Voltage-sensitivity at the human dopamine D 2S receptor is agonist-specific Kristoffer Sahlholm * , Daniel Marcellino, Johanna Nilsson, Kjell Fuxe, Peter Århem Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden article info Article history: Received 18 October 2008 Available online 5 November 2008 Keywords: Dopamine D 2 receptor Dopamine agonists Voltage-sensitivity Voltage-clamp G protein-coupled receptor Xenopus oocytes Phenethylamines Aminotetralins abstract Recently, we and others have shown that agonist potencies at some, but not all, G protein-coupled recep- tors are voltage-sensitive. Several of those studies employed electrophysiology assays in Xenopus oocytes with G protein-coupled potassium channels as a readout. Using this assay, we have now obtained evi- dence that voltage-sensitivity at the dopamine D 2S receptor is agonist-specific. Whereas the potency of dopamine at the D 2S receptor is decreased by depolarization, the potencies of b-phenethylamine, p- and m-tyramine are voltage-insensitive. Furthermore, both monohydroxylated and non-hydroxylated N,N-dipropyl-2-aminotetralin compounds are voltage-sensitive. Differential activation of G protein sub- types or differential ratios between effector and active G protein do not underlie this agonist-selective voltage-sensitivity. This is the first demonstration of voltage-sensitive and voltage-insensitive behaviour of different agonists acting via the same receptor. Ó 2008 Elsevier Inc. All rights reserved. Recently, voltage-sensitivity has been shown for the potencies of agonists to activate some G i - and G q -coupled receptors [1]. In studies utilizing the Xenopus oocyte expression system, significant shifts in the concentration–response relationships for ion channel activation at hyper- and depolarized membrane potentials were observed [2–5]. Ben-Chaim et al. [2 and 6] and Ohana et al. [3] demonstrated that the ligand potency shifts observed in these functional assays correlated with changes in radioligand binding affinity and with charge movement within the receptor. We have previously demonstrated that the potency of dopa- mine is reduced by depolarization when acting at D 2L ,D 2S , and D 4 receptors, but not at D 3 receptors [4 and 5]. Here, we investigate the voltage-sensitivities of a range of structurally related agonists activating G protein-coupled inward rectifier potassium (GIRK) channel currents via the human D 2S receptor. Three conserved serine residues in the fifth transmembrane segment (TM V) of dopamine receptors, corresponding to Ser193, Ser194, and Ser197 in the human D 2 receptor, play important roles in determining the binding affinity and efficacy of many hydroxylated dopamine agonists, including those exam- ined here [7–10]. Earlier studies delineating the requirements of monohydroxylated ligands to have stabilising interactions with these serine residues in the dopamine D 2S receptor identified a crucial role of a hydroxyl in the meta-(m) position as opposed to the para-(p) position [7,9,10] (see Fig. 1A). It was found that a p-hydroxyl alone does not increase binding affinity com- pared to non-hydroxylated ligands, and that an m-hydroxyl in- creases affinity, whereas both hydroxyls synergize to mediate binding. In the present study, we examined compounds in the phenethylamine and N,N-dipropyl-2-aminotetralin (DPAT) series to determine whether structural differences correlate with any differences in voltage-sensitivity. Materials and methods cDNA encoding human GIRK1, GIRK4, and pertussis toxin (PTX)- insensitive G ao1 C351I (UMR cDNA resource center, Rolla, MO; for other cDNA sources, see Acknowledgments) were all in pCDNA3.1. cDNA encoding the human dopamine D 2S receptor and the cata- lytic subunit of PTX were in pGEM4Z and pGEM-HE, respectively. The plasmids were linearized with NotI (GIRK1 and GIRK4), SalI (D 2S ), NheI (PTX-S1), and XbaI (G ao1 ). Xenopus oocytes were har- vested and injected with cRNA transcribed from the linearized plasmids as described [4]. One nanogram of each GIRK subunit cRNA and 5 ng or 20 pg of dopamine D 2S receptor cRNA, and when used, 3 ng of PTX-S1 and 15 ng of G ao1 C351I were injected per oocyte. The electrophysiological experiments were performed at room temperature using two-electrode voltage-clamp as described [4]. For recording of GIRK currents, a high-potassium solution (64 mM NaCl, 25 mM KCl, 0.8 mM MgCl 2 , 0.4 mM CaCl 2 , 15 mM HEPES, 1 mM acetic acid, pH 7.4) was used, giving a K + reversal po- tential of 40 mV. Single 80 or 0 mV pulses were applied from a holding potential of 40 mV to study current responses to D 2S receptor agonists. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.117 * Corresponding author. Fax: +46 8 34 95 44. E-mail address: kristoffer.sahlholm@ki.se (K. Sahlholm). Biochemical and Biophysical Research Communications 377 (2008) 1216–1221 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc