nav1.7 proteins carrying all combinations of these substitutions and studied their electrophysiological effects. Whereas the single amino acid substitutions do not affect the function of the ion channel, the full Neanderthal variant carrying all three substitutions, as well as the combination of V991L with D1908G, shows reduced inactivation, suggesting that peripheral nerves were more sensitive to painful stimuli in Neanderthals than in modern humans. We show that, due to gene flow from Neanderthals, the three Neanderthal substitutions are found in ∼0.4% of present-day Britons, where they are associated with heightened pain sensitivity.
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http://dx.doi.org/10.1016/j.cub.2020.06.045DOI Listing
September 2020

Leveraging a Low-Affinity Diazaspiro Orthosteric Fragment to Reduce Dopamine D Receptor (DR) Ligand Promiscuity across Highly Conserved Aminergic G-Protein-Coupled Receptors (GPCRs).

J Med Chem 2019 05 6;62(10):5132-5147. Epub 2019 May 6.

Department of Radiology , Perelman School of Medicine, University of Pennsylvania , Philadelphia , Pennsylvania 19104 , United States.

Previously, we reported a 3-(2-methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4 H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (1) compound with excellent dopamine D receptor (DR) affinity (DR K = 12.0 nM) and selectivity (DR/DR ratio = 905). Herein, we present derivatives of 1 with comparable DR affinity (32, DR K = 3.2 nM, DR/DR ratio = 60) and selectivity (30, DR K = 21.0 nM, DR/DR ratio = 934). Fragmentation of 1 revealed orthosteric fragment 5a to express an unusually low DR affinity ( K = 2.7 μM). Compared to piperazine congener 31, which retains a high-affinity orthosteric fragment (5d, DR K = 23.9 nM), 1 was found to be more selective for the DR among D- and D-like receptors and exhibited negligible off-target interactions at serotoninergic and adrenergic G-protein-coupled receptors (GPCRs), common off-target sites for piperazine-containing DR scaffolds. This study provides a unique rationale for implementing weakly potent orthosteric fragments into DR ligand systems to minimize drug promiscuity at other aminergic GPCR sites.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00412DOI Listing
May 2019

Dopamine receptor heteromers: biasing antipsychotics.

Future Med Chem 2018 12 6;10(23):2675-2677. Epub 2018 Dec 6.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

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http://dx.doi.org/10.4155/fmc-2018-0335DOI Listing
December 2018

Antipsychotics with similar association kinetics at dopamine D receptors differ in extrapyramidal side-effects.

Nat Commun 2018 09 3;9(1):3577. Epub 2018 Sep 3.

Department of Neuroscience, Karolinska Institutet, Stockholm, SE-17177, Sweden.

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http://dx.doi.org/10.1038/s41467-018-04489-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120954PMC
September 2018

The Beta-Arrestin-Biased Dopamine D2 Receptor Ligand, UNC9994, Is a Partial Agonist at G-Protein-Mediated Potassium Channel Activation.

Int J Neuropsychopharmacol 2018 12;21(12):1102-1108

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Background: Previous evidence suggests that UNC9994 is a beta-arrestin2-selective agonist at the dopamine D2 receptor, lacking ability both to activate and antagonize G protein-dependent signaling. However, this has only been reported by one laboratory using a single assay.

Methods: We used G protein-coupled inward rectifier potassium channel activation in Xenopus oocytes to investigate UNC9994-induced modulation of G protein-dependent signaling at dopamine D2 receptor and dopamine D3 receptor.

Results: At dopamine D2 receptor, UNC9994 induced G protein-coupled inward rectifier potassium channel currents that were 15% of the maximal response to dopamine, with an EC50 of 185 nM. At dopamine D3 receptor, the ligand elicited 89% of the maximal dopamine response with an EC50 of 62 nM. Pertussis toxin abolished G protein-coupled inward rectifier potassium channel activation. Furthermore, UNC9994 antagonized dopamine-induced G protein-coupled inward rectifier potassium channel activation at dopamine D2 receptor.

Conclusions: UNC9994 modulates G protein-coupled inward rectifier potassium channel channel activation via pertussis toxin-sensitive G proteins at dopamine D2 receptor and dopamine D3 receptor. These findings may have implications for the interpretation of data obtained with this ligand.
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http://dx.doi.org/10.1093/ijnp/pyy059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276031PMC
December 2018

Effects of the Dopamine Stabilizer, Pridopidine, on Basal and Phencyclidine-Induced Locomotion: Role of Dopamine D2 and Sigma-1 Receptors.

