The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperature.

ABSTRACT

Detailed information about the ligand-binding site of nicotinic acetylcholine receptors has emerged from structural and mutagenesis experiments. However, these approaches provide only static images of ligand-receptor interactions. Kinetic measurements of changes in protein function are needed to develop a more dynamic picture. Previously, we measured association and dissociation rate constants for competitive inhibition of current through embryonic muscle acetylcholine receptor channels at 25 degrees C. Little is known about competitive antagonism at physiological temperatures. Here, we performed measurements at 37 degrees C and used thermodynamics to estimate the energetics of antagonism. We used rapid solution exchange protocols to determine equilibrium and kinetics of inhibition of acetylcholine-activated currents in outside-out patches by (+)-tubocurarine, pancuronium and cisatracurium. Kinetic rates as high as 600 s(-1) were resolved by this technique. Binding was primarily enthalpy driven. The 12 degrees C increase in temperature decreased equilibrium antagonist binding by 1.7- to 1.9-fold. In contrast, association and dissociation rate constants increased 1.9- to 6.0-fold. Activation energies for dissociation were 90 +/- 6, 106 +/- 8 and 116 +/- 10 kJ mol(-1) for cisatracurium, (+)-tubocurarine and pancuronium, respectively. The corresponding apparent activation energies for association were 38 +/- 6, 85 +/- 6 and 107 +/- 13 kJ mol(-1). The higher activation energy for association of (+)-tubocurarine and pancuronium compared with cisatracurium is notable. This may arise from either a more superficial binding site for the large antagonist cisatracurium compared to the other ligands, or from a change in receptor conformation upon binding of (+)-tubocurarine and pancuronium but not cisatracurium. Differences in ligand desolvation and ligand conformation are not likely to be important.