Kinetics and mechanism of L-[3H]nicotine binding to putative high affinity receptor sites in rat brain.

The properties of high affinity nicotine-binding sites in rat brain were studied by monitoring the kinetics of L-[3H]nicotine binding to whole brain membrane preparations, including both total membranes and membrane subfractions. Although nicotine appeared to bind to a single class of sites, with an apparent equilibrium dissociation constant of 2-3 nM, the binding kinetics were biphasic at all temperatures and at all nicotine concentrations tested. An initial rapid binding process, with an association rate constant of around 0.02 min-1 nM-1 at 0 degree, was followed by a slower binding process. Both the rate and the proportion of binding that occurred by the slower process were dependent on the nicotine concentration. By comparison, the kinetics of dissociation were first order at all concentrations, with a rate constant of 0.04 min-1 at 0 degree. The rates of both association and dissociation increased significantly with temperature, but there was no changed in the apparent affinity of the sites. The same results were obtained with several different membrane preparations, including whole brain membrane preparations, detergent-permeabilized membranes, P-2 fractions, and synaptosomes. The results were found to be consistent with a two-state model. Analyses based on this model indicate that the binding sites can assume two different conformations, one having a high affinity (KD = 1 nM) and the other a low affinity (KD = 150 nM) for nicotine. It was estimated that approximately 60% of the sites are in the low affinity conformation in the absence of ligand. However, the evidence suggests that nicotine binding can facilitate a shift in the conformational equilibrium, favoring the high affinity state.