CNS Neurol Disord Drug Targets 2018 ;17(7):522-527

Unitat de Farmacologia, Departament Patologia i Terapeutica Experimental, Facultat de Medicina i Ciencies de la Salut, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Background: Pridopidine, a compound in clinical trials for Huntington's disease treatment, was originally synthesized as a dopamine D2 receptor (D2R) ligand, but later found to possess higher affinity for the sigma-1 receptor (S1R). However, the putative contributions of D2R and S1R to the behavioral profile of acutely administered pridopidine have not been investigated.

Objective: The present study sought to compare the effects of acute pridopidine on wild-type vs. D2R and S1R knockout mice, at high (60 mg/kg) and low (6 mg/kg) doses.

Method: Pridopidine effects on basal and phencyclidine-induced locomotor activity was measured in the open field test. Additionally, the actions of pridopidine on prepulse inhibition was measured in animals treated with saline or phencyclidine.

Results: Whereas inhibition of spontaneous and phencyclidine-induced locomotion was readily observed at 60 mg/kg pridopidine, neither locomotor stimulation in habituated mice, nor any effects on prepulse inhibition were detected upon pridopidine treatment. Surprisingly, inhibition of spontaneous locomotion was unaffected by both D2R and S1R deletion.

Conclusion: The present results suggest the involvement of additional targets, besides D2R and S1R, in mediating locomotor inhibition by pridopidine.
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http://dx.doi.org/10.2174/1871527317666180627103337DOI Listing
October 2019

Pridopidine Reverses Phencyclidine-Induced Memory Impairment.

Front Pharmacol 2018 10;9:338. Epub 2018 Apr 10.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Pridopidine is in clinical trials for Huntington's disease treatment. Originally developed as a dopamine D receptor (DR) ligand, pridopidine displays about 100-fold higher affinity for the sigma-1 receptor (sigma-1R). Interestingly, pridopidine slows disease progression and improves motor function in Huntington's disease model mice and, in preliminarily reports, Huntington's disease patients. The present study examined the anti-amnesic potential of pridopidine. Thus, memory impairment was produced in mice by administration of phencyclidine (PCP, 10 mg/kg/day) for 10 days, followed by 14 days' treatment with pridopidine (6 mg/kg/day), or saline. Finally, novel object recognition performance was assessed in the animals. Mice receiving PCP and saline exhibited deficits in novel object recognition, as expected, while pridopidine treatment counteracted PCP-induced memory impairment. The effect of pridopidine was attenuated by co-administration of the sigma receptor antagonist, NE-100 (10 mg/kg). Our results suggest that pridopidine exerts anti-amnesic and potentially neuroprotective actions. These data provide new insights into the therapeutic potential of pridopidine as a pro-cognitive drug.
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http://dx.doi.org/10.3389/fphar.2018.00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902730PMC
April 2018

Point mutation of a conserved aspartate, D69, in the muscarinic M receptor does not modify voltage-sensitive agonist potency.

Biochem Biophys Res Commun 2018 01 2;496(1):101-104. Epub 2018 Jan 2.

Department of Neuroscience, Retzius väg 8, Karolinska Institutet, SE-171 77, Stockholm, Sweden. Electronic address:

The muscarinic M receptor (MR) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of MR, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant MR. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type MR, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the MR.
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http://dx.doi.org/10.1016/j.bbrc.2018.01.005DOI Listing
January 2018

Highly Selective Dopamine D Receptor Antagonists with Arylated Diazaspiro Alkane Cores.

J Med Chem 2017 12 21;60(23):9905-9910. Epub 2017 Nov 21.

Department of Radiology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.

A series of potent and selective D receptor (DR) analogues with diazaspiro alkane cores were synthesized. Radioligand binding of compounds 11, 14, 15a, and 15c revealed favorable DR affinity (K = 12-25.6 nM) and were highly selective for DR vs DR (ranging from 264- to 905-fold). Variation of these novel ligand architectures can be achieved using our previously reported 10-20 min benchtop C-N cross-coupling methodology, affording a broad range of arylated diazaspiro precursors.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767125PMC
December 2017

Antipsychotic-Like Efficacy of Dopamine D Receptor-Biased Ligands is Dependent on Adenosine A Receptor Expression.

