Jp. Dilger et al., MECHANISMS OF BARBITURATE INHIBITION OF ACETYLCHOLINE-RECEPTOR CHANNELS, The Journal of general physiology, 109(3), 1997, pp. 401-414
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.