Ming-De Wang

Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, the adrenals, and the gonads and have potent and selective effects on the GABAA-receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, and testosterone are positive modulators of GABA_A-receptor in a non-genomic manner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α, 17α-diol (Adiol), and 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl^- currents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and picrotoxin binding sites. 3α5α-P and 3α5α-THDOC potentiate synaptic GABA_A-receptor function and activate δ-subunit containing extrasynaptic receptors that mediate tonic currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are GABA_A-receptor antagonists and induce activation-dependent inhibition of the receptor. The activities of neurosteroid are dependent on brain regions and types of neurons. In addition to the slow genomic action of the parent steroids, the non-genomic, and rapid actions of neurosteroids play a significant role in the GABA_A-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABA_A-receptor, mood changes, and cognitive functions.

Summary: 1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_Areceptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits inXenopusoocytes using the two‐electrode voltage‐clamp technique.2. Both Hα₁β₃δ and Hα₄β₃γ_2Sreceptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2Sreceptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively).3. Substituting the Hα₄subunit with Rα₄conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀and increasedI_max. When the Hα₄subunit was combined with the Rβ₃and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor.4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2Scombinations may contribute to functions of extrasynaptic GABA_Areceptors. The presence of the Rα₄subunit at recombinant GABA_Areceptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.

SUMMARY: In the present study, we compared the pharmacology, particularly neurosteroid modulation of the GABA_A receptor, between human and rat a₁b₂g_2L GABA_A receptors and between human receptors containing the long (L) and short (S) forms of the g₂‐subunit. We observed that maximum responses to GABA were significantly higher with the human a₁b₂g_2L receptor compared with the rat receptor. In terms of neurosteroid modulation, increases in the EC₁₅ response to GABA induced by 3a‐OH‐5b‐pregnan‐20‐one (3a5bP), 5a‐androstane‐3a,17b‐diol (3a5aADL) and 5a‐pregnane‐3a,20b‐diol (3a5a‐diol) were significantly greater for the rat compared with the human receptor. Responses to 30 mmol/L GABA were inhibited by 3b‐OH‐5a‐pregnan‐20‐one (UC1010) and 5b‐pregnan‐3b,20(R)‐diol (UC1020) to a greater degree for human and rat receptors, respectively. Responses to GABA + 3a5aTHDOC were inhibited by 5a‐pregnan‐3b,20(S)‐diol (UC1019) and pregnenolone sulphate to a greater degree for human and rat receptors, respectively. The GABA dose–response curves for human a₁b₂g_2S and a₁b₂g_2L receptors were identical. However, the maximum GABA‐evoked current, the direct gating effect of pentobarbital and the allosteric potentiation of the GABA EC₁₅ response by 3a5aTHDOC and 3a5bP were significantly higher with a₁b₂g_2S than a₁b₂g_2L receptors. Inhibition of the response to 30 mmol/L GABA by UC1010 and UC1020 was greater for a₁b₂g_2L and a₁b₂g_2S receptors, respectively. Inhibition of responses to 3a5aTHDOC + GABA by UC1019 and UC1010 was significantly higher for a₁b₂g_2L receptors. In conclusion, the site of activation by GABA and neurosteroid modulation differ between human and rat a₁b₂g_2L receptors, as well as between human receptors containing the L and S splice variants of the g₂‐subunit.

Aims:  We compared the antagonistic effects of state‐dependent gamma‐aminobutyric acid A (GABA_A) receptor blockers picrotoxin, Zn²⁺ and pregnenolone sulphate (PS) on GABA‐ and pentobarbital‐activated currents in recombinant rat GABA_A receptors in Xenopus oocytes.Methods:  Experiments were performed with wild type ratα1β2γ2L and α1β2 receptors, mutants α1V256Sβ2γ2L and α1β2A252Sγ2L receptors by the two‐electrode voltage‐clamp technique.Results:  In contrast to respective 3840‐ and 56‐fold increases in Zn²⁺ potencies to inhibit GABA‐ and pentobarbital‐activated currents in the α1β2 receptor, the corresponding potencies of PS remained unchanged in comparison with the α1β2γ2L receptor. A homologous mutation of the residue at 2′ position closest to the cytoplasmic end of the M₂ helix to serine on both α1 and β2 subunit, α1V256S and β2A252S, abolished the inhibition of GABA_A receptor by PS. In comparison with the wild type α1β2γ2L receptor, mutants α1V256Sβ2γ2L and α1β2A252Sγ2L receptors did not affect the Zn²⁺ inhibition. Furthermore, a significant increase in GABA potency was observed in the mutant α1V256Sβ2γ2L receptor (P < 0.05), but not the mutant α1β2A252Sγ2L receptor compared with the wild type receptor.Conclusions:  Pregnenolone sulphate was a γ2‐subunit independent inhibitor in the GABA_A receptor, whereas the Zn²⁺ antagonism was profoundly influenced by the γ2‐subunit. It is likely that the 2′ residue closest to the N‐terminus of the protein at M₂ helix on both α1 and β2 subunit are critical to the inhibitory actions of PS and the function of Cl⁻ channels. These results are consistent with the hypothesis that PS behaves as a Cl⁻ channel blocker that does not share with Zn²⁺, the coincident channel property in the GABA_A receptors.

Aim: The roles of α‐subunits on the gamma‐aminobutyric acid (GABA)‐site antagonism and pentobarbital actions were examined in rat recombinant GABA_A receptors in Xenopus oocytes. Methods: Experiments were performed with binary and ternary GABA_A receptors containing α1‐, α4‐ or α5‐subunit by the two‐electrode voltage‐clamp technique. Results: The potency of GABA was significantly higher in the α1β2, α4β2 and α5β2 receptors compared with the α1β2γ2L, α4β2γ2L and α5β2γ2L receptors. However, the α5β2 receptor possessed significantly lower GABA efficacy compared with the α5β2γ2L receptor. While the γ2‐subunit was essential to the potency of GABA, its influence on the apparent GABA‐site antagonism was less profound. The antagonist affinity constants (K_B) of bicuculline inhibition and slopes of Schild plots were similar between all types of ternary and binary receptors except α5β2 receptor which was not tested. The pK_Bs and IC₅₀s of the GABA‐site antagonism were not significantly different between the α1β2γ2L, α4β2γ2L and α5β2γ2L receptors. Bicuculline blocked pentobarbital‐activated currents in a reversible and non‐competitive manner with the α1β2γ2L, α4β2γ2L, and α5β2γ2L receptors, indicating an allosteric inhibition of the GABA‐site. No significant difference of bicuculline potencies in inhibiting GABA‐ and pentobarbital‐activated currents was found between the α1β2γ2L, α4β2γ2L and α5β2γ2L receptors. Conclusion: The GABA‐site antagonism does not depend on the subtype of α‐subunits. Similarly, pentobarbital activates ternary receptors composed of different α‐subunits in a bicuculline‐sensitive manner. The potencies of bicuculline to inhibit pentobarbital‐activated currents are identical with receptors containing α1, α4 or α5‐subunit. The α1β2 and α4β2 receptors possess higher GABA potencies compared with the α1β2γ2L and α4β2γ2L receptors.