NEUROSTEROID REGULATION OF GABA(A) RECEPTOR SINGLE-CHANNEL KINETIC-PROPERTIES OF MOUSE SPINAL-CORD NEURONS IN CULTURE

1. Single-channel kinetics of steroid enhancement of single gamma-aminobutyric acid(A) (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside-out patch-clamp recording technique. GABA (2 muM) and GABA (2 muM) plus androsterone (5alpha-androstan-3alpha-ol-17-one, AND, 10 nM-10 muM) or pregnanolone (5beta-pregnan-3alpha-ol-20-one, PRE, 100 nM-10 muM) 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 increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main-conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub-conductance level of about 20 ps. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main-conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main-conductance level. Time constants obtained from the GABA receptor channel open-duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open-duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration-dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 muM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel-closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids. 5. The average durations of GABA receptor channel bursts (groups of openings separated by closures greater than 5 ms) were increased by the steroids. The increased average burst durations were AND and PRE concentration-dependent, but time constants obtained from the GABA receptor channel burst-duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average burst durations was due to a shift in the proportion of bursts with shorter time constants to bursts with longer time constants. The steroids did not alter intrinsic burst properties of the GABA receptor channel, but rather, increased the likelihood of longer bursts which were comprised primarily of longer-duration openings. 6. The steroids also enhanced averaged currents resulting from the subconductance level. Qualitatively similar findings for the regulation of the open durations of the sub-conductance level were found. 7. These results suggest that AND and PRE enhanced GABA receptor current. The steroid enhancement of GABA receptor current was due to an increase in channel-opening frequency and an increase in the probability of opening of longer openings without altering the intrinsic dwell times or open time constants of the GABA receptor main-conductance channel. The mechanism for prolongation of average open and burst durations was similar to that described for barbiturates, but a significant change in channel-opening frequency has not been a prominent effect observed for barbiturates. Although it has been suggested that steroids and barbiturates bind to different sites and their differential effect on opening frequency seems to corroborate this, the mechanism for the observed prolongation of the GABA receptor main-conductance level was similar to that described for barbiturates. This suggests that these steroids and barbiturates may regulate the GABA receptor channel through at least one common effector mechanism.