On the Mechanism of Inhibition of the Nicotinic Acetylcholine Receptor by the Anticonvulsant MK-801 Investigated by Laser-Pulse Photolysis in the Microsecond-to-Millisecond Time Region

Abstract

The mechanism of inhibition of the muscle nicotinic acetylcholine receptor is of interest because of the many drugs which are known to modify its function. The laser-pulse photolysis technique, using a photolabile, biologically inert ligand (caged carbamoylcholine) for the nicotinic acetylcholine receptor, and BC3H1 cells have been used to investigate the mechanism of inhibition of the receptor by MK-801 [(+)-dizocilpine] in the microsecond-to-millisecond time region. MK-801 is an anticonvulsant and a known inhibitor of the N-methyl-D-aspartate and nicotinic acetylcholine receptors. Both the chemical kinetic and the single-channel current-recording measurements reported here indicate the existence of two inhibition processes, one occurring within 50 ms and the other within about 1 s of equilibration of the receptor with the inhibitor. Unless stated otherwise, here we characterize the receptor inhibition observed when MK-801 is equilibrated with the receptor for only 50 ms. We determined the effect of MK-801 on the concentration of the open receptor-channels and the apparent dissociation constant of the inhibitor from the closed-channel (KI(obs) = 180 microM) and open-channel ( = 950 microM) forms. Within a few milliseconds after inhibitor binding, decreases to about 100 microM, due to an inhibitor-induced isomerization to an inactive receptor form. A mechanism that incorporates the new results is proposed. It includes the formation of an ion-conducting receptor:inhibitor complex with a channel-opening equilibrium constant that is unfavorable compared to the open-channel receptor form in the absence of inhibitor. In the MK-801 concentration range of 0-500 microM, this mechanism accounts for the observed MK-801-induced decrease in the concentration of open channels. At high concentrations of carbamoylcholine, when the receptor is mainly in the open-channel form, the conducting receptor:inhibitor complex isomerizes to a nonconducting state with a rate constant of about 2400 s-1 for the forward reaction and 230 s-1 for the back reaction. It is shown that the proposed new mechanism, based on transient kinetic measurements, also accounts for the results of previous investigations with other inhibitors (procaine, cocaine), which were carried out under both pre-steady-state and equilibrium conditions. A compound that binds to the same regulatory site on the receptor as MK-801 but does not affect the channel-opening equilibrium constant may have considerable use in protecting an organism from the effects of abused drugs.

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

“…These results can be understood in the light of transient kinetic results obtained previously (6). The earlier results indicated that the muscle-type AChR in BC 3 H1 cells has two inhibitory sites for MK-801; one site equilibrates with MK-801 within 50 ms and the second site within 4 s (6) Fig. 1) to a receptor form that does not conduct ions was observed (6,9,29,30).…”

“…3 A and B), all of the inhibition by MK-801 was relieved in the presence of 2 M aptamer II-3. These results can be understood in the light of transient kinetic results obtained previously (6). The earlier results indicated that the muscle-type AChR in BC 3 H1 cells has two inhibitory sites for MK-801; one site equilibrates with MK-801 within 50 ms and the second site within 4 s (6) Fig.…”

“…II-C). For instance, in the case of MK801, k cl increases with increasing inhibitor concentration, whereas k op does not change (6). The simplest mechanism we can think of that is consistent with these (33).…”

“…The earlier results indicated that the muscle-type AChR in BC 3 H1 cells has two inhibitory sites for MK-801; one site equilibrates with MK-801 within 50 ms and the second site within 4 s (6) Fig. 1) to a receptor form that does not conduct ions was observed (6,9,29,30). Such a transition was first observed in single-channel current measurements (14).…”

“…For clarity, the desensitization reaction, which in the case of the AChR occurs in the 100-to 500-ms time region (44,45), and the binding of the inhibitor I to the unliganded receptor form are not shown. The relatively slow transitions of receptor͞inhibitor complexes to nonconductive forms (6,9,30) . This minimum mechanism is based on chemical kinetic measurements and on predictions it makes regarding the properties of ligands that will inhibit the receptor and of those that will not inhibit the receptor but will prevent the binding of inhibitors.…”