one-channel-cell-attached-patch-clamp-recording

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

University at Buffalo, SUNY

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.

Ifenprodil effects on GluN2B-containing glutamate receptors.

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.

Article	Year
Ifenprodil effects on GluN2B-containing glutamate receptors. Molecular pharmacology\| PubMed ID: 22936815	2012
Role of cross-cleft contacts in NMDA receptor gating. PloS one\| PubMed ID: 24278352	2013

Meaghan A. Paganelli

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