Opposing effects of N-ethylmaleimide on the affinity of carbachol for muscarinic cholinoceptors of guinea-pig atrium

1. 1. Inhibition of the binding of [ 3H]quinuclidinyl benzilate to homogenates of guinea pig right atrium (M2 receptors) by varying concentrations of carbachol was studied. 2. 2. Pretreatment of membranes with 5 × 10 −5 M N-ethylmaleimide at 2°C shifted the carbachol inhibition curve to the right, indicating decreased affinity of the receptor for carbachol. However pretreatment at 37°C moved the curve to the left. 3. 3. The ability of guanyl-5′-yl imidodiphosphate to reduce agonist affinity was largely eliminated by treatment with N-ethylmaleimide at both temperatures. 4. 4. Conflicting reports in the literature and the present results can be explained by invoking a model in which N-ethylmaleimide has a high affinity for a heat-labile site and a lower affinity for a heat-insensitive site. Reaction with the first site decreases agonist affinity, but at 37°C this site is largely inactivated and reaction with the second site, which leads to increased agonist affinity, predominates.