Lysine²⁰⁰ Located in the Fifth Transmembrane Domain of the Histamine H₁ Receptor Interacts with Histamine but Not with All H₁ Agonists

Abstract

Previously, we have shown that asparagine²⁰⁷ in the fifth transmembrane domain of the histamine H₁ receptor is crucial for the binding of the N^τ-nitrogen of the imidazole ring of histamine (Leurs et al., Biochem. Biophys. Res. Commun., 201, 295, 1994). In view of the potential interaction of the imidazole ring of histamine histamine with a binding site, formed by asparagine²⁰⁷ and lysine²⁰⁰, we mutated lysine²⁰⁰ in the fifth transmembrane domain of the histamine H₁ receptor to a nonfunctional alanine residue. This mutation did not affect the binding of the tested H₁ receptor antagonists bur resulted in a 5-fold lower affinity for histamine. The binding of other H₁ receptor agonists was not affected. In stably transfected CHO cells histamine was 55-fold less effective in activating the H₁Lys²⁰⁰Ala receptor (EC₅₀ = 66 μM) compared to the wild type H₁ receptor (EC₅₀ = 1.2 μM). Receptor activation by the 2-methyl and the 2-(3-bromophenyl)-analogues however was hardly affected by the mutation, indicating that the 2-substituent probably prevents the interaction with the lysine²⁰⁰ residue. Finally, the Lys²⁰⁰Ala mutation reduced the production of [³H]inositol phosphates, stimulated by the non-imidazole H₁ receptor agonist 2-pyridylethylamine. These data indicate that lysine²⁰⁰ interacts with the N^π-nitrogen of histamine and is important for the activation of the H₁ receptor by histamine and the non-imidazole agonist 2-pyridylethylamine.