Kinetics of binding of [3H]acetylcholine and [3H]carbamoylcholine to Torpedo postsynaptic membranes: slow conformational transitions of the cholinergic receptor

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Abstract
The kinetics of binding of [3H]acetylcholine (AcCh) and [3H]carbamoylcholine (Carb) to membrane-bound nicotinic receptor for Torpedo electric tissue were measured on the second time scale by rapid mixing and ultrafiltration. The concentration dependence of the association kinetics of agonist binding and the kinetics of ligand dissociation and receptor reisomerization following the removal of agonist were analyzed with a model. In the model, the binding was by a single population of receptors existing in the absence of agonist in 2 interconvertible conformations, one binding agonist weakly (R1) and the other binding with high affinity (R2). A computer simulation was used to determine values of rate and equilibrium constants [Keq] characterizing the ligand interactions with the 2 conformations and for the conformational equilibrium in the presence and absence of agonist. At 4.degree. C, R1/R2 = 4.5 and the half-time for isomerization for low to high affinity of unliganded receptor was equal to 200 s. For receptors occupied by AcCh or Carb, the half-time was reduced to .apprx. 4 s. For AcCh, the apparent Kd of the low- and high-affinity conformations were 800 4M and 2 nM, respectively (Keq = 8 nM), and for Carb the values were 30 .mu.M and 25 nM (Keq = 100 nM). The Kd dissociation rate constant of [3H]AcCh from R2 was 0.04/s. Alternate less satisfactory reaction models are discussed and compared with receptor conformational equlibria deduced by the use of other kinetic technqiues.
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