Desformylflustrabromine Potentiates High-Sensitivity α4β2 Receptors by Increasing Channel Opening Rate

Abstract

Desformylflustrabromine (dFBr) is a positive allosteric modulator of α4β2 neuronal nicotinic receptors (nAChRs). Investigations of mechanisms of nAChR modulators may facilitate the development of new therapeutic drugs that offset or correct errors in signaling associated with Alzheimer's disease, nicotine addiction, and other disorders of cholinergic signaling. We made steady-state, cell-attached patch-clamp recordings of 20 pS, high-sensitivity (HS) human α4β2 receptors stably expressed in HEK-293 cells. We developed 6-state gating models from data acquired with 1 μM ACh with and without 1 μM dFBr. Models included two sequential ligand-binding reactions leading to a desensitized state (C1↔C2↔C3↔D), and mono- and diliganded openings (O1 and O2) from C2 and C3, respectively. Rate constants governing transitions between connected states were obtained by fitting single-channel data to the model (constraints: C1→C2 = 2⋅C2→C3 and C3→C2=2⋅C2→C1) using a maximum likelihood method implemented in QUB. dFBr increased binding and unbinding rates for ACh (2.6- and 2-fold, respectively), the opening rate (C3→O2) for diliganded receptors (4.8-fold), and the (microscopic) desensitization rate (5.3-fold). Simulations of macroscopic responses to concentration-jumps (range: 0.1 μM - 100 μM) revealed that the enhanced rate into O2 yielded approximately 2 to 3-fold potentiation of peak currents (maximal Popen in 100 μM ACh = 0.29) despite the fact that potentiation was blunted by the enhanced rate of desensitization. Moreover, at ACh concentrations above 10 μM, our data are consistent with the interpretation that potentiation of HS receptor responses to dFBr arises not from modulation of ligand binding rates, but from an increase in the rate of channel opening, corresponding to a decrease in the latency to first opening and a reduction in the time to peak current (ACh 23 ms; dFBr 8 ms) of simulated macroscopic responses.