Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor
Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor
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DOI：
10.1074/jbc.m111.219071
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发表时间：
2011-06-10
影响因子：
4.8
通讯作者：
Cohen, Jonathan B.
中科院分区：
文献类型：
--
作者：
Hamouda, Ayman K.;Stewart, Deirdre S.;Cohen, Jonathan B.
已经开发了全身麻醉剂依托咪酯的光反应衍生物，以确定其在γ-氨基丁酸、A型和烟碱乙酰胆碱受体中的结合位点。一种这样的药物，[H-3] TDBzl-依托咪酯（4-[3-（三氟甲基）3 H-二氮杂环丙烯-3-基]苄基-[H-3]1-（1-苯乙基）-1H-咪唑-5-羧酸酯），作为电鳐nACh受体（nAChR）的正变构增效剂，并结合到γ-α亚基界面处跨膜结构域中的新位点。为了扩展我们对nAChR中变构调节剂结合位点位置的理解，我们现在表征第二种芳基二氮杂环丙烯依托咪酯衍生物TFD-依托咪酯（乙基-1-（1-（4-（3-三氟甲基）-3H-二氮杂环丙烯-3-基）苯乙基）1H-咪唑-5-羧酸酯）的相互作用。TFD-依托咪酯抑制乙酰胆碱诱导的电流，IC 50 = 4 μ M，而它抑制[H-3]苯环利定的结合电鳐nAChR离子通道在休息和脱敏状态，IC 50值分别为2.5和0.7 mM。与[H-3] TDBzl-依托咪酯类似，[H-3] TFD-依托咪酯结合于γ-α亚基界面的一个位点，光标记γ M3内的α M2-10（α Ser-252）和γ Met-295和γ Met-299，并结合于离子通道中的一个位点，光标记离子通道内腔的每个亚基M2螺旋内的氨基酸。此外，[H-3] TFD-依托咪酯以激动剂依赖性方式光标记δ亚基M2-M3环（δ Ile-288）和δ亚基跨膜螺旋束（δ M1内的δ Phe-232和δ Cys-236）内的氨基酸。在抑制乙酰胆碱反应的浓度下，TFD-依托咪酯不与离子通道阻滞剂竞争，这一事实表明，与γ-α亚基界面和/或δ亚基螺旋束内的位点结合介导了TFD-依托咪酯的抑制作用。这些结果还表明，γ-α亚基界面是电鳐nAChR负变构调节剂（TFD-依托咪酯）和正调节剂（TDBzl-依托咪酯）的结合位点。
Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in gamma-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [H-3]TDBzl-etomidate (4-[3-(trifluoromethyl)3H-diazirin-3-yl]benzyl-[H-3]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 gamma-alpha 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 mu M, whereas it inhibited the binding of [H-3]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 [H-3] TDBzl-etomidate, [H-3]TFD-etomidate bound to a site at the gamma-alpha subunit interface, photolabeling alpha M2-10 (alpha Ser-252) and gamma Met-295 and gamma Met-299 within gamma M3, 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, [H-3]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the delta subunit M2-M3 loop (delta Ile-288) and the delta subunit transmembrane helix bundle (delta Phe-232 and delta Cys-236 within delta M1). 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 gamma-alpha subunit interface and/or within delta subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the gamma-alpha subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).