Ayman K. Hamouda

Physostigmine and Galanthamine Bind in the Presence of Agonist at the Canonical and Noncanonical Subunit Interfaces of a Nicotinic Acetylcholine Receptor

Galanthamine and physostigmine are clinically used cholinomimetics that both inhibit acetylcholinesterase and also interact directly with and potentiate nAChRs. As with most nAChR-positive allosteric modulators, the location and number of their binding site(s) within nAChRs are unknown. In this study, we use the intrinsic photoreactivities of [3H]physostigmine and [3H]galanthamine upon irradiation at 312 nm to directly identify amino acids contributing to their binding sites in the Torpedo californica nAChR. Protein sequencing of fragments isolated from proteolytic digests of [3H]physostigmine- or [3H]galanthamine-photolabeled nAChR establish that, in the presence of agonist (carbamylcholine), both drugs photolabeled amino acids on the complementary (non-α) surface of the transmitter binding sites (γTyr-111/γTyr-117/δTyr172). They also photolabeled δTyr-212 at the δ-β subunit interface and γTyr-105 in the vestibule of the ion channel, with photolabeling of both residues enhanced in the presence of agonist. Furthermore, [3H]physostigmine photolabeling of γTyr-111, γTyr-117, δTyr-212, and γTyr-105 was inhibited in the presence of nonradioactive galanthamine. The locations of the photolabeled amino acids in the nAChR structure and the results of computational docking studies provide evidence that, in the presence of agonist, physostigmine and galanthamine bind to at least three distinct sites in the nAChR extracellular domain: at the α-γ interface (1) in the entry to the transmitter binding site and (2) in the vestibule of the ion channel near the level of the transmitter binding site, and at the δ-β interface (3) in a location equivalent to the benzodiazepine binding site in GABAA 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, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]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). TFD-etomidate inhibited acetylcholine-induced currents with an IC50 = 4 μm, whereas it inhibited the binding of [3H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC50 values of 2.5 and 0.7 mm, respectively. Similar to [3H]TDBzl-etomidate, [3H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [3H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).

Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

[3H]Chlorpromazine Photolabeling of the Torpedo Nicotinic Acetylcholine Receptor Identifies Two State-Dependent Binding Sites in the Ion Channel

Chlorpromazine (CPZ), a potent nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist, binds with higher affinity in the ion channel in the desensitized state than in the closed channel state and with low affinity to additional sites in nAChR-rich membranes. For nAChR equilibrated with agonist, we confirm previous reports that [(3)H]CPZ occupies a site near the cytoplasmic end of the M2 ion channel domain, photolabeling positions M2-2, M2-6, and/or M2-9 in each subunit. We find that [(3)H]CPZ also binds at the extracellular end of the channel, photolabeling amino acids at positions M2-16 (alpha,gamma), M2-17 (alpha,beta,delta), and M2-20 (alpha,beta,delta). The photolabeling at the cytoplasmic end of the channel is fully inhibitable by phencyclidine or proadifen, whereas neither drug inhibits [(3)H]CPZ photolabeling at the extracellular end, establishing that positively charged drugs can bind simultaneously at the cytoplasmic and extracellular ends of the ion channel. [(3)H]CPZ photolabeling is not detected in the transmembrane domain outside the ion channel, but it photolabels alphaMet-386 and alphaSer-393 in the cytoplasmic alphaMA helix. In the nAChR equilibrated with alpha-bungarotoxin to stabilize the nAChR in a closed state, [(3)H]CPZ photolabels amino acids at M2-5 (alpha), M2-6 (alpha,beta,delta), and M2-9 (beta,delta), with no labeling at M2-2. These results provide novel information about the modes of drug binding within the nAChR ion channel and indicate that within the nAChR transmembrane domain, the binding of cationic aromatic amine antagonists can be restricted to the ion channel domain, in contrast to the uncharged, allosteric potentiators and inhibitors that also bind within the delta subunit helix bundle and at subunit interfaces.

p-Trifluoromethyldiazirinyl-etomidate: a potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels

We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomidate, a novel photoactivable derivative of the stereoselective general anesthetic etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to etomidates, but stereoselectivity was reversed and attenuated. Relative to etomidate, TFD-etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((alpha1)(2)beta1delta1gamma1) and 5-HT(3A)R (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory alpha1beta2gamma2L GABA(A)Rs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [(3)H]TFD-etomidate photolabeled the alpha-subunit of the nAChR in a manner allosterically regulated by agonists and noncompetitive inhibitors. TFD-etomidates novel pharmacology is unlike that of etomidate derivatives with photoactivable groups in the ester position, which behave like etomidate, suggesting that it will further enhance our understanding of anesthetic mechanisms.

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies.

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.

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 [3H]R-mTFD-MPAB ([3H]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. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [3H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [3H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [3H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ–α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ–α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.

[3H]Epibatidine photolabels non-equivalent amino acids in the agonist binding Site of torpedo and α4β2 nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.

Photoaffinity Labeling the Agonist Binding Domain of α4β4 and α4β2 Neuronal Nicotinic Acetylcholine Receptors with [125I]Epibatidine and 5[125I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

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. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([3H]phencyclidine; IC50 = 4 μM) than in the resting state ([3H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([3H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [3H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [3H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [3H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.