Conductance of GABAA channels activated by pentobarbitone in hippocampal neurons from newborn rats

Abstract

Neurons were obtained from the CA1 region of the hippocampus of newborn rats and maintained in culture. Channels were activated by pentobarbitone in cell-attached, inside-out or outside-out patches, normally by applying pentobarbitone in flowing bath solution. Currents were outwardly rectifying and blocked by bicuculline, properties of GABAA channels in these cells. Maximum channel conductance increased as pentobarbitone concentration was increased to 500 microM but conductance then decreased as pentobarbitone concentration was raised further. The best fit of a Hill-type equation to the relationship between maximum channel conductance and pentobarbitone concentration (up to 500 microM) gave an EC50 of 41 microM, a maximum conductance of 36 pS and a Hill coefficient of 1.6. Bicuculline decreased the maximum conductance of the channels activated by pentobarbitone, with an IC50 of 224 microM. Diazepam increased channel conductance, with a maximum effect being obtained with 1 microM diazepam. Diazepam (1 microM) decreased the EC50 of the pentobarbitone effect on channel conductance from 41 microM to 7.2 microM and increased maximum conductance to 72 pS. We conclude that GABAA channel conductance is related to the concentration of the allosteric agonist pentobarbitone.

Figure 1. Single-channel currents activated directly by 100 μm pentobarbitone

Openings are upward in all traces and the dotted lines show the closed level. All-points histograms shown alongside traces were obtained from 20 s segments of data sampled at 10 kHz. A, currents recorded in an outside-out (o/o) patch (pipette potential, +80 mV) before (a) and after (b) exposing the patch to 100 μm pentobarbitone in the bath solution. B, currents recorded in an inside-out (i/o) patch (V_p =−60 mV) before (a) and after (b) exposing the patch to 100 μm pentobarbitone in the bath solution. C, currents recorded in a cell-attached (c/a) patch (V_p =−40 mV) before (a) and after (b) exposing the cell to 100 μm pentobarbitone in the bath solution. Channel activity disappeared when pentobarbitone was washed out of the bath (c).

Figure 2. Current-voltage characteristics of channels activated by pentobarbitone

Relationship between amplitude of single-channel currents (I (pA)) activated by 100 μm pentobarbitone and membrane potential (V (mV) =−V_p) recorded from an inside-out patch. In symmetrical chloride solutions (143 mM, •) the reversal potential was close to 0 mV. When the chloride concentration in the bath solution was changed to 30 mM (▪), the reversal potential shifted from 0 mV to −40 mV.

Figure 3. The effect of pentobarbitone concentration on single-channel currents

Records were all from the same outside-out patch at a pipette potential of +60 mV before (A) and during exposure to a range of pentobarbitone (PB) concentrations (B-F). All-points histograms shown alongside traces were constructed from 10 s segments of data sampled at 10 kHz. G, relationship between pentobarbitone concentration and channel conductance (γ). Average results were obtained from four cell-attached patches (▪), 39 inside-out patches (♦) and 15 outside-out patches (•). Data points represent the mean conductance ± 1 s.e.m. if larger than the symbol. H, all data from all patches were averaged and fitted using the Hill equation (eqn (1), Methods) over the range of 1−500 μm pentobarbitone. The maximum channel conductance was estimated to be 36 pS. The EC₅₀ and the Hill coefficient were 41 μm and 1.6 respectively.

Figure 4. Effect of bicuculline concentration on channels activated by pentobarbitone

Currents were activated in an outside-out patch by 100 μm pentobarbitone (V_p =+80 mV) and the patch was then exposed to varying concentrations of bicuculline (A, no bicuculline; B, C and D, 20 μm, 100 μm and 500 μm, respectively). The corresponding all-points histograms are from 10 s current records sampled at 10 kHz. E, frequency histogram of channel amplitude measured in another outside-out patch exposed to 100 μm pentobarbitone. Ordinate in E and F, number of observations. F, frequency histogram of channel amplitude measured in the same outside-out patch as in E exposed to 100 μm pentobarbitone plus 500 μm bicuculline. G, relationship between bicuculline concentration and average maximum conductance of channels activated by pentobarbitone. Currents were activated with 100 μm pentobarbitone in outside-out patches. Averaged data (n = 6) fitted with a simple binding equation (eqn (2), Methods) gave a bicuculline IC₅₀ of 224 μm. Vertical bars show ± 1 s.e.m.

Figure 5. Effect of diazepam on channels activated by pentobarbitone

Single-channel currents were recorded in an inside-out patch activated by 10 μm pentobarbitone (PB) (V_p =−40 mV) before (A) and after (arrow in B) exposure to 1 μm diazepam (DZ). There was a gradual increase in the single-channel conductance (traces in B and C are continuous). Thirty seconds later (D), two channels were opening in the patch.

Figure 6. Effect of diazepam concentration on maximum conductance of channels activated by pentobarbitone

A, low-conductance channels activated in an outside-out patch (V_p =+60 mV) with 20 μm pentobarbitone (PB). B, single-channel currents recorded in the same patch following exposure to 20 μm pentobarbitone plus 1 μm diazepam (DZ). C, currents recorded following exposure of the patch to 20 μm pentobarbitone + 10 μm diazepam. The corresponding all-points histograms on the right of each trace were obtained from 10 s current records sampled at 10 kHz. D, relationship between diazepam concentration and average maximum conductance of channels activated by 20 μm pentobarbitone measured in 10 patches (7 inside-out and 3 outside-out) in which channels were activated with 20 μm pentobarbitone and then exposed to a range of diazepam concentrations. Average maximum conductance in any patch was normalized to channel conductance measured before exposure to diazepam (γ (DZ/CON)). The line is the best fit of a third order polynomial. Vertical bars show ± 1 s.e.m.

Figure 7. Effect of diazepam on the relationship between pentobarbitone concentration and maximum channel conductance

A, data points show average maximum channel conductance (± 1 s.e.m.) for channels activated by pentobarbitone + 1 μm diazepam (19 patches; 17 inside-out and 2 outside-out). The continuous line is the best fit of eqn (1) to the points. The dashed line shows for comparison the line fitted to the data in the absence of diazepam from Fig. 3H. B, the dashed line has been scaled up to the same maximum as the continuous line to illustrate the change in pentobarbitone EC₅₀.