Single-channel properties of glycine receptors of juvenile rat spinal motoneurones in vitro

Abstract

An essential step in understanding fast synaptic transmission is to establish the activation mechanism of synaptic receptors. The purpose of this work was to extend our detailed single-channel kinetic characterization of alpha1beta glycine channels from rat recombinant receptors to native channels from juvenile (postnatal day 12-16) rat spinal cord slices. In cell-attached patches from ventral horn neurones, 1 mM glycine elicited clusters of channel openings to a single conductance level (41 +/- 1 pS, n = 12). This is similar to that of recombinant heteromers. However, fewer than 1 in 100 cell-attached patches from spinal neurones contained glycine channels. Outside-out patches gave a much higher success rate, but glycine channels recorded in this configuration appeared different, in that clusters opened to three conductance levels (28 +/- 2, 38 +/- 1 and 46 +/- 1 pS, n = 7, one level per cluster, all levels being detected in each patch). Furthermore, open period properties were different for the different conductances. As a consequence of this, the only recordings suitable for kinetic analysis were the cell-attached ones. Low channel density precluded recording at glycine concentrations other than 1 mM, but the 1 mm data allowed us to estimate the fully bound gating constants by global model fitting of the 'flip' mechanism of Burzomato and co-workers. Our results suggest that glycine receptors on ventral horn neurones in the juvenile rat are heteromers and have fast gating, similar to that of recombinant alpha1beta receptors.

Figure 1. Properties of glycine single channels recorded in the cell-attached configuration from rat spinal motoneurones in vitro

The traces in A show clusters of single-channel openings evoked by 1 mm glycine. The beginning of each cluster (indicated by the bar) is shown on an expanded time scale below each cluster. B shows the distribution of fitted amplitudes obtained in this patch at a transmembrane holding potential of 0 mV. Only openings longer than two filter rise times are included, and the distribution is fitted with a single Gaussian component. Means from such fits, for openings recorded at different transmembrane potentials, give the current–voltage plot shown for this patch in C. Data in the current–voltage plots were fitted to straight lines in order to estimate the (slope) conductance value for each patch. A summary of these results is shown in D.

Figure 2. The dwell time distributions of high-resolution recordings from native channels are similar to those obtained from recombinant α1β channels

The top row of graphs shows the open period (A) and the shut time (B) distributions for native GlyRs for a recording with optimal (30 μs) time resolution. Note the similarity with the data from recombinant α1β receptors expressed in HEK293 cells (C and D, data from Burzomato et al. 2004) recorded at the same resolution. There is good agreement between the values for the time constants for open periods and for the fastest component in the shut time distribution. E and F show distributions from a native patch with a less favourable signal-to-noise ratio, in which only events longer than 140 μs were resolved. Note the differences in the open and shut time distributions (E and F). These differences are only apparent and result from the distortion introduced by a greater number of missed events. Imposition of the same resolution on the patch shown in the top row produces distributions similar to those observed in the low-resolution native patch (details in the text).

Figure 3. Onset of channel activity during glycine application to an outside-out patch and block by strychnine: no channel openings were detected in this patch before glycine was applied

The start of glycine superfusion (indicated by the arrow) is accompanied by an increase in noise (agonist is superfused at a faster rate than control medium) and by a baseline drift (because of a change in bath level). Glycine reaches the patch after a short delay because of the distance between the inflow and the patch, which is held as close as possible to the surface in order to reduce noise. At first, several channels open simultaneously (line 3), but the onset of desensitization allows single clusters to be detected (line 4). Here, a low-amplitude cluster occurs on top of a high-amplitude one (double event on line 5, all taken from a continuous trace). Strychnine (1 μm) was applied at the end of the experiment and abolished glycinergic activity within 1 min from the onset of application (not shown). The slight increase in baseline noise results from progressive deterioration of the seal during the recording.

Figure 4. Glycine channels open to several conductance levels in outside-out patches

The traces in A (top row) show clusters of openings elicited by 1 mm glycine in the same patch. Three conductance levels are present but each cluster of openings only visits one level. The traces in the second row are expanded from the beginning of each cluster (bars). Inspection of these traces clearly shows that openings to the two lower levels have higher open channel noise and shorter open times. B and C show the distribution of amplitudes for the openings from the same patch. In B, the fitted amplitudes are shown (each opening is one event), whereas C is an open-point histogram, and effectively weights the contribution of each opening by its duration. The greater predominance of the largest conductance in C is due to the longer duration of its openings. D summarizes our findings across all seven patches. For each patch, the symbols show the values for each conductance obtained from the Gaussian fits to the fitted amplitude distributions (all patches were held at −100 mV). The bar chart on the right displays the overall proportion of clusters (out of the total of 237) that fell in each of the conductance levels.

Figure 5. In outside-out patches, GlyR openings to the largest conductance are longer than those to lower conductance levels

A and B show the dwell time distributions for spinal GlyRs recorded in the outside-out configuration and constructed including all conductance levels. C and D show conditional open period distributions, plotted for openings to the largest conductance (greater than 4 pA at −100 mV, D) or to the two smaller conductances (less than 4 pA, C). In D, the dashed line shows for reference the exponential fitted to the distribution in C (and vice versa), scaled to contain the same number of events. The longer duration of the larger openings is very apparent.

Figure 6. Estimation of the fully liganded gating rates for GlyRs recorded in the cell-attached configuration from spinal neurones in vitro

Data from the two best high-resolution recordings were fitted by maximum likelihood to the mechanism shown in D (the ‘flip’ mechanism of Burzomato et al. 2004). Since our native data were obtained at a single, saturating glycine concentration, only the fully bound gating rates (black in the mechanism) were estimated in the fit. Rate constants connecting the other states (grey) were constrained to the values estimated for recombinant α1β heteromeric GlyRs by Burzomato et al. (2004). A–C show that the values of rate constants optimized in this process describe the data adequately, in that they predict distributions of apparent open periods (A) and shut times (B) similar to the observed data (shown as histograms; note that the fit is to the actual sequence of openings and shuttings after idealization, not to the distributions shown here). Dashed lines show the distributions predicted for infinite resolution (i.e. no missed events). Note the profound effect of missing the very common short shut times on the apparent open time distribution. In the case of ‘infinite’ resolution, this would have a main exponential component with a time constant of 345 μs and a relative area of 99.3%. The mechanism and its fitted rates also adequately describe the shape of the correlation between mean open periods adjacent to gaps in the specified range (shown in C). Here, observations are shown as diamonds connected by dashed lines and the values calculated from the mechanism as filled circles connected by a solid line.