Abstract
Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.
Highlights
Drugs that act as positive allosteric modulators of GABAARs include benzodiazepines, which bind in the extracellular domain at a site equivalent to the transmitter binding sites but at a different subunit interface [8, 9], and general anesthetics of diverse chemical structure, including volatiles, neurosteroids, and intravenous agents such as etomidate and barbiturates [10, 11]
TDBzl-etomidate, a Positive Modulator of ACh Responses— We used two-electrode voltage clamp to compare the effects of TDBzl-etomidate and etomidate on the ACh current responses for Torpedo nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes (Fig. 2, A and B)
We demonstrate that [3H]TDBzl-etomidate acts as a positive allosteric nAChR modulator, and we identify its binding sites in the nAChR
Summary
Materials—Membranes rich in nAChRs, containing 1–2 nmol of [3H]acetylcholine (ACh) binding sites/mg of protein, were isolated from Torpedo californica electric organs (Aquatic Research Consultants, San Pedro, CA) as described previously [28]. For photolabeling on a preparative scale, the membrane suspensions (10 mg of protein/condition) at 2 mg/ml concentration were incubated in 6-cm plastic Petri dishes for 45 min at 4 °C with 1.25 M [3H]TDBzl-etomidate in the presence or absence of additional ligands. The gel bands containing the nAChR , ␥, and ␦ subunits were excised and passively eluted for 3 days into 12 ml of elution buffer (100 mM NH4HCO3, 0.1% SDS, 2.5 mM dithiothreitol). CDOCKER was used to dock 50 energy-minimized structures of TDBzl-etomidate (volume, 284 Å3) into potential binding sites in the nAChR TMD defined by a sphere of 15-Å radius centered within the ion channel or of 12-Å radius centered at each of the five subunit interfaces or within the helical bundle of the ␦ subunit. No stable binding was predicted at the ␦- interface or within the ␦ subunit helix bundle
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
