The local anesthetics proadifen and adifenin act at different conformational states of the nicotinic receptor

GUILLERMO SPITZMAUL; FERNANDA GUMILAR; JAMES DILGER; CECILIA BOUZAT

California.

Congreso; Biophysical Society 49 th Annual meeting; 2005

Biophysical Society

Local anesthetics act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR). Proadifen has been shown to increase desensitization of the AChR, but its mechanism of action as well as that of its analog, adiphenine, have not been elucidated. Single-channel and macroscopic-current recordings from cells transfected with adult muscle AChR reveal that both drugs increase desensitization by acting at different conformational states. At the single-channel level, both drugs decrease the frequency of opening events, without significant changes in the mean open time. At high ACh concentrations, proadifen does not affect cluster duration. In contrast, adiphenine significantly decreases cluster duration, such reduction being about 90% at 20 µM. Proadifen decreases the peak of macroscopic currents activated by rapid application of ACh to outside-out patches (IC50= 23 uM), without changing the decay rate due to desensitization. The onset of inhibition by proadifen is slower than recovery (rates are 0.19 +/- 0.5/S and and 0.34 +/- 0.07/s for onset and recovery, respectively). Adiphenine increases the decay rate without changing the peak current. For both drugs preincubation is necessary to exert their full actions, which are not dependent on membrane potential. Experiments with the E262 mutant AChR (a site labeled by meproadifen) show no differences in inhibition by proadifen but less sensitivity to inhibition by adiphenine. In summary, our results show that these chemically-related drugs modulate different conformational states of the AChR. Proadifen desensitizes AChRs that are in the closed state and adiphenine increases desensitization from the open state.