Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Abstract

) J. Neurosci. 26, 11599 -11605) azietomidate photolabeling of GABA A R ␣1Met-236 and ␤Met-286 (in ␣M1 and ␤M3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of ␤ and ␣ subunits (two copies per pentamer) is consistent with a GABA A R homology model based upon the structure of the nicotinic acetylcholine receptor and with recent ␣M1 to ␤M3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA A R subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA A R-modulating neurosteroids do not inhibit photolabeling of GABA A R ␣1Met-236 or ␤Met-286 but enhance labeling of ␣1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486 -489) are located at the subunit interface defined by our etomidate site model. GABA A3 receptors (GABA A R) are major mediators of brain inhibitory neurotransmission and participate in most circuits and behavioral pathways relevant to normal and pathological function (1). GABA A R are subject to modulation by endogenous neurosteroids, as well as myriad clinically important central nervous system drugs including general anesthetics, benzodiazepines, and possibly ethanol (1, 2). The mechanism of GABA A R modulation by these different classes of drugs is of major interest, including identification of the receptor amino acid residues involved in binding and action of the drugs.In the absence of high resolution crystal structures of drugreceptor complexes, the locations of anesthetic binding sites in GABA A Rs have been predicted based upon analyses of functional properties of point mutant receptors, which identified residues in the ␣ and ␤ subunit M1-M4 transmembrane helices important for modulation by volatile anesthetics (primarily ␣ subunit) and by intravenous agents, including etomidate and propofol (␤ subunit) (3-5). Position ␤M2-15, numbered relative to the N terminus of the helix, functions as a major determinant of etomidate and propofol potency as GABA modulators in vitro and in vivo (6 -8). By contrast, this residue is not implicated for modulation by the neurosteroids, potent endogenous modulators of GABA A R (9).Photoaffinity labeling, which allows the identification of residues in proximity to drug binding sites (10, 11), has been used to identify two GABA A R amino acids covalently modified by the etomidate analog [ 3 H]azietomidate (12): ␣1Met-236 within ␣M1 and ␤Met-286 within ␤M3. Photolabeling of these residues was inhibited equally by nonradioactive etomidate and enhanced proportionately by GABA present in the assay, consistent with the presence of these two residues in a common drug binding pocket that would be located at the interface between the ␤ and ␣ subunits in the transmembrane domain (12). Mutational analyses identi...

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

“…Thus, the ␣1-M1/␤2-M3 interface apparently contains overlapping sites for volatile anesthetics, propofol and etomidate. Hosie and Smart (25) also identified ␣1T237 and ␣1Q242 as determinants for neurosteroid sensitivity, although neurosteroids do not displace etomidate (41). Our cysteine protection results argue against alphaxalone contact with ␣1Met-236.…”

“…2, E and F). Supporting this interpretation, alphaxalone, which enhances GABA activation without blocking etomidate binding (41), accelerated pCMBS modification of ␣1M236C about 3-fold over the GABA-only reference rate (data not shown). Thus, etomidate probably reduced pCMBS modification rates much more than 10-fold in GABA-activated ␣1M236C␤2␥2L receptors.…”

The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

“…Etomidate is thought to bind at the lipidfacing ␣-␤ interface at the extracellular end of the transmembrane domain (Li et al, 2009), distal to the internal porefacing 6Ј positions; therefore, we expected it would not interfere with MMTS-induced persistent inhibition of GABAactivated currents. Indeed, whereas 1 M etomidate modulated ␣2(T6ЈC)␤2 receptors to an equivalent degree as wild type (Fig.…”

EVOLUTION IN CHEMOTHERAPY OF HUMAN AND ANIMAL HELMINTHIASES (review)

“…It is known that positive inotropic action of cardiotonic steroids is associated with inhibition of membrane Na,KATPase (118), along with the recently discovered antitumor effect (119). At the same time, it was observed modulation of activity of GABA A -receptor by neurosteroids (120). Therefore, the mechanism of anthelmintic action of these substances (inhibition of Na,KATPase or some other way) needs further investigation.…”