IN-VITRO SELECTION OF RNA MOLECULES THAT DISPLACE COCAINE FROM THE MEMBRANE-BOUND NICOTINIC ACETYLCHOLINE-RECEPTOR

The nicotinic acetylcholine receptor (AChR) controls signal transmissi on between cells in the nervous system. Abused drugs such as cocaine i nhibit this receptor. Transient kinetic investigations indicate that i nhibitors decrease the channel-opening equilibrium constant [Hess, G, P, & Grewer, C, (1998) Methods Enzymol, 291, 443-473], Can compounds b e found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of R NA ligands by exponential enrichment methodology and the AChR in Torpe do californica electroplax membranes were used to find RNAs that can d isplace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of hi gh-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membr ane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibit or, phencyclidine, After nine selection rounds, two classes of RNA mol ecules that bind to the AChR with nanomolar affinities were isolated a nd sequenced, Both classes of RNA molecules are displaced by phencycli dine and cocaine from their binding site on the AChR, Class I molecule s are potent inhibitors of AChR activity in BC(3)H1 muscle cells, as d etermined by using the whole-cell current-recording technique. Class I I molecules, although competing with AChR inhibitors, do not affect re ceptor activity in this assay; such compounds or derivatives may be us eful for alleviating the toxicity experienced by millions of addicts.