A neurosteroid potentiation site can be moved among GABA A receptor subunits

Neuroactive steroids are endogenous compounds that are potent and efficacious potentiators of GABA A receptors. Neuroactive steroids have been shown to act after interacting with amino acids in the first transmembrane‐spanning region (TM1 region) of the α subunit. We show that the TM1 region can be moved from the α1 subunit to other subunits of the GABA A receptor (the β2 or γ2 subunits) and confer potentiation. Our data show that neurosteroid potentiation does not require that the steroid interacts with a particular subunit of the GABA A receptor, nor that it interacts with a subunit that also binds GABA. Our data also indicate that a steroid binding site is located in the TM1 region. Abstract Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA A receptors. It has been proposed that a conserved glutamine residue in the first membrane‐spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single‐channel kinetics are similar for wild‐type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid.