As previously described, it has been firmly established that the activity of the inhibitory GABAaR is modulated by steroids, which exert a fast potentiation of this family of receptors. Comparable studies of native or reconstituted neuronal nAChRs have shown that steroids also modulate the activity of this member of the LGC superfamily. Electro-physiological recordings have illustrated that steroid action leads to a fast and reversible inhibition of the ACh-evoked currents (61-64). Because these studies were carried out on preparations that are known to express different nicotinic subtypes, it can be concluded that steroid inhibition of nAChRs is likely to be an ubiquitous mechanism for this family of LGCs.
Although diverse protocols can be employed to reveal such modulation, one of the most direct experimental paradigms consists of exposing the nAChRs to a steady concentration of ACh and, when the current is stabilized, adding onto it a short pulse of steroid (Fig. 2A). This method offers the advantage of allowing the determination of both the time course of inhibition and recovery, as well as the sensitivity of the receptor for a defined steroid. Studies of chick neuronal nAChRs reconstituted with the major brain subunits a4^2 have demonstrated that these receptors are inhibited by low concentrations of progesterone (PROG) and display an IC50 of about 6 ^M with an empirical Hill coefficient close to unity (64). The low empirical Hill coefficient was reported for all tested compounds and can be interpreted by the absence of cooperativity in the process of steroid inhibition. In addition, these experiments illustrate that the time course of inhibition is fast and reaches a steady state within a few seconds. Full recovery of this inhibition is observed within the same time frame (63,64).
The recent isolation and cloning of the human genes encoding for neuronal nAChRs, which correspond to the a4, |32, and a7 subunits, opens new possibilities for investigating the role of steroids on these receptors (29,65,66). Moreover, it was also shown that these subunits can be successfully reconstituted in Xenopus oocytes or transfected into cell lines (29,55,67). New experiments were thus designed to address the mode of action of steroids on these receptors. PROG effects on human a4^2 nAChR were first investigated using the coapplication protocols (Fig. 2A) and it was found that, as for the chick receptor, steroid exposure induces a fast and reversible inhibition of the ACh-evoked current. Plots of the current inhibition as a function of the logarithm of the PROG concentration reveal that the current is reduced by half for a concentration of PROG of about 4.75 ± 2.3 ^M (n = 4, [ACh] test pulse = 0.3 ^M). As expected from previous studies (64), increasing the agonist concentration by 10-fold induces no significant shift of the apparent inhibition.
Fig. 2. Human neuronal nAChRs are inhibited by progesterone in the \xM range. (A) Typical protocol of steroid application on human a4P2 nAChR reconstituted in Xenopus oocytes. Exposure of the receptor to a low ACh concentration elicits a steady current. Coapplications of progesterone at three different concentrations induce a fast reduction of the ACh-evoked current. Timing of the applications are symbolized by the bars. The cell was held throughout the experiment at -100 mV. (B) Dose-response inhibition of the human a4P2 nAChR for two ACh concentrations. Currents measured as in (A) are plotted as a function of the progesterone concentration. Continuous thick lines are best fits obtained for an allosteric model with respective values L = 32, Ka = 0.041 106 M, Kb = 0.394 106 M, n = 2 and KJ = 3.9 106 M, Kb = 0.35 10 M, m = 2 (Eq. 1 in Fig. 5). Thin dashed lines correspond to prediction made for a competitive inhibitor (81). (C) ACh dose-response of the human a7 nAChR expressed in a Xenopus oocytes. (D) Progesterone dose-response inhibition curve determined using prepulses. The experimental paradigm is illustrated by the traces presented in the inset. Peak inward currents are plotted as a function of the logarithm of the progesterone concentration. The thick continuous curve is the best fit obtained for an negative allosteric inhibitor using five equivalent binding sites (81). The dashed line was obtained using the same equation and same parameters L = 106, Ka = 3 106 M, Kb = 28.3 106 M, n = 5 and KJ = 2.7 106 M, Kb' = 0.024 106 M, m = 5 (Eq. 1 in Fig. 5) but with only two binding sites (m = 2). All data were recorded at -100 mV in Ba2 -containing medium to minimize contamination by calcium-activated chloride currents (95).
Given the very fast desensitization of receptor reconstituted with the a7 subunit (29,51,55), the experimental protocol employed for the a4^2 nAChR cannot be adapted to determine a possible effect of the steroids on the a7 homomeric nAChR. The experimental design that was retained to explore steroid, effects on this class of receptor is to apply, for a brief time, a prepulse of steroid, which is then followed by a test pulse of ACh. Experiments carried out under these conditions reveal that PROG also inhibits the ACh-evoked current of the human a7 receptor with a half-inhibition concentration at about 12 ^M. As distinct from the a4^2 nAchR, the a7 nAChR exhibits a steeper PROG dose-response inhibition curve and ACh-evoked currents are more strongly suppressed by 100 ^M progesterone. These receptor subtypes differences cannot be attributed to the specific experimental protocols used.
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