Pharmacological properties of 5-hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors

We studied the pharmacological properties of twenty-four 5-hydroxytryptamin e (5-HT)(4) receptor ligands known to act as antagonists on 5-HT4 receptors positively coupled to adenylyl cyclase endogenously expressed in mouse col liculi neurons. In COS-7 cells expressing human or mouse 5-HT4(a) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agoni sts, and inverse agonists. The majority of neutral antagonists belong to th e benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 2072 66, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated recep tors revealed that 1) the ratio between the efficiencies of the full agonis t 5-HT and the partial agonist RS 23597 was invariable when the receptor de nsity increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor de nsity but was dependent on receptor structure; 3) when the receptor concent ration increased, the EC50 values of the full agonist 5-HT were not modifie d and the increase in basal constitutive activity, as well as its stimulati on by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Gas . All these results indicate that in COS-7 cells, the coupling of the 5-HT4 receptor to adenylyl cyclase was linear with no indication of spare recept ors even at high receptor density (8 pmol/mg). These results are also in ac cordance with a precoupling between the activated receptor (f(R*)) and aden ylyl cyclase. Such observations allowed us to use the two-state model to ca lculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*] ). We found that J was a receptor structural characteristic, independent of receptor density.