PT - JOURNAL ARTICLE AU - Kazuhiko Takahashi AU - Shigeru Tokita AU - Hidehito Kotani TI - Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors AID - 10.1124/jpet.103.053249 DP - 2003 Oct 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 213--218 VI - 307 IP - 1 4099 - http://jpet.aspetjournals.org/content/307/1/213.short 4100 - http://jpet.aspetjournals.org/content/307/1/213.full SO - J Pharmacol Exp Ther2003 Oct 01; 307 AB - Constitutive activation of G-protein-coupled receptors is a well recognized phenomenon, and G-protein-coupled receptor antagonists have been found to possess inverse agonist activity. Constitutive activation of histamine H3 receptor is recently documented in in vivo as well as in recombinant receptor systems in vitro. Several H3 antagonists have been shown to act as inverse agonists and such profiles of H3 antagonists have been implicated in their pharmacological functions. Here we report the construction and characterization of a highly constitutive active H3 receptor (MT6), in which the 357 alanine residue was converted to lysine (A357K). We generated a series of mutated H3 receptors and their functions were examined in human embryonic kidney (HEK) 293 cells. Among them, induced mutation at the amino acid 357 position (A357K) showed a dramatically enhanced response to thioperamide-induced cAMP accumulation compared with the cells expressing wild-type (WT) H3 receptors, suggesting that the mutation rendered receptors to high constitutive activity. We further characterized by ligand binding assays using membrane fractions, and Ki values of imetit (agonist) and proxyfan (partial agonist) against the MT6 receptors were lower compared with those observed in WT H3 receptors. In contrast, H3 antagonists (thioperamide, ciproxifan, and GT2016) with inverse agonism displayed increased Ki values against the MT6 receptors (2.5- to 5.8-fold), demonstrating more a prominent effect of inverse agonists to the constitutive active receptor. Taken together, these data suggested that A357K mutation in the H3 receptor increased the population of active state receptors that preferably binds to agonists than inverse agonists, which could be termed as a constitutively active mutant of H3 receptor. The American Society for Pharmacology and Experimental Therapeutics