Voltage sensitivity of dopamine D2-like receptors

Abstract

G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotransmitter, and sensory functions. These receptors are important drug targets; in fact, about 27 % of prescribed drugs are GPCR ligands. The dopamine D2 receptor is prominently expressed within the CNS as two distinct isoforms; D2L (long isoform) and D2S (short isoform). The former is mainly expressed postsynaptically, whereas the latter functions primarily as an inhibitory auto- and heteroreceptor. The D2 receptor is of considerable pharmacological interest, as it constitutes the main target for most antiparkinsonian and antipsychotic drugs in clinical use. While many ion channels have long been known to be voltage sensitive, this property has not been attributed to GPCRs until quite recently. As a notable example, the muscarinic M2 receptor was shown to display depolarization-induced decreases in agonist binding and functional potency. M2 receptor voltage sensitivity has been implicated in the autoreceptor function of this GPCR, by permitting rapid control of neurotransmitter release kinetics by membrane voltage.

The present work investigated the voltage sensitivities of the three D2-like dopamine receptors; D2, D3, and D4. The bulk of the experiments were carried out in Xenopus oocytes heterologously expressing D2-like receptors with G protein-coupled inwardly rectifying potassium channel (GIRK) subunits. GIRK channels are activated by Gβγ subunits from inhibitory G proteins and were used as readout of receptor activation. It was found that dopamine potency was reduced by depolarization to a similar extent at both isoforms of the D2 receptor. However, at the dopamine D3 receptor dopamine potency was not significantly affected, while a weak, albeit significant potency decrease was observed at the dopamine D4 receptor. Moreover, in mammalian cells expressing fluorescent G protein subunits, changes in inter-subunit Förster Resonance Energy Transfer (FRET) were used as readout of D2S receptor activation. Determination of dopamine concentration-response relationships in single cells under simultaneous patch clamp revealed similar depolarization-induced potency shifts as when studying GIRK channel activation in oocytes. Furthermore, radioligand binding experiments carried out on oocytes in hyperpolarizing vs. depolarizing buffer established that dopamine binding is reduced by depolarization. Interestingly, the effect of voltage was different for different agonists at the D2S receptor, including efficacious, high-affinity antiparkinsonian agonists. This agonist-specificity did not reflect selective signalling via distinct G protein subtypes. However, contacts between agonist hydroxyl groups and receptor serine residues, as well as between the agonist amine group and a conserved aspartate residue, were found to be important for the voltage induced potency shift of phenethylamine agonists, such as dopamine.

In conclusion, the findings presented in this thesis suggest that the dopamine D2-like receptors are differentially affected by voltage. At the D2S receptor, specific agonist- receptor interactions determine the effect of the receptor’s voltage sensitivity on agonist potency and efficacy. This information demonstrates the relevance of GPCR voltage sensitivity to dopaminergic signalling, reveals new details about the mechanism of voltage sensitive agonism, and points to the possibility of using differentially voltage- modulated agonists to investigate the relevance of this phenomenon in native tissue.