Pharmacological Characterization of the Zebrafish (Danio Rerio) Histamine H1 Receptor Reveals the Involvement of the Second Extracellular Loop in the Binding of Histamine.

Abstract

The zebrafish (Danio rerio) histamine H₁ receptor gene (zfH₁R) was cloned in 2007 and reported to be involved in fish locomotion. Yet, no detailed characterization of its pharmacology and signaling properties have so far been reported. In this study, we pharmacologically characterized the zfH₁R expressed in HEK-293T cells by means of [³H]-mepyramine binding and G protein-signaling assays. The zfH₁R [dissociation constant (K_D), 0.7 nM] displayed similar affinity for the antagonist [³H]-mepyramine as the human histamine H₁ receptor (hH₁R) (K_D, 1.5 nM), whereas the affinity for histamine is 100-fold higher than for the human H₁R. The zfH₁R couples to Gα_q/11 proteins and activates several reporter genes, i.e., NFAT, NFϰB, CRE, VEGF, COX-2, SRE, and AP-1, and zfH₁R-mediated signaling is prevented by the Gα_q/11 inhibitor YM-254890 and the antagonist mepyramine. Molecular modeling of the zfH₁R and human H₁R shows that the binding pockets are identical, implying that variations along the ligand binding pathway could underly the differences in histamine affinity instead. Targeting differentially charged residues in extracellular loop 2 (ECL2) using site-directed mutagenesis revealed that Arg210^45x55 is most likely involved in the binding process of histamine in zfH₁R. This study aids the understanding of the pharmacological differences between H₁R orthologs and the role of ECL2 in histamine binding and provides fundamental information for the understanding of the histaminergic system in the zebrafish. SIGNIFICANCE STATEMENT: The use of the zebrafish as in vivo models in neuroscience is growing exponentially, which asks for detailed characterization of the aminergic neurotransmitter systems in this model. This study is the first to pharmacologically characterize the zebrafish histamine H₁ receptor after expression in HEK-293T cells. The results show a high pharmacological and functional resemblance with the human ortholog but also reveal interesting structural differences and unveils an important role of the second extracellular loop in histamine binding.