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, 518 (Pt 1), 55-70

Properties of Single NMDA Receptor Channels in Human Dentate Gyrus Granule Cells

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Properties of Single NMDA Receptor Channels in Human Dentate Gyrus Granule Cells

D N Lieberman et al. J Physiol.

Abstract

1. Cell-attached single-channel recordings of NMDA channels were carried out in human dentate gyrus granule cells acutely dissociated from slices prepared from hippocampi surgically removed for the treatment of temporal lobe epilepsy (TLE). The channels were activated by L-aspartate (250-500 nM) in the presence of saturating glycine (8 microM). 2. The main conductance was 51 +/- 3 pS. In ten of thirty granule cells, clear subconductance states were observed with a mean conductance of 42 +/- 3 pS, representing 8 +/- 2 % of the total openings. 3. The mean open times varied from cell to cell, possibly owing to differences in the epileptogenicity of the tissue of origin. The mean open time was 2.70 +/- 0.95 ms (range, 1.24-4.78 ms). In 87 % of the cells, three exponential components were required to fit the apparent open time distributions. In the remaining neurons, as in control rat granule cells, two exponentials were sufficient. Shut time distributions were fitted by five exponential components. 4. The average numbers of openings in bursts (1.74 +/- 0.09) and clusters (3.06 +/- 0.26) were similar to values obtained in rodents. The mean burst (6.66 +/- 0.9 ms), cluster (20.1 +/- 3.3 ms) and supercluster lengths (116.7 +/- 17.5 ms) were longer than those in control rat granule cells, but approached the values previously reported for TLE (kindled) rats. 5. As in rat NMDA channels, adjacent open and shut intervals appeared to be inversely related to each other, but it was only the relative areas of the three open time constants that changed with adjacent shut time intervals. 6. The long openings of human TLE NMDA channels resembled those produced by calcineurin inhibitors in control rat granule cells. Yet the calcineurin inhibitor FK-506 (500 nM) did not prolong the openings of human channels, consistent with a decreased calcineurin activity in human TLE. 7. Many properties of the human NMDA channels resemble those recorded in rat hippocampal neurons. Both have similar slope conductances, five exponential shut time distributions, complex groupings of openings, and a comparable number of openings per grouping. Other properties of human TLE NMDA channels correspond to those observed in kindling; the openings are considerably long, requiring an additional exponential component to fit their distributions, and inhibition of calcineurin is without effect in prolonging the openings.