Mol Neurobiol 2018 Jun 5;55(6):4952-4958. Epub 2017 Aug 5.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, 08907, Barcelona, Spain.

Dopamine D receptor (DR) activation triggers both G protein- and β-arrestin-dependent signaling. Biased DR ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of DR to heteromerize with adenosine A receptor (AR) has been associated to DR agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the AR dependence of DR/β-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between β-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of DR/AR homomers and heteromers, we demonstrated that the DR agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when AR/DR heteromers were expressed. Subsequently, the role of AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and AR mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in AR mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that DR/AR heteromerization facilitates DR β-arrestin recruitment, and furthermore, reveal a pivotal role for AR in the antipsychotic-like activity of the β-arrestin-biased DR ligand, UNC9994.
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http://dx.doi.org/10.1007/s12035-017-0696-yDOI Listing
June 2018

The role of beta-arrestin2 in shaping fMRI BOLD responses to dopaminergic stimulation.

Psychopharmacology (Berl) 2017 Jul 5;234(13):2019-2030. Epub 2017 Apr 5.

Institute for Biomedical Engineering, University and ETH Zurich, Wolfgang-Pauli-Str. 27, 8093, Zurich, Switzerland.

Rationale: The dopamine D receptor (DR) couples to inhibitory G proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied DR to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo.

Objectives: The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies DR-dependent responses.

Methods: fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the DR antagonist, eticlopride.

Results: Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes.

Conclusions: The prolonged striatal response decay rates in KO animals might reflect impaired DR desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of DR.
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http://dx.doi.org/10.1007/s00213-017-4609-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486931PMC
July 2017

The fast-off hypothesis revisited: A functional kinetic study of antipsychotic antagonism of the dopamine D2 receptor.

Eur Neuropsychopharmacol 2016 Mar 14;26(3):467-76. Epub 2016 Jan 14.

Department of Neuroscience, Karolinska Institutet, Retzius väg 8, SE-171 77 Stockholm, Sweden.

Newer, "atypical" antipsychotics carry a lower risk of motor side-effects than older, "typical" compounds. It has been proposed that a ~100-fold faster dissociation from the dopamine D2 receptor (D2R) distinguishes atypical from typical antipsychotics. Furthermore, differing antipsychotic D2R affinities have been suggested to reflect differences in dissociation rate constants (koff), while association rate constants (kon) were assumed to be similar. However, it was recently demonstrated that lipophilic accumulation of ligand in the cell interior and/or membrane can cause underestimation of koff, and as high-affinity D2R antagonists are frequently lipophilic, this may have been a confounding factor in previous studies. In the present work, a functional electrophysiology assay was used to measure the recovery of dopamine-mediated D2R responsivity from antipsychotic antagonism, using elevated concentrations of dopamine to prevent the potential bias of re-binding of lipophilic ligands. The variability of antipsychotic kon was also reexamined, capitalizing on the temporal resolution of the assay. kon was estimated from the experimental recordings using a simple mathematical model assumed to describe the binding process. The time course of recovery from haloperidol (typical antipsychotic) was only 6.4- to 2.5-fold slower than that of the atypical antipsychotics, amisulpride, clozapine, and quetiapine, while antipsychotic kons were found to vary more widely than previously suggested. Finally, affinities calculated using our kon and koff estimates correlated well with functional potency and with affinities reported from radioligand binding studies. In light of these findings, it appears unlikely that typical and atypical antipsychotics are primarily distinguished by their D2R binding kinetics.
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http://dx.doi.org/10.1016/j.euroneuro.2016.01.001DOI Listing
March 2016

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells.

J Am Chem Soc 2015 Dec 17;137(50):15892-8. Epub 2015 Dec 17.

Departmento de Química, Universidad de Burgos , 09001 Burgos, Spain.

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.
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http://dx.doi.org/10.1021/jacs.5b09970DOI Listing
December 2015

G protein-gated inwardly rectifying potassium channel subunits 1 and 2 are down-regulated in rat dorsal root ganglion neurons and spinal cord after peripheral axotomy.

Mol Pain 2015 Jul 22;11:44. Epub 2015 Jul 22.

School of Life Science and Technology, Harbin Institute of Technology, 150001, Harbin, China.

