Single-channel analysis of an NMDA receptor possessing a mutation in the region of the glutamate binding site.

Recombinant NR1a/NR2A(T671A) N-methyl-D-aspartate (NMDA) receptor-channels, which carry a point mutation in the putative glutamate binding site that reduces glutamate potency by around 1000-fold, have been expressed in Xenopus laevis oocytes and their single-channel properties examined using patch-clamp recording techniques. Shut time distributions of channel activity were fitted with a mixture of five exponential components. The first three components in each distribution were considered to occur within a channel activation as they exhibited little or no dependence on agonist concentration. Bursts of single-channel openings were defined by a critical gap length with a mean of 5.65 +/- 0.37 ms. Shut intervals with a duration longer than this value were considered to occur between separate bursts of channel openings. Distributions of the lengths of bursts of openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer in the channel recordings and had means of 7.71 +/- 1.1 and 37.7 +/- 4.3 ms. The overall probability of a channel being open during a burst was high (mean 0.92 +/- 0.01). Brief concentration jumps (1 ms) of 10 mM glutamate were applied to outside-out patches so that a comparison between the macroscopic current relaxation and steady-state single-channel activity evoked by glutamate could be made. The decay of such macroscopic currents was fitted with a single exponential component with a mean of 32.0 +/- 3.53 ms. The good agreement between macroscopic current decay following brief agonist exposure and the value for the slowest component of the burst length distribution suggests that the bursts of openings that we identified in steady-state recordings represent individual activations of recombinant NR1a/NR2A(T671A) NMDA receptor-channels. A new way of displaying geometric distributions is suggested, and the utility of a modified definition of the 'probability of being open within a burst' is discussed. The single-channel data that we present in this paper support further the idea that the point mutation T671A in the NR2A NMDA receptor subunit affects mainly the ability of glutamate to remain bound to these channels.

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors.

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).

The effect of the oxytocin antagonists, CAP 476 and F327, on calcium mobilisation in single cultured human myometrial cells.

OBJECTIVE: To investigate the mechanism of action of the oxytocin (OT) antagonists, CAP 476 and F327. DESIGN: A prospective descriptional study. SUBJECTS: Women undergoing caesarean section at term or hysterectomy. INTERVENTIONS: Myometrial cells were cultured from uterine biopsies. MAIN OUTCOME MEASURES: Intracellular calcium ([Ca2+]i), determined in single cells. RESULTS: Application of OT caused a transient increase in [Ca2+]i. CAP 476 abolished and F327 reduced the response to OT but neither reduced the [Ca2+]i transient induced by cell depolarisation with 120 mmol K+. CAP 476 did not reduce transients caused by prostaglandin E2. F327 reduced the frequency of repetitive [Ca2+]i transients occurring during continuous application of OT. CONCLUSIONS: The results demonstrate that the antagonists reduce the effect of OT and that their action is relatively specific. Their mechanism of action as clinical tocolytic agents is discussed.