In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.
The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.
Ulrich
H
H
Section of Biochemistry, Molecular and Cell Biology, Cornell University Ithaca, NY 14853-2703, USA.
Ippolito
J E
JE
Pagán
O R
OR
Eterović
V A
VA
Hann
R M
RM
Shi
H
H
Lis
J T
JT
Eldefrawi
M E
ME
Hess
G P
GP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003042:Cocaine / Q000378:metabolism*; D016384:Consensus Sequence; D004557:Electric Organ / Q000378:metabolism; D013287:Illicit Drugs / Q000493:pharmacokinetics; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D012313:RNA / Q000378:metabolism* / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D014101:Torpedo
D013287:Illicit Drugs; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011978:Receptors, Nicotinic; D012313:RNA; C012058:tenocyclidine; D003042:Cocaine; D010622:Phencyclidine