Changes in channel properties of acetylcholine receptors during the time course of thiol chemical modifications

Abstract

The changes occurring during chemical modification of thiol groups in single acetylcholine receptor (AChR) channels of BC3H-11 cells were examined by the patch-clamp technique in the "cell-attached" configuration. Treatment with either 1 mM or 5 mM dithiothreitol or with 5 mM N-ethylmaleimide (NEM) does not cause significant changes in the conductance and mean open time of the channels. However, reduction with dithiothreitol followed by alkylation with NEM produces modifications of AChRs. Under these conditions, channels activated by 2 microM acetylcholine show decreased open times (about 15-fold shorter for the most-modified AChRs) and a slight reduction in single-channel current. Both changes are dependent on the time of exposure and concentration of NEM. The rate of occurrence of openings, however, changes little during NEM treatment. When reduced and alkylated AChRs are activated by 100 microM acetylcholine, clusters of short openings separated by silent periods of about 1 s are observed. The channel-open probability, determined for openings within a cluster, is decreased by about 10-fold when compared with control receptors. The observations at high agonist concentration indicate that the modified AChR is still able to undergo desensitization.