Functional activation by central monoamines of human dopamine D4 receptor polymorphic variants coupled to GIRK channels in Xenopus oocytes

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Abstract

We studied the functional activation of different polymorphic variants of the human dopamine D4 receptors by the three major central monoamines, dopamine, noradrenaline and serotonin. Dopamine D4 receptors carrying two (D4.2), four (D4.4) or seven (D4.7) repeats within the third intracellular domain were co-expressed with G protein-regulated inwardly rectifying potassium channels (GIRK1) in frog oocytes. All the dopamine D4 receptor variants coupled to oocyte Gi/o proteins and modulated co-expressed GIRK1 channels. Monoamine-induced responses were detected as increases in voltage-clamp recorded GIRK1 currents. Dopamine, noradrenaline as well as serotonin stimulated dopamine D4 receptors. Dose-response analysis showed that dopamine and noradrenaline are full agonists whereas serotonin acted as partial agonist. Dopamine was 5-fold more potent on D4.2 and D4.7 (EC50 = 1 nM) than on D4.4 (EC50 = 5 nM) suggesting that the actions of dopamine and therapeutic drugs on dopamine D4 receptors might vary among individuals depending on their repertoire of expressed alleles. In contrast, noradrenaline and serotonin did not discriminate among dopamine D4 receptor variants (EC50 NA = 50 nM, EC50 5-HT = 1.5 μM). All monoamine effects were blocked by the specific dopaminergic D4 antagonist (S)-(−)-4-[4-[2-(Isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide (PNU101387). Sequence analyses of dopamine D4 receptors and related monoamine receptors revealed that dopamine D4 receptors have most aminoacidic residues necessary for binding of dopamine, noradrenaline and serotonin. Our data indicate that dopamine D4 receptors can be pharmacologically stimulated by any the three major central monoamines.

Introduction

The dopamine D4 receptor is a member of the G protein-coupled receptors superfamily (Neve, 2005, Van Tol et al., 1991) that mediates changes in neuronal excitability and synaptic plasticity in the brain (Rubinstein et al., 2001, Wang et al., 2003, Wang et al., 2002), and is predominantly expressed in the brain prefrontal cortex (Ariano et al., 1997, Mrzljak et al., 1996), where it is thought to play a major role in the control of integrative functions underlying the organization of complex behaviors (Fuster, 2001, Goldman-Rakic, 1995b, Rubinstein et al., 1997). Dopamine D4 receptor activation may trigger multiple intracellular pathways including inhibition of cAMP synthesis (Seamans and Yang, 2004) and modulation of G protein-regulated ion channels (Lavine et al., 2002, Pillai et al., 1998, Werner et al., 1996).

An outstanding feature of the human dopamine D4 receptor is its highly polymorphic nature, particularly in the third cytoplasmic domain where a variable number of 48 bp repeats and single nucleotide polymorphisms account for more than 35 different alleles already detected in the population (Grady et al., 2003). Variants carrying four tandem repeats (D4.4) account for approximately 64% of all human alleles, whereas the other two most abundant variants are D4.7 (20%) and D4.2 (8%) (Chang et al., 1996). Many studies have attempted to correlate polymorphic human dopamine D4 receptor variants with psychiatric diseases and personality traits and some of them found that D4.7 variants are associated with attention deficit and hyperactivity disorder (ADHD) and with the personality trait novelty seeking (Ebstein et al., 1996, Faraone et al., 2001, Grady et al., 2003, Paterson et al., 1999). Because the third intracellular domain of G protein-coupled receptors interact with its cognate G proteins and other intracellular signaling molecules (Neve, 2005), numerous studies have also analyzed the functional and pharmacological properties of several human polymorphic dopamine D4 receptor variants. For example, the ability of the various human dopamine D4 receptor variants to bind dopamine, and to induce dopamine-mediated inhibition of adenylyl-cyclase activity or stimulation of GTPγS binding, was evaluated in transfected cell lines (Asghari et al., 1995, Czermak et al., 2006, Jovanovic et al., 1999). Dopamine D4 receptor-mediated modulation of inwardly rectifying potassium channels (GIRK) was also demonstrated in heterologous expression systems (Werner et al., 1996). However, the functional coupling between different dopamine D4 receptor polymorphic variants and GIRK has not been studied yet.

