Effects of Allosteric Ligands on the Gating of Single Channel Currents in BC₃H-1 Cells

Abstract

The effects of three allosteric ligands of the acetylcholine receptor (tetracaine, (-)N-allylnormetazocine [ANMC, SKF-10047], and phencyclidine [PCP]) on the single channel currents of the nicotinic acetylcholine receptor (AChR) of BC₃H-1 cells obtained from the American Type Culture Association were examined. At micromolar concentrations these agents acted as noncompetitive blockers of acetylcholine (ACh) activated currents. In the presence of ACh alone, the distributions of apparent open times and burst durations were fit by double exponential functions: τ₁=.46 ±.03 ms, τ₂ = 14.4 ±.9 ms for the open time distributions, and τ₁=.45 ±.02 ms, τ₂ = 15.5 ± 1.2 ms for burst durations. The relative area of the distributions represented by the short duration events decreased as a function of the concentration of ACh, representing 71 ± 3% of the area at 20 nM ACh and only 26 ± 8% at 2 µM ACh for the open time distributions. In the presence of 100 nM ACh, tetracaine, PCP, and ANMC decreased apparent mean channel open times with IC₅₀ ’s of 2.5 ±.5 µM, 5.0 ±.5µM, and 3.0 ±.5µM, respectively. The effects of tetracaine were inconsistent with a model for sequential block of open channels. The number of fast closures decreased in the presence of increasing concentrations of tetracaine, and the ratio of long and short events remained constant at all concentrations of tetracaine. This suggests that, in the presence of tetracaine, events were more likely to occur in isolation and that, at concentrations which more effectively blocked long duration events, the channel was not required to pass through the same open state repeatedly in order to return to the normal closed state. In addition, tetracaine acted as an agonist at concentration greater than 75 µM. In the presence of ANMC, in the range of concentration near the IC₅₀ for the block of long events, the ratio of long to short events increased substantially. ANMC also introduced a new component into the distribution of closed times, τ=13 ms. The area represented by this component increased with the concentration of ANMC and may represent the time that the channel is blocked by ANMC. The effects of ANMC seem more consistent with a mechanism of sequential block. Block by PCP also influenced the ratios of long and short events. A new component of the closed time distributions appeared that was several seconds long and increased with increasing concentration of PCP. In conclusion, we distinguish two types of channel block with these ligands: one consistent with sequential block of open channels and another which may allow the channel to pass into other state during the block.