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 is 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 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, 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 ≥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 μM pentobarbital.
Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels
Address correspondence to Dr. James P. Dilger, Department of Anesthesiology, University at Stony Brook, Stony Brook, NY 11794-8480. Fax: 516-444-2907; E-mail: [email protected]
This research was supported in part by a grant from the National Institute of General Medical Sciences (GM 42095); Klinik für Anästhesiologie, Universität Bonn; Department of Anesthesiology, University at Stony Brook; and an institutional grant from the School of Medicine, University at Stony Brook.
A preliminary report of these results has appeared in abstract form (Boguslavsky, R., and J.P. Dilger. 1995. Biophys. J. 68:A377).
Abbreviations used in this paper: ACh, acetylcholine; AChR, ACh receptor; Barb, barbital; PB, pentobarbital.
This estimate comes from comparison of single channel burst frequency with peak macroscopic currents measured on the same patch(Liu, Y., and J.P. Dilger, unpublished data).
For the data shown in Fig. 2 with 100 μM PB, the long closed component increased to 140 ms. However, subsequent return to control conditions showed the long closed time to be 140 ms. We assume that, in this patch, there was a rundown in channel activity between the first control and 100 μM PB runs. In patches that did not exhibit any rundown, 100 μM PB did not affect the long closed time. We did observe that 250 and 500 μM PB tended to decrease the long closed interval.
The predicted closed duration histogram for scheme SIII has four components. The longest represents the time between bursts and is not a part of the gap duration. We plotted the weighted average of the remaining three components in Fig. 4,C. We defined a burst in scheme SIII as sojourns starting in state O and ending in state C. The predicted burst duration histogram has three components but the briefest component has a negligible area. The remaining two components are separated by a factor of three or more. Because the measured burst duration histograms have two components and we have been considering the longer of the two observed components, we plotted the longest predicted burst duration component in Fig. 4,A. The predicted number of openings per burst for scheme SIII has a single component, and this is plotted in Fig. 4 B.
James P. Dilger, Rebecca Boguslavsky, Martin Barann, Tamir Katz, Ana Maria Vidal; Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels . J Gen Physiol 1 March 1997; 109 (3): 401–414. doi: https://doi.org/10.1085/jgp.109.3.401
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