Kinetic and structural determinants for GABA-A receptor potentiation by neuroactive steroids

Abstract

Endogenous neurosteroids and synthetic neuroactive steroid analogs are among the most potent and efficacious potentiators of the mammalian GABA-A receptor. The compounds interact with one or more sites on the receptor leading to an increase in the channel open probability through a set of changes in the open and closed time distributions. The endogenous neurosteroid allopregnanolone potentiates the α1β2γ2L GABA-A receptor by enhancing the mean duration and prevalence of the longest-lived open time component and by reducing the prevalence of the longest-lived intracluster closed time component. Thus the channel mean open time is increased and the mean closed time duration is decreased, resulting in potentiation of channel function. Some of the other previously characterized neurosteroids and steroid analogs act through similar mechanisms while others affect a subset of these parameters. The steroids modulate the GABA-A receptor through interactions with the membrane-spanning region of the receptor. However, the number of binding sites that mediate the actions of steroids is unclear. We discuss data supporting the notions of a single site vs. multiple sites mediating the potentiating actions of steroids.

Keywords: Receptor; channel; kinetics.; patch clamp.

Fig. (1)

Potentiation of the α1β2γ2L GABA-A receptor by allopregnanolone. (A) Whole-cell recordings from a HEK cell expressing wild-type α1β2γ2L GABA-A receptors. The cell was exposed to 5 µM GABA in the absence and presence of 1 µM allopregnanolone. 5 µM GABA corresponds to approximately EC20 in the activation concentration-effect curve. 1 µM allopregnanolone corresponds to a maximally potentiating concentration. (B) Sample single-channel currents from cell-attached patches exposed to 50 µM GABA (top trace) or GABA + 1 µM allopregnanolone (bottom trace). Openings are shown as downward deflections. The open and closed time histograms from the respective patches are shown next to the data traces. Only single-channel clusters, i.e., data resulting from the activation of a single receptorchannel, were used in the analysis. Allopregnanolone potentiates the α1β2γ2L GABA-A receptor by enhancing the mean duration and prevalence of the longest of the three open time components (red line in open time histograms) and reducing the prevalence of the longest of the three intracluster closed time components (red line in closed time histograms). For GABA, the open times were 0.11 ms (21 %), 2.8 ms (75 %) and 8 ms (4 %), and the closed times were 0.14 ms (64 %), 1 ms (13 %) and 12 ms (22 %). For GABA + allopregnanolone, the open times were 0.39 ms (32 %), 2.8 ms (32 %) and 12 ms (35 %), and the closed times were 0.12 ms (83 %), 1 ms (14 %) and 10 ms (3 %). The data shown in the figure have not been published previously.

Fig. (2)

PTotal potentiation by steroids is dominated by the effect on the prevalence of long intracluster closed times. The curves show total potentiation (black line) and potentiation under conditions where only the duration (green) or prevalence of long openings (blue), or the prevalence of long closed times (red) is affected by the steroid. The calculations were carried out based on the Po and effective opening rate data from the rat α1β2γ2L receptor expressed in HEK cells [33]. The dashed line represents control response. The steroid effect was calculated in the presence of EC20 GABA, and the extent of steroid effect on each of the kinetic parameters was held similar to that seen in the presence of 1 µM allopregnanolone [24]. The mean duration of OT3 was 7.3 ms in the absence of steroid and 14.1 ms in the presence of a saturating concentration of steroid. The prevalence of OT3 was 13 % in the absence of steroid and 38 % in the presence of a saturating concentration of steroid. The prevalence of CT3 was 27 % in the absence of steroid and 5 % in the presence of a saturating concentration of steroid. The EC₅₀-s for each of the kinetic effects of the steroid were held at 100 nM. The data shown in the figure have not been published previously.

Fig. (3)

Mode switching in the presence of 11-TFMPD3α5αP. (A) A sample single-channel cluster from a cell-attached patch exposed to 50 µM GABA + 50 nM 11-TFMPD3α5αP. For illustrative purposes channel openings are shown as upward deflections. The locations of mode switches between episodes of high and low open probability (Po) were selected by eye. (B) The mean duration of low Po episodes decreases as the steroid concentration is increased. (C) The probability of a receptor being in the low Po mode is reduced at higher steroid concentrations. The parameter was calculated as the mean fraction of time spent in the low Po mode. (D) Structure of the steroid analog 11- TFMPD3α5αP. The data shown in the figure have not been published previously.