Background: Increased nociceptive neuronal excitability underlies chronic pain conditions. Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. The members of the family of G protein-gated inwardly rectifying potassium (GIRK) channels, GIRK1-4, have been implicated in modulating excitability. Here, we investigated the expression and distribution of GIRK1 and GIRK2 in normal and injured dorsal root ganglia (DRGs) and spinal cord of rats.

Results: We found that ~70% of the DRG neurons expressed GIRK1, while only <10% expressed GIRK2. The neurochemical profiles of GIRK1- and GIRK2-immunoreactive neurons were characterized using the neuronal markers calcitonin gene-related peptide, isolectin-B4 and neurofilament-200, and the calcium-binding proteins calbindin D28k, calretinin, parvalbumin and secretagogin. Both GIRK subunits were expressed in DRG neurons with nociceptive characteristics. However, while GIRK1 was widely expressed in several sensory neuronal subtypes, GIRK2 was detected mainly in a group of small C-fiber neurons. In the spinal dorsal horn, GIRK1- and -2-positive cell bodies and processes were mainly observed in lamina II, but also in superficial and deeper layers. Abundant GIRK1-, but not GIRK2-like immunoreactivity, was found in the ventral horn (laminae VI-X). Fourteen days after axotomy, GIRK1 and GIRK2 were down-regulated in DRG neurons at the mRNA and protein levels. Both after axotomy and rhizotomy there was a reduction of GIRK1- and -2-positive processes in the dorsal horn, suggesting a presynaptic localization of these potassium channels. Furthermore, nerve ligation caused accumulation of both subunits on both sides of the lesion, providing evidence for anterograde and retrograde fast axonal transport.

Conclusions: Our data support the hypothesis that reduced GIRK function is associated with increased neuronal excitability and causes sensory disturbances in post-injury conditions, including neuropathic pain.
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http://dx.doi.org/10.1186/s12990-015-0044-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511542PMC
July 2015

Pridopidine selectively occupies sigma-1 rather than dopamine D2 receptors at behaviorally active doses.

Psychopharmacology (Berl) 2015 Sep 11;232(18):3443-53. Epub 2015 Jul 11.

Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Rationale: Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding.

Objectives: The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats.

Methods: Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer (11)C-SA4503 (sigma-1R) or (11)C-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with (11)C-raclopride. Cerebral (11)C-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V T) calculated by Logan graphical analysis. (11)C-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model.

Results: Cunningham-Lassen plots indicated sigma-1R occupancies of 57 ± 2 and 85 ± 2% after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44-66%) reduction of (11)C-raclopride binding was only observed at 60 mg/kg pridopidine.

Conclusions: At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D2Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity.
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http://dx.doi.org/10.1007/s00213-015-3997-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537502PMC
September 2015

Sigma-2 receptor binding is decreased in female, but not male, APP/PS1 mice.

Biochem Biophys Res Commun 2015 May 18;460(2):439-45. Epub 2015 Mar 18.

Department of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., St. Louis, MO 63110, USA. Electronic address:

The sigma-2 receptor is a steroid-binding membrane-associated receptor which has been implicated in cell survival. Sigma-2 has recently been shown to bind amyloid-β (Aβ) oligomers in Alzheimer's disease (AD) brain. Furthermore, blocking this interaction was shown to prevent or reverse the effects of Aβ to cause cognitive impairment in mouse models and synaptic loss in neuronal cultures. In the present work, the density of sigma-2 receptors was measured in a double transgenic mouse model of amyloid-β deposition (APP/PS1). Comparisons were made between males and females and between transgenic and wt animals. Sigma-2 receptor density was assessed by quantitative autoradiography performed on coronal brain slices using [(3)H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methyl-benzamide ([(3)H]RHM-1), which has a 300-fold selectivity for the sigma-2 receptor over the sigma-1 receptor. The translocator protein of 18 kDa (TSPO) is expressed on activated microglia and is a marker for neuroinflammation. TSPO has been found to be upregulated in neurodegenerative disorders, including AD. Therefore, in parallel with the sigma-2 autoradiography experiments, we measured TSPO expression using the selective radioligand, [(3)H]PBR28. We also quantified Aβ plaque burden in the same animals using a monoclonal antibody raised against aggregated Aβ. Sigma-2 receptor density was significantly decreased in piriform and motor cortices as well as striata of 16-month old female, but not male, APP/PS1 mice as compared to their wt counterparts. [(3)H]PBR28 binding and immunostaining for Aβ plaques were significantly increased in piriform and motor cortices of both male and female transgenic mice. In striatum however, significant increases were observed only in females.
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http://dx.doi.org/10.1016/j.bbrc.2015.03.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818091PMC
May 2015

Typical and atypical antipsychotics do not differ markedly in their reversibility of antagonism of the dopamine D2 receptor.

