Molecular analysis of the histamine H3-receptor

The histamine H3 receptor (H3R) is a biogenic amine receptor that belongs to family I of G protein-coupled receptors (GPCRs). During the past two decades, the H3R has gained much interest in academia and industry. The H3Rs is predominantly localized in the brain and regulates the release of histamine as well as other neurotransmitters into the synaptic cleft via negative feedback mechanisms. Thus, H3Rs serve as presynaptic auto- or heteroreceptors. H3Rs play an important role in processes like cognition and the sleep-wakecycle. Numerous ligands targeting H3R have been developed as pharmacological tools or potential therapeutics. Some of them show unexpected and pleiotropic effects. The currently available data are not sufficient to explain this uncommon behaviour. The aim of this thesis was to investigate the detailed molecular mechanisms of some yet unexplained H3R-ligand effects. Therefore, sensitive baculovirus/Sf9 cell-based assay systems to analyze human H3R (hH3R) and rat H3R (rH3R) on a molecular level, were established. It is known that certain imidazole-containing H3R-ligands like proxyfan are functionally selective, i. e. activate only specific pathways mediated by H3R. In this work, the detailed G protein coupling-profile of H3R was investigated and various imidazole-based ligands were examined. We did not obtain evidence for differences in the G protein coupling profile of the H3R or functional selectivity of any of the compounds assayed. Possible reasons for the discrepancies between the results and data obtained from the literature are discussed. These “negative” results cannot be attributed to unsuitability of our expression system for exclusion of ligand functional selectivity. However, our system is not suitable to definitely exclude protean agonism, a special case of functional selectivity, at H3R, since that would require a systematic and precise variation of receptor-to-G protein stoichiometries. Extensive systematic studies under clearly defined experimental conditions are required to reconcile the discrepancies. Thus, presently, a specific and generally applicable mechanistic explanation for the previously observed pleiotropic effects of proxyfan cannot yet be provided. Additionally, despite a very high sequence homology of hH3R and rH3R, there are substantial pharmacological species differences. Imoproxifan is an inverse agonist at rH3R, but almost full agonist at hH3R. We have shown that hH3R and rH3R expressed in Sf9 cells both couple similarly to defined Gi/Go-protein heterotrimers and display similar constitutive activities. We show species-differences in pharmacological properties of imoproxifan and offer an explanation on the molecular basis for these differences. Most importantly, we introduce novel active state models of hH3R and rH3R that are suitable to explain the efficacy of H3R ligands. Two amino acid residues between hH3R and rH3R cause the reversal in efficacy of imoproxifan due to substantial differences in the electrostatic potential surfaces of the binding pockets. Monovalent ions differentially affect GPCR signalling by as yet poorly understood mechanisms. In particular, Na+ is known as universal allosteric GPCR modulator. However, it is unknown how Na+ - ions exert the effects and whether it is not the counter-ion, which is responsible for the salt effects. Therefore, the H3R was used as a model system to study the effect of various monovalent ions. Moreover, a highly conserved aspartate in TM II, thought to be an interaction site of Na+ - ions in GPCRs, was mutated to asparagine. It turned out that most probably both, cation and anion, exert a modulatory effect on GPCR/G proteincoupling. Monovalent cations may stabilize an inactive H3R-state via interaction with the conserved aspartate in TM II, while anions may increase the affinity of G proteins for GDP and thus, indirectly affect their interaction with H3R. Interestingly, NaCl differentially affects the G protein coupling-profile of H3R. NaCl selectively abolished constitutive signalling of H3R only in the presence of Gαi3. Obviously, the conserved aspartate in TM II is a key residue for H3R/Gαi3 protein-activation. The latter result suggests that H3R/G protein-coupling interfaces may differ even between closely related subunits. In conclusion, this thesis provides new insight into the molecular mechanisms of H3R function, G protein-coupling and species-differences. The availability of a sensitive H3R test system will improve the development of new histamine receptor ligands, especially with respect to selectivity over the structurally related H4R, and contribute to a better understanding of ligand effects. Most importantly, a gap was filled regarding the interaction of H3R with specific G protein α-subunits, an until now insufficiently investigated area