Tryptophan and Cysteine Mutations in M1 Helices of α1β3γ2L γ-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site.

Background: γ-Aminobutyric acid type A (GABAA) receptors mediate important effects of intravenous general anesthetics. Photolabel derivatives of etomidate, propofol, barbiturates, and a neurosteroid get incorporated in GABAA receptor transmembrane helices M1 and M3 adjacent to intersubunit pockets. However, photolabels have not been consistently targeted at heteromeric αβγ receptors and do not form adducts with all contact residues.

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor.

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM.

Mutations at beta N265 in γ-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.

Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α- inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15') on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding.

Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR.

Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors.

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site.

State-dependent etomidate occupancy of its allosteric agonist sites measured in a cysteine-substituted GABAA receptor.

A central axiom of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. However, measuring agonist affinity in inactive receptors is confounded by concomitant activation. We identified a cysteine substituted mutant γ-aminobutyric acid type A (GABAA) receptor with unique characteristics allowing the determination of allosteric agonist site occupancy in both inactive and active receptors.

Two etomidate sites in α1β2γ2 γ-aminobutyric acid type A receptors contribute equally and noncooperatively to modulation of channel gating.

Background: Etomidate is a potent hypnotic agent that acts via γ-aminobutyric acid receptor type A (GABA(A)) receptors. Evidence supports the presence of two etomidate sites per GABA(A) receptor, and current models assume that each site contributes equally and noncooperatively to drug effects. These assumptions remain untested.

p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol.

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like propofol, the compound potentiates inhibitory GABA(A) receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABA(A) receptors.

Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate).

Photoactivated 3-azioctanol irreversibly desensitizes muscle nicotinic ACh receptors via interactions at alphaE262.

3-Azioctanol is a photoactivatable analogue of octanol that noncompetitively inhibits nicotinic acetylcholine receptors (nAChRs). Photolabeling studies using [3H]-3-azioctanol in Torpedo nAChR identified alphaE262 as a site of desensitization-dependent incorporation. However, it is unknown whether photolabeling of alphaE262 causes functional effects in nAChRs and what other roles this residue plays in gating, desensitization, and channel block.

[3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure.

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain.

Mapping the structural requirements for nicotinic acetylcholine receptor activation by using tethered alkyltrimethylammonium agonists and antagonists.

A molecule as simple in structure as tetramethylammonium gates the nicotinic acetylcholine receptor (nAChR) with high efficacy. To compare the structure of the nAChR transmitter binding site in the open channel state with that of the ACh binding protein, we determined the efficacy of nAChR gating by -S(CH(2))(n)N(CH(3))(3)(+) (n = 1-4) tethered to substituted cysteines at positions in the alpha subunits or gamma and delta subunits predicted to contribute to the ACh binding sites in mutant Torpedo nAChRs expressed in Xenopus oocytes. For tethered thiocholine [-S(CH(2))(2)N(CH(3))(3)(+)], we previously reported that within alpha195-201 gating was observed only at alphaY198C while at alphaY93C it acted as an antagonist.