SINGLE CHANNELS ACTIVATED BY HIGH-CONCENTRATIONS OF GABA IN SUPERIOR CERVICAL-GANGLION NEURONS OF THE RAT

1. Single-channel currents evoked by high concentrations of GABA (10-2000 mu-M) have been analysed to investigate the characteristics of GABA(A) receptor channels in outside-oup patches from rat sympathetic neurones. When high concentrations of GABA were applied to a patch, channel openings occurred in prolonged clusters (3.8 +/- 3.7 s (mean +/- S.D.) at 50 mu-M-GABA) consisting, on average, of 350 apparent openings per cluster. Individual clusters were separated by long silent intervals. 2. Channel openings were to many (often ill-defined) conductance states (range 7-36 pS), but the most frequently observed conductance level was approximately 30 pS, (29.6 +/- 0.34 pS). Only these clusters during which the channel was open to this main state conductance for at least 95% of the cluster open time were used in the analysis of probability of being open. Other less frequently observed conductance levels were 15-18 and 22-23 pS, while levels of 33-36 and 7-9 pS were occasionally, but reliably, observed. 3. Bursts within clusters were defined as a series of openings separated by closed intervals shorter than some critical value, t(c). At 50 mu-M-GABA the mean burst length was 439 +/- 434 ms (+/- S.D., t(c) = 50 ms). 4. The probability of being open, p(o), during bursts within clusters has been analysed as a function of GABA concentration. As expected, increasing the concentration of GABA resulted in an overall increase in p(o). However, for a given agonist concentration there was a wide spread in p(o), far greater than that predicted for a population of identical and independent receptor channels (demonstrated by comparison with simulated channel activity). 5. The wide range of p(o) values at a particular concentration of GABA was not due to inappropriate selection of t(c). At 50 mu-M-GABA the range of p(o) values was similar for t(c) of 20-1000 ms, although the overall mean p(o) became lower (0.64 rather than 0.81). 6. On the basis of simulated channel activity, it appears that most clusters which do not contain multiple openings, arise from the activity of one individual channel. Furthermore, there was no detectable tendency for gaps between bursts to be shorter in the middle of a cluster than at its ends. Therefore it is unlikely that variability in p(o) arose from overlapping activity of two or more channels. 7. The values for p(o), mean open time, and mean shut time for bursts within the same cluster, and between different clusters, were compared by a randomization test. This was to determine whether the wide variability in p(o) could plausibly occur if all the open and closed intervals came from the same population. Values of p(o) were found to differ not only between one channel and another (i.e. between clusters), but also for the same channel at different times (i.e. between bursts within a cluster). 8. The heterogeneity of GABA receptor channel properties results, at least in part, from one type of receptor channel that changes its activity with time. However, heterogeneity resulting from the presence of several subtypes of GABA receptor channels cannot be excluded.