Procaine rapidly inactivates acetylcholine receptor from Torpedo and competes with agonist for inhibition sites

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Abstract
The relationship between the high-affinity procaine channel inhibition site (apparent dissociation constant Kp .simeq. 200 .mu.M) and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86Rb+ quenched-flux assays at 4.degree.C in native AChR-rich vesicles on which 50.sbd.60% of ACh activation sites were blocked with .alpha.-bungarotoxin (.alpha.-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations (10 .mu.M.sbd.10 mM) alone, AChR undergoes one phase of inactivation (fast desensitization, rate = kd) in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting (> 10 mM) ACh concentrations [Forman and Miller (1988) Biophys. J. 54, 149.sbd.158] .sbd. rapid inactivation (rate = kr) complete in 30.sbd.75 ms is followed by fast desensitization at the same kd observed without procaine. The dependence of kr on [procaine] is consistent with a bimolecular association between procaine and its AChR site with kon = 2.5 .times. 105 M-1 s-1, koff = 36 s-1, and Kp = 145 .+-. 36 .mu.M. Inhibition of AChR function by mixtures of procaine (up to 12Kp) plus self-inhibiting concentrations of ACh or suberyldicholine ([SubCh] up to 13 .times. the 50% self-inhibiting agonist concentration, KB) was studied by reducing the level of .alpha.-BTX block in vesicles. The apparent KB increased in the presence of procaine, and the apparent Kp increased linearly with [SubCh], indicating mutually exclusive actions at a common AChR site. Our data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and kr''s dependence on [ACh] in the channel-activating range closely parallels that of 86Rb+ flux response to ACh.

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