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

1. Recombinant NR1a/NR2A(T671A) N-methyl-D-aspartate (NMDA) receptor-channe ls, which carry a point mutation in the putative glutamate binding site tha t reduces glutamate potency by around 1000-fold, have been expressed in Xen opus laevis oocytes and their single-channel properties examined using patc h-clamp recording techniques. 2. Shut time distributions of channel activity were fitted with a mixture o f five exponential components. The first three components in each distribut ion were considered to occur within a channel activation as they exhibited little or no dependence on agonist concentration. 3. 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 ope nings. Distributions of the lengths of bursts of openings were fitted with a mixture of four exponential components. The longest two components carrie d the majority of the charge transfer in the channel recordings and had mea ns of 7.71 +/- 1.1 and 37.7 +/- 4.3 ms. The overall probability of a channe l being open during a burst was high (mean 0.92 +/- 0.01). 4. Brief concentration jumps (1 ms) of 10 mM glutamate were applied to outs ide-out patches so that a comparison between the macroscopic current relaxa tion and steady-state single-channel activity evoked by glutamate could be made. The decay of such macroscopic currents was fitted with a single expon ential component with a mean of 32.0 +/- 3.53 ms. 5. The good agreement between macroscopic current decay following brief ago nist exposure and the value for the slowest component of the burst length d istribution suggests that the bursts of openings that we identified in stea dy-state recordings represent individual activations of recombinant NR1a/NR 2A(T671A) NMDA receptor-channels. 6. A new way of displaying geometric distributions is suggested, and the ut ility of a modified definition of the 'probability of being open within a b urst' is discussed. 7. The single-channel data that we present in this paper support further th e idea that the point mutation T671A in the NR2A NMDA receptor subunit affe cts mainly the ability of glutamate to remain bound to these channels.