Cys-Loop Receptor Pharmacology: Alterations in Agonist or Receptor Structure may Provide Insight into Receptor Function

The Cys-loop family of LGICs plays a crucial role in chemical synaptic transmission throughout the CNS and includes receptors that are activated by acetylcholine (Ach), serotonin (S-HT), γ-aminobutyric acid (GABA) and glycine (Gly). The Cys-loop family of LGICs were named after a characteristic highly conserved signature sequence of 13 amino acid residues flanked by cysteines that is present in each family member. This receptor family is an area of great interest for multiple disease processes and are targets for clinically significant pharmacotherapies.;Alternatively, by changing the receptor's structure via site-directed mutagenesis it is possible to elucidate the role that individual amino acids may play in agonist interactions. Cys-loop receptors are dynamic proteins that undergo agonist-induced conformational changes. Predictions from homology models of the α1β2γ2 GABAAR suggest binding loops C and F come into close proximity upon interaction with agonist. We performed site-directed mutagenesis creating the mutant receptor αV205Cβ2V223Cγ2, predicting that, upon interaction with agonist, these two cysteine residues would come into close proximity to form a disulphide bond locking the C-loop in the capped position. Recombinant receptors were expressed in Xenopus oocytes but we were unsuccessful in trapping the C-loop in the capped conformation, suggesting our predictions of residue location or residue motion may be inaccurate.;In addition, we investigated the role of a conserved arginine residue in binding loop D at the synaptic α1β2γ2 and extrasynaptic α4β3δ GABAARs. Homology modeling and ligand docking studies predict the residue α1R94 may play a larger role in stabilizing GABA than THIP in the binding pocket whereas the homologues residue α4R100 may playa larger role in stabilizing THIP than GABA. We performed the charge conserving α1R94K and α4R100K mutations and carried out functional studies of the effect of GABA and THIP on recombinant wild-type and mutant receptors expressed in Xenopus oocytes. Our data suggest that this arginine residue plays a larger role in the stabilization of THIP than GABA in the binding pocket of each receptor. In addition, there is a decrease in the efficacy of THIP observed in the α1R94Kβ2γ2 receptor relative to its wild-type. However, there is no decrease in the efficacy of THIP observed at the α4R100Kβ3δ receptor relative to its wild-type receptor.;Recent advances in the elucidation of receptor structure have led to an abundance of work attempting to describe the dynamic properties responsible for receptor activation. To investigate receptor-agonist interactions, alterations in the structure of the agonist or receptor may be introduced. Here we manipulate the structure of the ligand while observing the binding and functional properties of a series of bischoline agonists at recombinant Torpedo nAChRs expressed in Xenopus oocytes. Bischoline compounds show an increase in apparent affinity and potency with increasing interonium length. However, bischoline compounds shorter than 16 Å display weak partial agonist properties at the Torpedo nAChR.