The relationship between agonist occupation and the permeability response of the cholinergic receptor revealed by bound cobra alpha-toxin

Abstract

The decrement in functional capacity of the nicotinic receptor on intact BC3H-1 cells has been simultaneously compared with the fractional occupation of the receptor by cobra alpha-toxin. A parabolic, concave inward relationship between the fractional occupation of receptors by alpha-toxin and the decrement in permeability response is observed when the latter is tested over a range of agonist concentrations. Since alpha-toxin binding appears equivalent at each site on the receptor, the observed relationship is accommodated by a model where activation of a permeability response requires agonist occupation of two toxin-binding sites per functional receptor. Furthermore, the binding of alpha-toxin and agonist appears to be mutually exclusive, but occupation of either of the two sites by alpha-toxin is sufficient to block the functional capacity of the receptor. Consistent with this model, when a major fraction of sites is occupied by alpha-toxin, the concentration dependence for either carbamylcholine-mediated activation or desensitization of the remaining functional receptors is not detectably altered and retains positive cooperativity. In contrast, progressive occupation of the available sites by alpha-toxin leads to a decrease in apparent affinity and a corresponding loss of positive cooperation for agonist occupation functions generated upon instantaneous or following equilibrium exposure to the agonist. At high degrees of fractional occupancy to alpha-toxin, where the dominant species capable of binding agonist would contain a single bound toxin molecule, the Hill coefficient for the equilibrium occupation function for full agonists falls from a value of 1.4 to 0.7. By contrast, the binding isotherms for antagonists which typically exhibit values less than 1.0 are not altered following fractional irreversible occupation by alpha-toxin. Thus, the two binding sites on the receptor oligomer are not intrinsically equivalent for the binding of agonists and reversible antagonists. A scheme for desensitization of the receptor is presented which incorporates both nonequivalence in the two agonist binding-sites and the maintenance of symmetry in the receptor states undergoing transitions.