Mutations of the GABA-A Receptor α1 Subunit M1 Domain Reveal Unexpected Complexity for Modulation by Neuroactive Steroids

Abstract

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues ␣1Asn407/Tyr410 in the M4 transmembrane domain and residue ␣1Gln241 in the M1 domain. We examined the role of residues in the ␣1 subunit M1 domain in the modulation of the rat ␣1␤2␥2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the ␣1Q241W or ␣1Q241L mutations were insensitive to (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P), albeit with different underlying mechanisms. The ␣1Q241S mutant was potentiated by 3␣5␣P, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the ␣1Q241L mutation had no effect on channel potentiation by (3␣,5␣)-3-hydroxymethylpregnan-20-one, but mutation of the neighboring residue, ␣1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5␣-pregnan-20-one) potentiated the wild-type receptor but not the ␣1Q241L mutant. The findings are consistent with a model in which the ␣1Ser240 and ␣1Gln241 residues shape the surface to which steroid molecules bind.Potentiating neurosteroids are among the most efficacious modulators of the mammalian GABA-A receptor having potential applications as anxiolytics, anticonvulsants, sedatives, and anesthetics. Recent work has given significant insights into the functional and structural mechanisms of steroid actions. Potentiating steroids [e.g., (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P) and (3␣,5␤)-3-hydroxypregnan-20-one (3␣5␤P)] act on the GABA-A receptor by modifying the channel open and closed times, leading to an increase in the open probability of the channel, enhanced macroscopic peak current, and a slower current decay when exposure to agonist is terminated. The putative steroid binding site is located in the membrane-spanning regions of the ␣ subunit of the receptor, extending from the ␣1Gln241 residue in the M1 membrane-spanning region to the residues ␣1Asn407 and ␣1Tyr410 in the M4 domain (Hosie et al., 2006). Mutations that reduce the H-bonding ability of these residues reduce receptor potentiation by both 5␣-and 5␤-reduced steroids. It was proposed that a common interaction site mediates the effects of the two classes of steroids, the ␣1Gln241 residue acting as an H-bond acceptor to the 3␣-hydroxyl group of the steroid molecule and the ␣1Asn407/Tyr410 residues interacting with the ketone group in the side chain on the D ring of steroids (Hosie et al., 2006). Subsequent studies showed that mutations that disrupt channel potentiation by steroids also affect modulation by a tricyclic benz[e]indene neurosteroid analog (Li et al., 2006), enantiomers of natural steroids (Li et al., 2007a), and the marine cembranoid eupalmerin acetate (Li et al., 2008), suggesting that the site may function as a common interaction site for a number of GABA-A receptor modul...

Single-Channel Analysis of Ethanol Enhancement of Glycine Receptor Function

“…This would have the effect of prolonging the duration of GlyR activation at the synapse, and this is consistent with ethanol producing leftward shifts of glycine concentration-response curves. The effect of ethanol on select parameters of channel function is similar to the specificity exhibited by other modulators such as zinc (Laube et al, 2000) or neurosteroids (Akk et al, 2008) that interact with receptors at defined binding sites.…”

“…For example, barbiturates and neurosteroids enhance GABA A receptor function primarily by increasing the mean channel open time; thus, they act to stabilize an open state of the receptor (Steinbach and Akk, 2001;Akk et al, 2008). Zinc is a positive modulator of GlyR function at low micromolar concentrations.…”

Occupation of Either Site for the Neurosteroid Allopregnanolone Potentiates the Opening of the GABAA Receptor Induced from Either Transmitter Binding Site

“…Although the exact structural determinants of steroid binding are unknown, the actions of potentiating steroids are strongly reduced or eliminated by mutations to specific residues in the M1 and M4 transmembrane domains in the ␣1 subunit (Hosie et al, 2006;Akk et al, 2008;Li et al, 2009). Specifically, the ␣1Q241L mutation abolishes potentiation by the steroid allopregnanolone (Hosie et al, 2006;Akk et al, 2008). The GABA A receptor contains two ␣ subunits and, presumably, two binding sites for steroids.…”

Cysteine Substitutions Define Etomidate Binding and Gating Linkages in the α-M1 Domain of γ-Aminobutyric Acid Type A (GABAA) Receptors

“…Residues in ␣-M1 have previously been linked to GABA A receptor modulation by barbiturates (23,24) and neurosteroids (25,26) as well as gating transduction between extracellular GABA sites and the transmembrane channel (23,27,28). Cysteine substitution and modification studies (21,22) have provided clues to some side chain orientations and evidence of rearrangements during gating for the pre-M1 and outer ␣-M1 domain from ␣Ile-223 to ␣Met-236.…”