Multiple roles for the first transmembrane domain of GABAA receptor subunits in neurosteroid modulation and spontaneous channel activity

Abstract

Neurosteroids exert potent physiological effects by allosterically modulating synaptic and extrasynaptic GABA(A) receptors. Some endogenous neurosteroids, such as 3alpha, 21-dihydroxy-5beta-pregnan-20-one (5alpha, 3alpha-THDOC), potentiate GABA(A) receptor function by interacting with a binding pocket defined by conserved residues in the first and fourth transmembrane (TM) domains of alpha subunits. Others, such as pregnenolone sulfate (PS), inhibit GABA(A) receptor function through as-yet unidentified binding sites. Here we investigate the mechanisms of PS inhibition of mammalian GABA(A) receptors, based on studies of PS inhibition of the UNC-49 GABA receptor, a GABA(A)-like receptor from Caenorhabditis elegans. In UNC-49, a 19 residue segment of TM1 can be mutated to increase or decrease PS sensitivity over a 20-fold range. Surprisingly, substituting these UNC-49 sequences into mammalian alpha(1), beta(2), and gamma(2) subunits did not produce the corresponding effects on PS sensitivity of the resulting chimeric receptors. Therefore, it is unlikely that a conserved PS binding pocket is formed at this site. However we observed several interesting unexpected effects. First, chimeric gamma2 subunits caused increased efficacy of 5alpha, 3alpha-THDOC potentiation; second, spontaneous gating of alpha(6)beta(2)delta receptors was blocked by PS, and reduced by chimeric beta(2) subunits; and third, direct activation of alpha(6)beta(2)delta receptors by 5alpha, 3alpha-THDOC was reduced by chimeric beta(2) subunits. These results reveal novel roles for non-alpha subunits in neurosteroid modulation and direct activation, and show that the beta subunit TM1 domain is important for spontaneous activity of extrasynaptic GABA(A) receptors.

Figure 1

Sequence, GABA sensitivity, and PS inhibition of GABA_A receptors (A) Amino acid sequences of GABA_A receptor subunits and UNC-49 subunits. The swapped TM1 region is indicated by a bar above the residues, the α subunit glutamine necessary for neurosteroid potentiation is indicated with an arrow, and the UNC-49C residues that can be swapped into UNC-49B to alter PS sensitivity are indicated with triangles (black indicates residues that increase UNC-49B PS inhibition, white indicates a residue that reduces PS inhibition of UNC-49B). (B) GABA concentration-response curves for wild-type and chimeric α₁β₂γ₂ receptors (left) and α₆β₂δ receptors (right; n=4 for α₁β₂γ₂ and α₆β_2×δ, n=3 for all others). (C) Representative traces of wild-type and chimeric α₁β₂γ₂ GABA_A receptors exposed to EC₅₀ GABA alone, or EC₅₀ GABA plus 10 μM PS. Bars above traces indicate duration of drug exposure. (D) PS concentration-response curves for wild-type and chimeric α₁β₂γ₂ receptors. (E) Plot of PS IC₅₀ values for all combinations of wild-type and chimeric α₁β₂γ₂ receptors (* P < 0.05 Student's T test, # P < 0.05 Mann-Whitney test, compared to wild-type).

Figure 2

5α, 3α-THDOC modulation of α₁β₂γ₂ GABA_A receptors A) Representative traces of wild-type and chimeric α₁β₂γ₂ GABA_A receptors exposed to EC₂₀ GABA alone, or EC₂₀ GABA plus 1 μM 5α, 3α-THDOC. (B) Concentration-response plots for 5α, 3α-THDOC potentiation of EC₂₀ GABA currents. The bell-shaped concentration-response curves are separated into the rising phase (top plot) and the falling phase (lower plot). The 1.0 μM data point is shown in both plots. (C) Comparison of potentiation by 1 μM 5α, 3α-THDOC of all wild-type and chimeric combinations (n = 6 for each data point, * P < 0.05, # P = 0.05 Mann-Whitney test, compared to wild-type).

Figure 3

Inhibition of α₆β₂δ receptors by PS and other inhibitors (A) Representative traces of wild-type α₆β₂δ and α₆β_2×δ receptors exposed to EC₅₀ GABA or EC₅₀ GABA plus 10 μM PS. The wild-type, but not the chimera receptor showed large outward currents during preapplication. (B) Concentration-response curve for PS inhibition of GABA-evoked currents from α₆β₂δ (n=6) and α₆β_2×δ (n=5) receptors. (C) Magnitude of PS-evoked outward current relative to the EC₅₀ GABA current for selected subunit combinations (α₁-containing receptors are also shown for comparison; n ≥ 5 for each data point). (D) Traces from oocytes expressing wild-type α₆β₂δ receptors exposed to a series of inhibitors: 10 μM PS, 10 μM picrotoxinin, 500 μM furosemide, and 100 μM Zn²⁺. Each row represents an independent cell; EC₅₀ GABA traces are shown for comparison. (E) Same as D, for α₆β_2×δ receptor-expressing cells.

Figure 4

5α, 3α-THDOC direct activation and potentiation of α₆β₂δ receptors (A) Wild-type α₆β₂δ (left) or chimeric α₆β_2×δ (right) receptors were exposed to EC₂₀ GABA or EC₂₀ GABA plus 1 μM 5α, 3α-THDOC. Concentration-response plots of 5α, 3α-THDOC direct activation (as a proportion of EC₂₀ GABA current; B) and potentiation of EC₂₀ GABA currents (C). As in Fig. 2B, the rising and falling phases of the bell-shaped curves are plotted separately (upper and lower traces, respectively). Statistical significance (wild-type vs. chimera; P < 0.05) is indicated by * (Student's T test) or # (Mann-Whitney test); n = 9 for wild-type, n=3 for chimera.