Kinetics of open channel block by penicillin of single GABAA receptor channels from mouse spinal cord neurones in culture.

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1. Reduction by penicillin of single gamma-aminobutyric acidA (GABAA) receptor currents from somata of mouse spinal cord neurones in culture was investigated using the excised outside-out patch-clamp recording technique. 2. GABA (2 microM) alone or with penicillin (100-5000 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage-clamped at -75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were decreased in the presence of penicillin. 3. GABA receptor currents were recorded with at least two conductance states, a more frequent or main-conductance state of about 27 pS and a less frequent sub-conductance state of about 19-20 pS. The conductances of the two states were unchanged in the presence of penicillin. The kinetic properties of the main-conductance state were analysed and are summarized below. 4. Penicillin produced a concentration-dependent reduction of GABA receptor open properties by reduction of average GABA receptor channel open duration and an increase in channel opening frequency. 5. Penicillin shifted frequency histograms of GABA receptor channel open durations to shorter durations in a concentration-dependent manner. Three exponential functions were required to fit best the frequency histograms of open durations, suggesting that the channel had at least three open states. Penicillin produced a concentration-dependent reduction in the time constants obtained from the open duration frequency histograms. 6. Frequency histograms of GABA receptor channel closed durations could be fitted with five to seven exponential functions, suggesting that the channel had multiple closed states. In the presence of increased concentration of penicillin, there was a reduction in the relative frequency of brief gaps and the appearance of new closed time constants. 7. With increased penicillin concentration, GABA receptor channel burst frequency was unchanged, burst durations were increased, the number of openings per burst was increased and the per cent time open within a burst was decreased. 8. The results suggested that penicillin produced simple open channel blockade of the GABA receptor channel. However, the experimental results also suggested that the association with and, perhaps, the dissociation of the blocker from its binding site were dependent upon the kinetic state of the open channel. Penicillin had faster association and slower dissociation rates when the channel was in unstable, brief open kinetic state than when the channel was in a more stable, longer open kinetic state. Possible models for penicillin reduction of single GABA receptor currents were simulated by computer and analysed.(ABSTRACT TRUNCATED AT 400 WORDS)

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