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Comparative Study
. 1998 Jul 1;510 ( Pt 1)(Pt 1):1-18.
doi: 10.1111/j.1469-7793.1998.001bz.x.

Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors

D J Wyllie  1 , P Béhé, D Colquhoun
Affiliations
Comparative Study

Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors

D J Wyllie et al. J Physiol. .

Erratum in

  • J Physiol (Lond) 1998 Nov 1;512(Pt 3):939

Abstract

1. We have expressed recombinant NR1a/NR2A and NR1a/NR2D N-methyl-D-aspartate (NMDA) receptor channels in Xenopus oocytes and made recordings of single-channel and macroscopic currents in outside-out membrane patches. For each receptor type we measured (a) the individual single-channel activations evoked by low glutamate concentrations in steady-state recordings, and (b) the macroscopic responses elicited by brief concentration jumps with high agonist concentrations, and we explore the relationship between these two sorts of observation. 2. Low concentration (5-100 nM) steady-state recordings of NR1a/NR2A and NR1a/NR2D single-channel activity generated shut-time distributions that were best fitted with a mixture of five and six exponential components, respectively. Individual activations of either receptor type were resolved as bursts of openings, which we refer to as 'super-clusters'. 3. During a single activation, NR1a/NR2A receptors were open for 36 % of the time, but NR1a/NR2D receptors were open for only 4 % of the time. For both, distributions of super-cluster durations were best fitted with a mixture of six exponential components. Their overall mean durations were 35.8 and 1602 ms, respectively. 4. Steady-state super-clusters were aligned on their first openings and averaged. The average was well fitted by a sum of exponentials with time constants taken from fits to super-cluster length distributions. It is shown that this is what would be expected for a channel that shows simple Markovian behaviour. 5. The current through NR1a/NR2A channels following a concentration jump from zero to 1 mM glutamate for 1 ms was well fitted by three exponential components with time constants of 13 ms (rising phase), 70 ms and 350 ms (decaying phase). Similar concentration jumps on NR1a/NR2D channels were well fitted by two exponentials with means of 45 ms (rising phase) and 4408 ms (decaying phase) components. During prolonged exposure to glutamate, NR1a/NR2A channels desensitized with a time constant of 649 ms, while NR1a/NR2D channels exhibited no apparent desensitization. 6. We show that under certain conditions, the time constants for the macroscopic jump response should be the same as those for the distribution of super-cluster lengths, though the resolution of the latter is so much greater that it cannot be expected that all the components will be resolvable in a macroscopic current. Good agreement was found for jumps on NR1a/NR2D receptors, and for some jump experiments on NR1a/NR2A. However, the latter were rather variable and some were slower than predicted. Slow decays were associated with patches that had large currents.

