Mechanisms of barbiturate inhibition of acetylcholine receptor channels

We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital.