Pharmacological characterization of seven human histamine H₃ receptor isoforms

The histamine H₃ receptor (H₃R) regulates as a presynaptic G protein-coupled receptor the release of histamine and other neurotransmitters in the brain, and is consequently a potential therapeutic target for neuronal disorders. The human H₃R encodes for seven splice variants that vary in the length of intracellular loop 3 and/or the C-terminal tail but are all able to induce heterotrimeric G_i protein signaling. The last two decades H₃R drug discovery and lead optimization has been exclusively focused on the 445 amino acids-long reference isoform H₃R-445. In this study, we pharmacologically characterized for the first time all seven H₃R isoforms by determining their binding affinities for reference histamine H₃ receptor agonists and inverse agonists. The H₃R-453, H₃R-415, and H₃R-413 isoforms display similar binding affinities for all ligands as the H₃R-445. However, increased agonist binding affinities were observed for the three shorter isoforms H₃R-329, H₃R-365, and H₃R-373, whereas inverse agonists such as the approved anti-narcolepsy drug pitolisant (Wakix®) displayed significantly decreased binding affinities for the latter two isoforms. This opposite change in binding affinity of agonist versus inverse agonists on H₃R-365 and H₃R-373 is associated with their higher constitutive activity in a cAMP biosensor assay as compared to the other five isoforms. The observed differences in pharmacology between longer and shorter H₃R isoforms should be considered in future drug discovery programs.