Kinetic mechanism of NMDA receptor inhibition by the ketamine metabolite 2R,6R-hydroxynorketamine.

Abstract

2R,6R-hydroxynorketamine (2R,6R-HNK) is a metabolite of the fast-acting dissociative anesthetic ketamine (KET). It is currently in clinical trials for the treatment of major depressive disorder due to its strong safety profile at larger doses. The endogenous therapeutic target of 2R,6R-HNK is unknown. We reported recently that like the parent compound ketamine, 2R,6R-HNK reduces NMDA receptor currents with an open-channel block mechanism which is voltage-dependent, pH-dependent, and competitive with Mg2+, and has ∼100-fold lower efficacy (IC50, 46 µM) than KET (IC50, 0.5 µM) at pH 7.2 and −70 mV. We used electrophysiology, kinetic analyses, and kinetic modeling to examine the effect of 2R,6R-HNK on the reaction mechanism of NMDA receptors. We recorded on-cell single-channel currents from HEK293 cells expressing GluN1/GluN2A receptors (and GFP) with pipettes containing (in mM): 150 NaCl, 2.5 KCl, 1 EDTA, 10 HEPES, pH 7.2 (NaOH), agonists glutamate (1) and glycine (0.1), and applied +100 mV (membrane potential ∼−120 mV). Relative to activity recorded in the absence of 2R,6R-HNK (control, n=7), activity recorded with 50 µM 2R,6R-HNK (n=7) displayed a lower open probability (Po), shorter openings (MOT), and longer closed durations (MCT). Fitting these data with state models allowed us to optimize rate constants for the kinetic model. In the future, we will test these models by simulating macroscopic responses and recording whole cell currents in similar conditions.