Abstract
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). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]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, [(3)H]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).
Highlights
The Torpedo nACh receptor (nAChR) is composed of four homologous subunits (␣2␥␦) that assemble around an axis perpendicular to the membrane, forming a cation-conducting channel
TFD-etomidate, a Potent nAChR Inhibitor—For Torpedo nAChR expressed in Xenopus oocytes, TFD-etomidate inhibited currents elicited by 10 M ACh with an IC50 of 4 M, i.e. a potency 5-fold greater than etomidate (IC50 ϭ 20 M) (Fig. 2A), as reported previously [21]
Etomidate for the ACh binding sites or for a binding site in the ion channel, we examined the effect of TFD-etomidate on the equilibrium binding to Torpedo nAChR-rich membranes of [3H]ACh or the channel blockers [3H]tetracaine and [3H]PCP, which bind in the ion channel preferentially in the resting and desensitized states, respectively [30, 31] (Fig. 2B)
Summary
Materials—Torpedo nAChR-rich membranes were isolated from Torpedo californica electric organs (Aquatic Research Consultants, San Pedro, CA) as described previously [23]. [3H]TFD-etomidate Photolabeling—Torpedo nAChR-rich membranes at 2 mg of protein/ml in Torpedo physiological saline supplemented with 1 mM oxidized glutathione were incubated at room temperature for 40 min with 1 M [3H]TFDetomidate in the absence or presence of other drugs. For the  subunit digest, fragments beginning near the N termini of M2 and M4 were isolated for sequence analysis by rpHPLC from material eluted from gel bands with apparent molecular masses of ϳ10 and ϳ7 kDa, respectively [27]. Photolabeling efficiency in cpm/pmol at a specific residue was calculated by (cpmx Ϫ cpm(x Ϫ 1)/5IoRx. To chemically isolate during sequence analysis the nAChR subunit fragments produced by V8 protease that begin at Thr273, ␥Thr-276, or ␦Thr-281 within the M2-M3 loops [16], sequencing filters were treated with o-phthalaldehyde (OPA) before cycle 6 of Edman degradation, which contains a proline (Thr-278, ␥Thr-281, or ␦Thr-286, respectively). For the binding site in the ␦ subunit helix bundle, 30 orientations were contained in a volume of 340 Å3 bounded by amino acids in M1, M3, M4, and the M2-M3 loop
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