Desensitization of acetylcholine receptor by the local anesthetic proadifen

CECILIA BEATRIZ BOUZAT; G SPITZMAUL,; F GUMILAR,; J P DILGER,

Baltimore

Congreso; 48th Annual Meeting Biophysical Society; 2004

Biophysical Society

There are several mechanisms by which noncompetitive inhibitors of nicotinic acetylcholine receptors (AChR) decrease channel open probability. The local anesthetic proadifen is often used to desensitize AChRs by inducing a state of high agonist affinity. A binding site for proadifen has been identified at the extracellular end of the M2 domain (J Biol Chem. 267:10489-99, 1992), but themechanism of action of proadifen has not yet been elucidated. We made single channel and macroscopic current recordings from HEK-293 cells transfected with adult muscle AChR in the presence of proadifen. Single-channel recordings on cell-attached patches reveal a profound decrease in the frequency of openings events, such reduction being about 90% at 20 µM. No significant changes in the mean open time or in the duration of clusters elicited by highACh concentrations were observed. These observations suggest that proadifen is affecting mainly the closed state. In macroscopic currents activated by rapid application of ACh to outside-out patches, proadifen decreases the peak current (IC50= 15 µM) without changing the decay rate due to desensitization. Preincubation with proadifen is necessary and sufficient for inhibition; noadditional inhibition is observed if proadifen is present in both preincubating and ACh-containing solutions. Onset of inhibition appears to be slower than recovery from inhibition. Combining results at 15 and 30 µM, we measured rates of 0.19±0.05/s and 0.34±0.07/s for onset and recovery respectively. These results suggest that proadifen desensitizes AChRs that are in the closed state,has relatively slow kinetics and neither affects open AChRs nor acts as an open-channel blocker of the AChR. Preliminary experiments with the αE262A,K mutant AChR (a site labeled by meproadifen) show no differences in inhibition by proadifen.