Noncompetitive inhibitors reach their binding site in the acetylcholine receptor by two different paths

Electron spin resonance was used to contrast the accessibility of tertiary and quaternary local anesthetics to their high affinity binding site in the desensitized acetylcholine receptor (AChR). The time dependence of agonist addition on the association of spin-labeled local anesthetics with the nicotinic AChR-enriched membranes from Torpedo californica was studied. Preincubation of AChR-enriched membranes with agonist for more than a few minutes before the addition of C6SLMel, a quaternary amine local anesthetic, resulted in substantial reduction in the initial association of the label with the receptor. The time-dependent reduction in the initial association of the label with the receptor is modeled by an exponential function having a rate constant of approximately 0.2 min-1. In contrast, agonist preincubation did not produce a comparable decrease in the association of C6SL, a tertiary amine analog, with the AChR. These findings show that whereas the affinity of either anesthetic for the AChR is dependent on the presence of agonist, for C6SLMel the timing of agonist addition is an important factor in determining the rate of anesthetic association with the receptor. Our results are concerned with the desensitized receptor at an early phase, when the average open-channel time limits the anesthetic binding to the receptor. We interpret our results by a model in which the cationic local anesthetic reaches its high affinity binding site in the receptor by an aqueous path that is accessible only when the channel is open. On the other hand, anesthetic in its uncharged form is not restricted only to the aqueous path of access. An additional path, probably through the lipid bilayer, allows uncharged forms of anesthetics to reach the high affinity binding site in the AChR even when the aqueous path is closed. During the "open state" of the receptor both cationic and uncharged anesthetics have access to the high affinity site through the aqueous path. However, after this open state, the channel opens only intermittently. The rapidly decreasing open time results in the time-dependent reduction in the binding of cationic anesthetics. This model is consistent with the open channel hypothesis of anesthetic binding to the AChR immediately after agonist stimulation; however, our model also includes an additional hydrophobic path of access for uncharged and reversibly charged anesthetics.