NMDA receptors mediate excitatory neurotransmission in brain and spinal cord and play a pivotal role in the neurological disease state of chronic pain, which is caused by central sensitization. Bupivacaine is the indicated local anesthetic in caudal, epidural, and spinal anesthesia and is widely used clinically to manage acute and chronic pain. In addition to blocking Na ϩ channels, bupivacaine affects the activity of many other channels, including NMDA receptors. Importantly, bupivacaine inhibits NMDA receptor-mediated synaptic transmission in the dorsal horn of the spinal cord, an area critically involved in central sensitization. We used recombinant NMDA receptors expressed in HEK293 cells and found that increasing concentrations of bupivacaine decreased channel open probability in GluN2 subunit-and pH-independent manner by increasing the mean duration of closures and decreasing the mean duration of openings. Using kinetic modeling of one-channel currents, we attributed the observed current decrease to two main mechanisms: a voltagedependent "foot-in-the-door" pore block and an allosteric gating effect. Further, the inhibition was state-independent because it occurred to the same degree whether the drug was applied before or after glutamate stimulation and was mediated by extracellular and intracellular inhibitory sites, via hydrophilic and hydrophobic pathways. These results predict that clinical doses of bupivacaine would decrease the peak and accelerate the decay of synaptic NMDA receptor currents during normal synaptic transmission. These quantitative predictions inform possible applications of bupivacaine as preventative and therapeutic approaches in chronic pain.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patchclamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechanosensitive channel PIEZO1. This method can provide data regarding the channel's conductance properties and the temporal sequence of openclosed conformations that make up the channel's activation mechanism, thus helping to understand their functions in health and disease. Video LinkThe video component of this article can be found at

N-Methyl-D-aspartate (NMDA) receptors are glutamate-and glycine-gated channels that mediate fast excitatory transmission in the central nervous system and are critical to synaptic development, plasticity, and integration. They have a rich complement of modulatory sites, which represent important pharmacological targets. Ifenprodil is a well tolerated NMDA receptor inhibitor; it is selective for GluN2B-containing receptors and has neuroprotective effects. The mechanism by which ifenprodil inhibits NMDA receptor responses is not fully understood. The inhibition is incomplete and noncompetitive with other known NMDA receptor agonists or modulators, although reciprocal effects have been reported between ifenprodil potency and that of extracellular ligands including glutamate, glycine, zinc, protons, and polyamines. Recent structural studies revealed that ifenprodil binds to a unique site at the interface between the extracellular N termini of GluN1 and GluN2B subunits, supporting the view that interactions with other extracellular modulators are indirect. In this study, we examined how ifenprodil affects the gating reaction of NMDA receptors in conditions designed to minimize actions by contemporaneous ligands. We found that ifenprodil decreased NMDA receptor equilibrium open probability by raising an energetic barrier to activation and also by biasing the receptor toward low open probability gating modes. These results demonstrate intrinsic effects of ifenprodil on NMDA receptor stationary gating kinetics and provide means to anticipate how ifenprodil will affect receptor responses in defined physiological and pathological circumstances.

In response to brief glutamate exposure, NMDA receptors produce excitatory currents that have sub-maximal amplitudes and characteristically slow kinetics. The activation sequence starts when glutamate binds to residues located on the upper lobe of extracellularly located ligand-binding domains (LBDs) and then contacts lower lobe residues to bridge the cleft between the two hinged lobes. This event stabilizes a narrow-cleft LBD conformation and may facilitate subsequent inter-lobe contacts that further stabilize the closed cleft. Agonist efficacy has been traced to the degree of agonist-induced cleft-closure and may also depend on the stability of the closed-cleft conformation. To investigate how cross-cleft contacts contribute to the amplitude and kinetics of NMDA receptor response, we examined the activation reaction of GluN1/GluN2A receptors that had single-residue substitutions at the interface between LBD lobes. We found that side-chain truncations at residues of putative contact between lobes increased glutamate efficacy through independent additive mechanisms in GluN1 and GluN2A subunits. In contrast, removing side-chain charge with isosteric substitutions at the same sites decreased glutamate efficacy. These results support the view that in GluN1/GluN2A receptors’ natural interactions between residues on opposing sides of the ligand-binding cleft encode the stability of the glutamate-bound closed-cleft conformations and limit the degree of cleft closure, thus contributing to the sub-maximal response and emblematically slow NMDA receptor deactivation after brief stimulation.

To investigate the role of the N-terminal do-mains (NTDs) in NMDA receptor signaling we used kinetic analyses of one-channel currents and compared the reaction mechanism of re-combinant wild-type GluN1/GluN2A and GluN1/GluN2B receptors with those observed for NDT-lacking receptors. We found that trun-cated receptors maintained the fundamental gat-ing mechanism characteristic of NMDA recep-tors, which includes a multi-state activation se-quence, desensitization steps, and mode transi-tions. This result establishes that none of the functionally-defined NMDA receptor activation events require the NTD. Notably, receptors that lacked the entire NTD layer retained isoform-specific kinetics. Together with previous reports, these results demonstrate that the entire gating machinery of NMDA receptors resides within a core domain that contains the ligand-binding and the channel-forming transmembrane domains, whereas the NTD and C-terminal layers serve modulatory functions, exclusively