PREreview of Divergent mechanisms of steroid inhibition in the human ρ1 GABA_Areceptor

The manuscript by Chen Fan and colleagues details a comprehensive study of the homomeric ρ1 GABA receptor, which was previously known as the type C GABA receptor but has been re-classified into type A GABA receptor given their closely related molecular architectures.  The authors study the receptor in complexes with by two different steroid molecules using cryo-EM, site-directed mutagenesis, electrophysiology, and molecular dynamics simulations.

One of the steroids is β-estradiol, an estrogen steroid hormone and the primary female sex hormone. It is known to exert inhibitory effects on ρ1 GABA receptor, but the molecular mechanism of this inhibition remains elusive. The authors have determined homomeric ρ1 GABA receptor structures in a complex with β-estradiol, with and without GABA. In doing so, they capture a new binding pocket of β-estradiol between the GABA-binding extracellular domain and the pore-lining transmembrane domain, which is specific to the ρ-type GABA receptor because a critical phenylalanine residue in a pocket (F283) is substituted with a tyrosine residue in the α1, β2, β3, γ2 subunits. The binding of β-estradiol blocks the transduction of GABA-induced ECD rotations to the transmembrane domain, thus providing a mechanism for inhibition of ρ-type GABA receptor by β-estradiol.

The second steroid is pregnenolone sulfate (PS), one of the first neurosteroids discovered, and one that modulates several receptors in the brain, including the type A GABA receptor. In 2023, a joint study from the Lindahl lab and the Hibbs lab proposed that PS inhibits the canonical α1β2γ2 GABA receptor by binding to the ion pore and serving as a pore blocker. In this study, the authors have determined homomeric ρ1 GABA receptor structures in complex with PS, with and without GABA. In agreement with the previous report, they observed density in the pore attributed to PS. Given the ambiguity in modeling the PS molecule into the density map, the authors used molecular dynamics simulation to explore its binding pose and binding energetics. In addition, they showed with electrophysiology recording that the inhibition by the charged PS was dependent on the membrane potential, which is consistent with it being a pore blocker.

Together, this study presents a significant advance in our understanding of the different modes of steroid modulation of GABA receptors. In particular, the β-estradiol binding pocket opens avenues for drug developments specific to the ρ-type GABA receptors, which could be further facilitated by identification of receptor subtypes from native tissues.

Major points:

1.     PS was modeled as “sulfate-up” in the PS-GABA structure (Figure 3F), which was supported by the all-atom molecular dynamics (MD) simulations. However, the MD simulations presumably had a chloride ion placed near the -2’ of the ion pore in addition to the PS molecule. Could the author provide more details of their MD simulations, particularly about criteria used to position the chloride ion at the -2’ position of the ion pore?  Did the authors explore the possibility of not placing a chloride ion at the -2’ position, especially in the context of the sulfate ‘down’ orientation of PS?

2.     In Figure 3G, the M2 helices of the α1β2γ2 structure appear to be ‘higher’ in the Z axis than those from the ρ1 structure. This displacement of the M2 helices may alter ones understanding of the overlay of the PS molecules. Could the authors explain how the structure superimposition was done?

Minor points:

1.     On page 4, the first paragraph of the Result section, it should be “30 μM” rather than “30 μm.”

2.     On page 4, “the side chains of S334 and R337 are positioned to make hydrophobic and π-orbital interactions with E2 rings A and D, respectively.” What is the meaning of “π-orbital” interaction and do the authors mean, instead, ‘cation- π’ interaction?

3.     For Figure 2E, the currents measured with oocytes should be plotted as “Relative Currents” rather than “Relative Po”.

4.     In Figure 3B, an increased current exceeding the previously elicited current was observed during the wash out of PS. However, in Figure S7F, such increase in current was not observed during the wash out the PS when the experiment was conducted at higher centration of GABA. Could the authors explain this difference?

Competing interests

The authors declare that they have no competing interests.