Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture.

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Abstract
1. Barbiturate regulation of the kinetic properties of .gamma.-aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole-cell and excised outside-out patch-clamp recording techniques. 2. GABA (2 .mu.M), GABA (2 .mu.M) plus phenobarbitone (PbB) (500 .mu.M) and GABA (2 .mu.M) plus pentobarbitone (PB) (50 .mu.M), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at -75 mV and the chloride equilibrium potential was 0 mV. GABA receptor channel currents were increased by PhB and PB. 3. Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 16.5 pS. The conductances of the two states were unchanged by the barbiturates. 4. The main conductance state kinetics were analysed. GABA alone or with the barbiturates gated the channel open singly and in groups of openings. 5. The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times. 6. Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (O1, O2, O3). The time constants for the exponential functions (0.9, 2.7 and 7.8 ms, respectively) were unchanged by the barbiturates. The increases in mean open times and the shifts of the open-time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and O2) and to an increase in the relative frequency of occurrence of the longest open state (O3). 7. Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states. None of the time constants nor areas of the exponetial functions were significantly changed by the barbiturates. 8. For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms. For GABA (2 .mu.M), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, suggesting that the channel has three burst states (B1, B2, B3). The B1 burst state was probably a single opening to the O1 open state while the B2 and B3 burst states were probably composed of multiple openings to the O2 and O3 open states. With both barbiturates, the relative frequency of occurrence of the two shortest burst states (B1 and B2) was reduced and that of the longest burst state (B3) was increased. The time constant of the B3 burst state was increased slightly by the barbiturates. 9. We suggest that barbiturates increase GABA receptor channel current by increasing the relative frequency of occurrence of the long (B3) bursts which contain multiple long openings and decreasing the relative proportion of shorter (B1 and B2) bursts which contain shorter openings. This results in an increase in GABA receptor channel apparent mean open time and mean burst duration. Barbiturates do not measurably alter the closing rates of the open states, and therefore, do not alter their mean open times. Rather, barbiturates alter the rate constants which regulate entry into the three open states, such that entry into the longest, stable open state (O3) is favoured over the two shorter and less stable open states (O2 and O2).

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