Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors

Background: The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is blocked by ketamine, and this action likely contributes to ketamine's anesthetic and analgesic properties. Previous studies suggest that ketamine occludes the open channel by binding to a site located within the channel pore. This hypothesis was examined by investigating the effects of ketamine on single-channel currents from NMDA receptors. Methods: The cell-attached and outside-out configurations of the patch clamp technique were used to study NMDA-activated currents recorded from cultured mouse hippocampal neurons. Results: In cell-attached patches, NMDA evoked currents that had an apparent mean open time (tau(o)) of 3.26 ms. The probability of at least one channel being open (Po') was 0.058. The addition of ketamine (0.1 mu M or 1 mu M) to the pipette solution decreased Po' to 53% and 24% of control values, respectively. At 1 mu M ketamine, this reduction was due to a decrease in both the frequency of channel opening and the mean open time (44% and 68% of control values, respectively). Ketamine did not influence channel conductance and no new components were required to fit the open- or closed-duration distributions. Ketamine (50 mu M), applied outside the recording pipette, reduced the opening frequency of channels recorded In the cell-attached configuration. This observation suggests that ketamine gained access to a binding site by diffusing across the hydrophobic cell membrane. In outside-out patches, ketamine potency was lower than that observed in cell-attached patches: 1 mu M and 10 mu M ketamine reduced Po' to 63% and 34% of control values, respectively, and this reduction was due primarily to a decrease in the frequency of channel opening with little change in mean open time. Conclusions: These observations are consistent with a model whereby ketamine inhibits the NMDA receptor by two distinct mechanisms: (1) Ketamine blocks the open channel and thereby reduces channel mean open time, and (2) ketamine decreases the frequency of channel opening by an allosteric mechanism.