Another level of complexity in studies of dopamine D4 receptor pharmacology and function derives from the interactions that exist between different monoamines and their receptors. Particularly, it was demonstrated that noradrenaline and adrenaline bind dopamine D4 receptors with high affinity and induce dopamine D4 receptor-mediated inhibition of adenylyl-cyclase (Czermak et al., 2006, Lanau et al., 1997, Newman-Tancredi et al., 1997) and that different human recombinant serotonin receptors can be activated by dopamine (Oz et al., 2003, Woodward et al., 1992).

Diverse crossed effects were also reported for dopaminergic, adrenergic or serotonergic agonists and antagonists, even for drugs showing high degrees of selectivity. In addition, it is well known that atypical antipsychotics, like clozapine, show high affinities for dopamine D2 receptors, dopamine D4 receptors and serotonin 5-HT2 receptors (Stockmeier et al., 1993, Van Tol et al., 1991). This level of ligand-receptor promiscuity may have important pharmacological consequences, for example during treatments with psychotherapeutic drugs.

The effects of serotonin on dopamine D4 receptors has not been investigated before, neither the actions of noradrenaline to modulate GIRK currents through dopamine D4 receptor activation. In the present study, we used electrophysiological recording in Xenopus laevis oocytes, to test the ability of the three major central monoaminergic neurotransmitters to stimulate the most abundant human dopamine D4 receptor variants and modulate GIRK currents through oocyte Gi/o proteins.

Section snippets

RNA preparation, oocyte isolation and injection

Plasmids encoding the different human dopamine D4 receptor variants: D4.2 (αξ), D4.4 (αβθξ) and D4.7 (αβηεβεξ) (greek characters are used in order to define the kind of repeat involved in protein structure) (Jovanovic et al., 1999) and the rat GIRK1, were provided by colleagues (see acknowledgments). Full-length cDNAs, cloned in pcDNA3 (Invitrogen), were used to in vitro transcribe cRNA with the mMessage mMachine transcription kit (Ambion, Austin, TX, USA). cRNA solutions (0.2–0.4 μg/μl) were

Stimulation of human dopamine D4 receptors by dopamine, noradrenaline and serotonin increases GIRK currents

Ionic currents were first recorded in oocytes expressing only GIRK1 cRNA (see Materials and Methods). GIRK1 channels undergo substantial activation at − 100 mV (Werner et al., 1996). Oocytes exposed to a high K+ solution displayed currents that developed relatively fast (Fig. 1A; first trace), were sensitive to Ba2+ and showed a distinct voltage dependence, all characteristic features of GIRK-mediated inward K+ currents. As shown in the same record, applications of dopamine did not induce

Discussion

In this study we carried out a functional analysis of the effects of the three major central monoamines on human dopamine D4 receptor polymorphic variants expressed in Xenopus laevis oocytes. The principal findings of this study are: 1) the three most abundant human dopamine D4 receptor polymorphic variants coupled to and modulated co-expressed GIRK1 channels. 2) Serotonin, in addition to dopamine and noradrenaline, was able to positively modulate GIRK channels gating through dopamine D4.2,

Acknowledgements

We thank Dr. Hubert H. Van Tol for dopamine D4 receptors cDNAs and Dr. Eduardo Perozo for GIRK cDNA. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (D.J.C., M.R.); Universidad de Buenos Aires (D.J.C., M.R.); CONICET (DJC) and Howard Hughes Medical Institute (M.R.). We also thank Pew Foundation and IBRO for support. C.W. is a Doctoral Fellow of the YPF-Repsol Foundation.

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