Int J Neuropsychopharmacol 2014 Jan 30;17(1):149-55. Epub 2013 Sep 30.

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. clozapine and amisulpride) are related to their ∼100-fold faster dissociation from dopamine D2 receptors (D2R) compared with typical antipsychotics (e.g. haloperidol and chlorpromazine). Fast dissociation would entail rapidly reversible antagonism; however, this has not been thoroughly studied using functional assays. We compared the reversibilities of D2R antagonism by 17 compounds using an electrophysiological method to measure dopamine-evoked potassium channel activation via D2R. Varying rates and amplitudes of D2R response recovery were observed following antagonist removal. Whereas recovery rates differed 15-fold between atypical drugs, recovery from clozapine and amisulpride antagonism was, unexpectedly, less than twofold faster than from chlorpromazine. The recovery amplitude correlated with calculated water solubility and lipid/water distribution coefficients, suggesting variable drug partitioning into cell membranes. Our data do not support the notion that the rate of reversibility of D2R antagonism is what distinguishes atypical from typical antipsychotics.
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http://dx.doi.org/10.1017/S1461145713000801DOI Listing
January 2014

Voltage sensitivities and deactivation kinetics of histamine H₃ and H₄ receptors.

Biochim Biophys Acta 2012 Dec 2;1818(12):3081-9. Epub 2012 Aug 2.

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH(3)(445) and hH(3)(365) isoforms of the human histamine H₃ receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H₄ receptor, which is expressed mainly on hematopoietic cells. We found that the hH(3)(445) receptor is the most sensitive to voltage, whereas the hH(3)(365) and H(4) receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH(3)(445) and hH(3)(365) isoforms, with the hH(3)(365) isoform being five to six-fold slower than the hH(3)(445) receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH₄ receptor. The differences in voltage sensitivities and deactivation kinetics between the hH(3)(445), hH(3)(365), and H₄ receptors might be relevant to their respective physiological roles.
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http://dx.doi.org/10.1016/j.bbamem.2012.07.027DOI Listing
December 2012

The role of RGS protein in agonist-dependent relaxation of GIRK currents in Xenopus oocytes.

Biochem Biophys Res Commun 2011 Nov 29;415(3):509-14. Epub 2011 Oct 29.

Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden.

G protein coupled inward rectifier K(+) channels (GIRK) are activated by the G(βγ) subunits of G proteins upon activation of G protein coupled receptors (GPCRs). Receptor-stimulated GIRK currents are known to possess a curious property, termed "agonist-dependent relaxation," denoting a slow current increase upon stepping the membrane voltage from positive to negative potentials. Regulators of G protein signaling (RGS) proteins have earlier been implicated in this phenomenon since RGS coexpression was required for relaxation to be observed in heterologous expression systems. However, a recent study presented contrasting evidence that GIRK current relaxation reflects voltage sensitive agonist binding to the GPCR. The present study re-examined the role of RGS protein in agonist-dependent relaxation and found that RGS coexpression is not necessary for the relaxation phenomenon. However, RGS4 speeds up relaxation kinetics, allowing the phenomenon to be observed using shorter voltage steps. These findings resolve the controversy regarding the role of RGS protein vs. GPCR voltage sensitivity in mediating agonist-dependent relaxation of GIRK currents.
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http://dx.doi.org/10.1016/j.bbrc.2011.10.106DOI Listing
November 2011

Agonist-specific voltage sensitivity at the dopamine D2S receptor--molecular determinants and relevance to therapeutic ligands.

Neuropharmacology 2011 Oct-Nov;61(5-6):937-49. Epub 2011 Jul 5.