Figures

Figure 1
Figure 1
Conductance of single human NMDA channels A, single-channel currents activated by L-aspartate and glycine in a cell-attached patch. Examples of channel openings (mean open time, 4.78 ms) are shown as downward deflections from the closed state (c) to two different conductance levels (o1 and o2). The dotted lines indicate the two current levels identified from the fit to the amplitude distribution shown on the right. The lower traces marked a and b in the left-hand panel are expansions of the corresponding segments marked on the raw traces shown in the upper panel. The vertical calibration bar is the same for both sets of traces. The channel spends less than 10 % of the time in the lower (45 pS) conductance state, while the larger (58 pS) conductance level is occupied over 90 % of the time. The right-hand panel is an all-points amplitude histogram for open events from the same patch, binned according to the time (s) spent in each 0.05 pA bin. Gaussian fits to the open state amplitudes are shown separately and also as a sum. B, relationship between single-channel current amplitudes (I) and patch potential (V). The mean single-channel current was estimated in this cell from Gaussian fits to the amplitude distribution for the main conductance state obtained at each membrane potential. The continuous line gives the least-squares fit, with a 49.6 pS slope conductance (γ) and a reversal potential (Vrev) of 5.4 mV. The error bars represent the s.d. of the fitted Gaussian distributions at each of the patch potentials. The mean open time for this cell is 3.93 ms.
Figure 2
Figure 2
Variability of mean open times across patients and the stability of recordings from a given patch A, cumulative probability distribution of mean apparent open times recorded in 30 granule cells from 10 TLE patients. The sigmoidal shape of the distribution suggests the presence of a single normally distributed population. Note the large variance of open times among different patients, but also within cells obtained from the same patient (e.g. patient F). All patients had marked hippocampal sclerosis upon pathological examination of the resected tissue specimens. B, stability plots of the mean open time and Popen in a cell with a mean open time close to the average of the overall distribution. The mean (±s.d.) of the 9440 openings recorded in this cell over a 15 min period was 2.99 ± 4.98 ms. The mean open times and the values for Popen were calculated over 10 s epochs corresponding to a single bin in the plots. The lines represent 6 point (1 min) running averages.
Figure 3
Figure 3
Distributions of shut times and open times Each cell was chosen because its mean open time falls at three distinct points along the range of the recorded values. The top panels are taken from a cell with a 1.24 ms mean open time, which was the lowest measured. The middle panels are from a cell with a 2.55 ms mean, which is close to the 2.7 ms mean value of the pooled data. The bottom panels are from the cell also shown in Fig. 1A with the longest mean open time measured in our recordings. The panels depict the shut (left) and open (right) dwell-time histograms. The values were log-binned at 9 bins per decade and are shown on a square root abscissa. The time constants and the relative areas for each distribution are indicated in each panel. Note that there are 5 exponential components in each of the shut time distributions. The number of exponential components in the open time distributions is 2 for the channel with the shortest mean opening, but increases to 3 for the other channels.
Figure 4
Figure 4
Voltage dependence of open durations The mean apparent open times are plotted as a function of membrane potential for the openings underlying the larger conductance state events used to generate the current-voltage relationship in the cell shown in Fig. 1B. Because of the exponential distribution of the open times, the large variances have been omitted for clarity from the figure. The decline in mean open time at positive membrane potentials may result in part from the increase in patch noise at these depolarized potentials. Note, however, that there is a steep linear relationship between open time and voltage near resting membrane potentials (-90 to -60 mV).
Figure 5
Figure 5
Distribution of burst and cluster durations The panels depict the burst durations (left), and cluster durations (right) from the same cells shown in Fig. 3. The values were log-binned at 9 bins per decade and are shown on a square root abscissa. The time constants and the relative areas for each distribution are indicated in each panel. The number of exponential components in the burst time distributions is 2 for the channel with the shortest mean opening, but increases to 3 for the other channels. Similarly, the number of exponential components in the distribution of clusters increases from 3 to 4.
Figure 6
Figure 6
Distribution of the number of apparent openings in bursts and clusters The left, middle and right panels in this figure are taken from openings observed in the same cells shown in Figs 3 and 5. A, the distribution of the number of apparent openings per burst is fitted with either one or two geometric components. The fitted geometric components are shown as continuous lines superimposed on the histograms. B, the distribution of the number of apparent openings per cluster are fitted with two geometric components. The mean and relative percentage for each component are indicated for both A and B.
Figure 8
Figure 8
Mean time constants (left) and areas (right) of the exponential components describing conditional open time distributions The apparent open times were taken from distributions II-VI of the adjacent shut time ranges indicated in Fig. 7A. For each range of shut times, three exponential components were fitted to the distributions of adjacent open times. The three open time constants (left) for the fast (□), intermediate (○) and slow (▵) components and their respective areas (right) are plotted against the average values for each of the five ranges of adjacent shut times.
Figure 7
Figure 7
Adjacent dwell-time distributions A, relationship between the mean durations of adjacent open and shut intervals. The graph shows the mean open time for each average adjacent shut time range for the channel shown in Fig. 1A. The openings were separated into groups based on the length of the adjacent closed times. The six ranges used (I, 50-100 μs; II, 100 μs to 1 ms; III, 1-10 ms; IV, 10-100 ms; V, 100 ms to 1 s; VI, 1-10 s) show that long openings occur adjacent to brief closures, while brief openings occur adjacent to long closures. The continuous straight line gives the linear regression (r= 0.91), while the other two lines indicate the 95 % confidence intervals. B, three-dimensional plot demonstrating the relationship between adjacent open and shut intervals. The most frequently observed occurrence is of brief shut times adjacent to long openings. This large number of long openings accounts for the prolonged mean open time. Note the relative absence of long openings flanked by long closed times that separate the superclusters.
Figure 9
Figure 9
Pharmacological inhibition of the Ca2+-calmodulin-dependent protein phosphatase calcineurin by FK-506 fails to prolong NMDA channel openings in human TLE neurons A, raw records of cell-attached channel openings taken from a cell before and after addition of FK-506 to the bath. Scale bars represent 25 ms/5 pA. B, the mean open times and the mean burst durations recorded in the 7 cells are plotted such that each point's abscissa is defined by the value of the dwell time measured during the control period (‘In control’), while the ordinate represents the dwell time obtained in the presence of FK-506 (‘In FK-506′). A linear regression of the form f (x) =ax was fitted to the points, and the values of the regression coefficients are indicated. The values of a are also given (±s.e.m.). Neither is significantly different from 1.0, indicating no change induced by FK-506 (also see Table 1) in mean open times and mean burst durations. The dotted lines correspond to the 95 % confidence intervals of the fits.

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