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

Neurosteroids exert potent physiological effects by allosterically modulating synaptic and extrasynaptic GABAA receptors. Some endogenous neurosteroids, such as 3α, 21-dihydroxy-5β-pregnan-20-one (5α, 3α-THDOC), potentiate GABAA receptor function by interacting with a binding pocket defined by conserved residues in the first and fourth transmembrane (TM) domains of α subunits. Others, such as pregnenolone sulfate (PS), inhibit GABAA receptor function through as-yet unidentified binding sites. Here we investigate the mechanisms of PS inhibition of mammalian GABAA receptors, based on studies of PS inhibition of the UNC-49 GABA receptor, a GABAA-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 α1, β2, and γ2 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 γ2 subunits caused increased efficacy of 5α, 3α-THDOC potentiation; second, spontaneous gating of α6β2δ receptors was blocked by PS, and reduced by chimeric β2 subunits; and third, direct activation of α6β2δ receptors by 5α, 3α-THDOC was reduced by chimeric β2 subunits. These results reveal novel roles for non-α subunits in neurosteroid modulation and direct activation, and show that the β subunit TM1 domain is important for spontaneous activity of extrasynaptic GABAA receptors.