Alpha7 Nicotinic Receptors: Intrinsic Kinetics and Modulation by PNU 120596

Abstract

We investigated the kinetics of choline evoked currents, and the effect of the positive modulator PNU 120596 on alpha7 nicotinic acetylcholine receptors expressed by GH4C1 cells (obtained from Siena Biotech SpA) in whole-cell and outside-out patch-clamp experiments. using a theta-tube system for rapid application of agonists, we measured the dependence of current kinetics on the solution exchange rate. By extrapolation and by kinetic modeling we estimated the intrinsic kinetics of the receptor: what would be the amplitude and kinetics of choline-evoked currents at instantaneous agonist application. In the presence of PNU 120596 the single channel mean open time is drastically prolonged, this allowed us to determine the ratio of simultaneously open channels using nonstationary fluctuation analysis. By determining the approximate number of channels in a patch, we could determine the peak open probability of 10 mM choline-evoked currents in the absence of the modulator (0.0333 ± 0.0056), as well as the open probability in the presence of 10 mM choline and 10 μM PNU 120596 (0.632 ± 0.065). We performed kinetic experiments to determine the affinity of PNU 120596 to three different conformational states of the receptor: resting state, desensitized state and a slowly developing second desensitized state. We found that PNU 120596 was ineffective at both the resting and the slow desensitized states, while it bound to the desensitised state and re-activated the receptors. We investigated the nature of cooperativity between the agonist and the modulator. We found that while the agonist increases the apparent affinity of the modulator only by inducing desensitized conformation (which is preferred by the modulator), the modulator induces a true increase of agonist affinity probably by allosterically affecting the conformation of the agonist binding site itself.