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

The mechanism of inhibition of the muscle nicotinic acetylcholine receptor is of interest because of the many drugs which are known to modify its func tion. The laser-pulse photolysis technique, using a photolabile, biological ly inert ligand (caged carbamoylcholine) for the nicotinic acetylcholine re ceptor, and BC(3)H1 cells have been used to investigate the mechanism of in hibition 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 rep orted here indicate the existence of two inhibition processes, one occurrin g within 50 ms and the other within about 1 s of equilibration of the recep tor with the inhibitor. Unless stated otherwise, here we characterize the r eceptor inhibition observed when MK-801 is equilibrated with the receptor f or only 50 ms. We determined the effect of MK-801 on the concentration of t he open receptor-channels and the apparent dissociation constant of the inh ibitor from the closed-channel (K-I(obs) = 180 mu M) and open-channel (K-I( obs) = 950 mu M) forms. Within a few milliseconds after inhibitor binding, K-I(obs) decreases to about 100 mu M, due to an inhibitor-induced isomeriza tion to an inactive receptor form. A mechanism that incorporates the new re sults is proposed. It includes the formation of an ion-conducting receptor: inhibitor complex with a channel-opening equilibrium constant that is unfav orable compared to the open-channel receptor form in the absence of inhibit or. In the MK-801 concentration range of 0-500 mu M, this mechanism account s for the observed MK-801-induced decrease in the concentration of open cha nnels. At high concentrations of carbamoylcholine, when the receptor is mai nly in the open-channel form, the conducting receptor:inhibitor complex iso merizes to a nonconducting state with a rate constant of about 2400 s(-1) f or the forward reaction and 230 s(-1) for the back reaction. It is shown th at the proposed new mechanism, based on transient kinetic measurements, als o accounts for the results of previous investigations with other inhibitors (procaine, cocaine), which were carried out under both pre-steady-state an d equilibrium conditions. A compound that binds to the same regulatory site on the receptor as MK-801 but does not affect the channel-opening equilibr ium constant may have considerable use in protecting an organism from the e ffects of abused drugs.