Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801

Nicotinic acetylcholine receptors (AChR) belong to a family of proteins tha t form ligand-gated transmembrane ion channels. They are involved in the fa st transmission of signals between cells and the control of intercellular c ommunication in the nervous system. A variety of therapeutic agents and abu sed drugs, including cocaine, inhibit the AChR and monoamine transporters a nd interfere with nervous system function. Here we describe a mechanism-bas ed approach to prevent this inhibition. We had previously developed prestea dy-state kinetic (transient kinetic) techniques, with microsecond-to-millis econd time resolutions, for investigations of reactions on cell surfaces th at allow one to determine the effects of inhibitors not only on the channel -opening probability but also on the opening and closing rates of the AChR channel. The transient kinetic measurements led to two predictions. (i) Lig ands that bind to a regulatory site on the closed-channel conformation of t he AChR with higher affinity than to the site on the open-channel form shif t the equilibrium toward the closed-channel form, thereby inhibiting the re ceptor. (ii) Ligands that bind to a regulatory site with an affinity for th e open conformation equal to or higher than their affinity for the dosed co nformations are expected not to inhibit the receptor and to displace inhibi tors. The identification of such ligands in a combinatorial library of RNA ligands is reported. The implication of this approach to other protein-medi ated reactions in which an inhibitor changes the equilibrium between active and inactive conformations is discussed.