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Figures

Figure 1
Figure 1. Steady-state activations of NR1a/NR2A and NR1a/NR2D channels activated by low concentrations of glutamate
A, a continuous 25 s recording of NR1a/NR2A steady-state channel activity evoked by 100 nM glutamate (+ 20 μM glycine). Activations of the channel are seen to occur in runs or trains of openings that we term ‘super-clusters’. B, a continuous 25 s recording of NR1a/NR2D channel activity evoked by 30 nM glutamate (+ 20 μM glycine). It is clear that NR1a/NR2D super-clusters can last for several seconds, but the probability of the channel being open within an activation is lower than that seen for NR1a/NR2A activations. In each case, the horizontal bars above each series of openings indicates the time between the first opening and last closing of the activation.
Figure 2
Figure 2. Shut-time distributions for NR1a/NR2A and NR1a/NR2D channels
A, shut-time distribution for glutamate-activated NR1a/NR2A channels (100 nM + 20 μM glycine, same experiment as shown in Fig. 1A) fitted with a mixture of five exponentials with means (and areas) of 30 μs (40 %), 732 μs (26 %), 8.31 ms (22 %), 23.9 ms (6 %) and 3211 ms (6 %). The fit predicts 4302 events and 2960 were included in the distribution, which has an overall mean of 183 ms. B, shut-time distribution for NR1a/NR2D channels activated by glutamate (30 nM + 20 μM glycine, same experiment as illustrated in Fig. 1B). The distribution is fitted with a mixture of six exponentials with means of 75 μs (30 %), 414 μs (14 %), 7.02 ms (11 %), 43.3 ms (24 %), 294 ms (18 %) and 4737 ms (3 %); 4568 events were predicted by the fit and 3734 events were included. The overall mean of the distribution is 225 ms.
Figure 3
Figure 3. Super-cluster distributions for NR1a/NR2A and NR1a/NR2D channels
A, distribution of all NR1a/NR2A super-clusters (eight experiments were pooled). The distribution is fitted with a mixture of six exponentials with means of 42 μs (39 %), 0.380 ms (8 %), 1.88 ms (8 %), 4.08 ms (14 %), 40.6 ms (17 %) and 201 ms (14 %). 1403 events were included in the fit while 1875 events were predicted. The overall mean of the distribution is 35.8 ms. B, distribution of all NR1a/NR2D super-clusters (four experiments were pooled). Again the distribution is fitted with six exponentials with means of 71 μs (8 %), 1.03 ms (18 %), 4.71 ms (14 %), 65.6 ms (6 %), 1405 ms (31 %) and 5174 ms (23 %). 647 events were included in the fit and 689 events were predicted. The overall mean super-cluster length predicted by the fit is 1602 ms.
Figure 4
Figure 4. Alignment of NR1a/NR2A super-clusters
A, examples of five NR1a/NR2A super-clusters from the experiment illustrated in Figs 1A and 2A. The tcrit in this experiment was 88 ms. Note the example of a ‘high Popen’ super-cluster (upper trace) and a super-cluster composed of only two apparent openings (middle trace). B, current obtained from the alignment and averaging of 1403 NR1a/NR2A super-clusters. The white dashed line shows the fit of these data with a sum of exponential components with time constants fixed at the values from the super-cluster distribution shown in Fig. 3A. The peak of this trace is truncated in this panel to allow clearer illustration of the slow decay of the current. C, expanded time-base illustration of the peak current to show the initial decay of the current fitted again with time constants obtained from the super-cluster length distribution fit shown in Fig. 3A. In B, the time constants are shown together with their respective relative areas.
Figure 5
Figure 5. Alignment of NR1a/NR2D super-clusters
A, examples of NR1a/NR2D super-clusters from an experiment that gave a tcrit of 1019 ms (same experiment as Figs 1B and 2B). Like their NR1a/NR2A counterparts, NR1a/NR2D super-clusters can be comprised of only a few openings (third trace) but in contrast to NR1a/NR2A super-clusters, they can last for many seconds. Each of these traces show 35 s of activity. They also show periods of ‘high Popen’ (bottom trace). The inset below the final super-cluster shows a 200 ms section of one of these ‘high Popen’ periods on an expanded time-base. Overall, however, the Popen of NR1a/NR2D super-clusters is about 10-fold less than NR1a/NR2A super-clusters. B, average of 647 NR1a/NR2D super-clusters fitted with a sum of exponential components where the time constants have been fixed to those obtained from the fit of the distribution shown in Fig. 3B. The peak of this trace is truncated in this panel to allow clearer illustration of the slow decay of the current. This trace is digitally filtered at 50 Hz for illustration.
Figure 6
Figure 6. NR1a/NR2A macroscopic current responses to brief and prolonged exposure to agonist
A, two individual responses obtained from an outside-out patch following a 1 ms pulse of 1 mM glutamate (in the presence of 20 μM glycine). Single-channel currents are clearly visible in these traces. B, the average of fifty such jumps fitted with the sum of three exponential components. C, two individual records obtained following a 4 s application of glutamate (1 mM). The channel activity declines during the prolonged application of agonist. D, average of sixty such sweeps. The decay of the current to a steady-state level is fitted with a single exponential with a time constant of 510 ms. All recordings were made at −100 mV.
Figure 7
Figure 7. NR1a/NR2D macroscopic current responses to brief and prolonged exposure to agonist
A, average of ten jumps recorded from an outside-out patch containing NR1a/NR2D receptor channels following a 1 ms pulse of 1 mM glutamate (+ 20 μM glycine). The off-relaxation of this current is well fitted with the sum of two exponentials, with time constants of 89.8 ms (rising) and 5162 ms (decay). B, average of ten sweeps illustrating that during prolonged agonist exposure, currents mediated by NR1a/NR2D channels are sustained, without visible desensitization. C, the same NR1a/NR2D-mediated current shown in A, but on an expanded time-base to illustrate the slower kinetics of this current when compared with NR1a/NR2A-mediated responses (same trace as shown in Fig. 6B). Currents have been scaled to have similar peaks. All recordings were made at −100 mV.
Figure 8
Figure 8. NR1a/NR2A and NR1a/NR2D macroscopic jumps fitted with time constants obtained from super-cluster distributions
A, ‘average’ NR1a/NR2D macroscopic current obtained following a 1 ms pulse of 1 mM glutamate. Data from five individual jump experiments on NR1a/NR2D receptor channels were pooled and averaged. The dashed white line shows the best least-squares fit of these data by a sum of four exponentials where the time constants were fixed at the values obtained from the super-cluster distribution shown in Fig. 3B. B, plot of the magnitude of the peak current versus the value of the slowest decay time constant for macroscopic jump experiments on outside-out patches containing NR1a/NR2A channels. It appears that patches giving the smallest currents possess the fastest decay time constants. C, an example of a macroscopic current (peak current, ≈10 pA) recorded following a 1 ms pulse of 1 mM glutamate, which was well fitted by a sum of four exponentials with time constants fixed to the values obtained from the fit of NR1a/NR2A super-cluster lengths (Fig. 3A). D, an example of a macroscopic current that had a large peak current (≈ 300 pA) and is fitted poorly by the sum of four exponentials with the same set of time constants as used in C.
Scheme 1
Scheme 1
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