MECHANISMS OF BARBITURATE INHIBITION OF ACETYLCHOLINE-RECEPTOR CHANNELS

We used patch clamp techniques to study the inhibitory effects of pent obarbital and barbital on nicotinic acetylcholine receptor channels fr om BC3H-1 dens. single channel recording from outside-out patches reve als that both drugs cause acetylcholine-activated channel events to oc cur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM bar bital reduces the apparent single channel current by 15%. Both barbitu rates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activat ed by rapid perfusion of 300 mu M acetylcholine to outside-out patches . The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, K-i = 32 mu M, n = 1.09; for barb ital, K-i = 1900 mu M, n = 1.24. Inhibition is voltage independent. Th e kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the K-i). Pentobarbi tal binds greater than or equal to 10-fold more tightly to open channe ls than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbi turates reveal that they do not compete for a single binding site on t he acetylcholine receptor channel protein, but the binding of one barb iturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed stat es of the AChR and block the flow of ions through the channel. An addi tional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 mu M pentobarbital.