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Voltage sensitivity has been demonstrated for some GPCRs. At the dopamine D(2S) receptor, this voltage sensitivity is agonist-specific; some agonists, including dopamine, exhibit decreased potency at depolarized potentials, whereas others are not significantly affected. In the present study, we examined some of the receptor-agonist interactions contributing to these differences, and investigated how dopamine D(2S) receptor voltage sensitivity affects clinically used dopamine agonists. GIRK channel activation in voltage-clamped Xenopus oocytes was used as readout of receptor activation. Structurally distinct agonists and complementary site-directed mutagenesis of the receptor's binding site were used to investigate the role of agonist-receptor interactions. We also confirmed that the depolarization-induced decrease of dopamine potency in GIRK activation is correlated by decreased binding of radiolabeled dopamine, and by decreased potency in G protein activation. In the mutagenesis experiments, a conserved serine residue as well as the conserved aspartate in the receptor's binding site were found to be important for voltage sensitive potency of dopamine. Furthermore, the voltage sensitivity of the receptor had distinct effects on different therapeutic D(2) agonists. Depolarization decreased the potency of several compounds, whereas for others, efficacy was reduced. For some agonists, both potency and efficacy were diminished, whereas for others still, neither parameter was significantly altered. The present work identifies some of the ligand-receptor interactions which determine agonist-specific effects of voltage at the dopamine D(2S) receptor. The observed differences between therapeutic agonists might be clinically relevant, and make them potential tools for investigating the roles of dopamine D(2) receptor voltage sensitivity in native tissue.
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http://dx.doi.org/10.1016/j.neuropharm.2011.06.022DOI Listing
February 2012

Evidence for oligomerization between GABAB receptors and GIRK channels containing the GIRK1 and GIRK3 subunits.

Eur J Neurosci 2010 Oct 16;32(8):1265-77. Epub 2010 Sep 16.

Unitat de Farmacologia (4102), Departament Patologia i Terapèutica Experimental, Facultat de Medicina-Bellvitge, Universitat de IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

The stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABA(B) ) receptors, activates G protein-gated inwardly-rectifying K(+) (GIRK) channels, which influence membrane excitability. There is now evidence suggesting that G protein-coupled receptors and G protein-gated inwardly-rectifying K(+) [GIRK/family 3 of inwardly-rectifying K(+) (Kir3)] channels do not diffuse freely within the plasma membrane, but instead there are direct protein-protein interactions between them. Here, we used bioluminescence resonance energy transfer, co-immunoprecipitation, confocal and electron microscopy techniques to investigate the oligomerization of GABA(B) receptors with GIRK channels containing the GIRK3 subunit, whose contribution to functional channels is still unresolved. Co-expression of GABA(B) receptors and GIRK channels in human embryonic kidney-293 cells in combination with co-immunoprecipitation experiments established that the metabotropic receptor forms stable complexes with GIRK channels. Using bioluminescence resonance energy transfer, we have shown that, in living cells under physiological conditions, GABA(B) receptors interact directly with GIRK1/GIRK3 heterotetramers. In addition, we have provided evidence that the receptor-effector complexes are also found in vivo and identified that the cerebellar granule cells are one neuron population where the interaction probably takes place. Altogether, our data show that signalling complexes containing GABA(B) receptors and GIRK channels are formed shortly after biosynthesis, probably in the endoplasmic reticulum and/or endoplasmic reticulum/Golgi apparatus complex, suggesting that this might be a general feature of receptor-effector ion channel signal transduction and supporting a channel-forming role for the GIRK3 subunit.
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http://dx.doi.org/10.1111/j.1460-9568.2010.07356.xDOI Listing
October 2010

Cocaine produces D2R-mediated conformational changes in the adenosine A(2A)R-dopamine D2R heteromer.

Biochem Biophys Res Commun 2010 Apr 20;394(4):988-92. Epub 2010 Mar 20.

Department of Neuroscience, Karolinska Institutet, Retzius väg 8, Stockholm 17177, Sweden.

Adenosine A(2A) receptors (A(2A)Rs) and dopamine D(2) receptors (D(2)Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A(2A)Rs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A(2A)R and D(2L)R formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D(2)R homodimers and in A(2A)R-D(2)R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET(max) between A(2A)R-D(2L)R heterodimers and D(2L)R homodimers, but not A(2A)R homodimers, indicating its effect on D(2)R. Cocaine was evaluated with regard to D(2)R binding using a human D(2L)R stable expressing CHO cell line and was found to produce an increase in the affinity of hD(2L)R for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPgammaS. However, cocaine failed to modulate D(2)R agonist-induced inhibition of cAMP in stable hD(2L)R CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D(2L)R, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D(2)R homodimers and A(2A)R-D(2)R heterodimers.
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http://dx.doi.org/10.1016/j.bbrc.2010.03.104DOI Listing
April 2010