TY - JOUR T1 - Time-Resolved Photolabeling of the Nicotinic Acetylcholine Receptor by [<sup>3</sup>H]Azietomidate, an Open-State Inhibitor JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1084 LP - 1095 DO - 10.1124/mol.108.054353 VL - 75 IS - 5 AU - David C. Chiara AU - Filbert H. Hong AU - Enrique Arevalo AU - S. Shaukat Husain AU - Keith W. Miller AU - Stuart A. Forman AU - Jonathan B. Cohen Y1 - 2009/05/01 UR - http://molpharm.aspetjournals.org/content/75/5/1084.abstract N2 - Azietomidate is a photoreactive analog of the general anesthetic etomidate that acts as a nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist. We used rapid perfusion electrophysiological techniques to characterize the state dependence and kinetics of azietomidate inhibition of Torpedo californica nAChRs and time-resolved photolabeling to identify the nAChR binding sites occupied after exposure to [3H]azietomidate and agonist for 50 ms (open state) or at equilibrium (desensitized state). Azietomidate acted primarily as an open channel inhibitor characterized by a bimolecular association rate constant of k+ = 5 × 105 M-1 s-1 and a dissociation rate constant of &lt;3s-1. Azietomidate at 10 μM, when perfused with acetylcholine (ACh), inhibited the ACh response by ∼50% after 50 ms; when preincubated for 10 s, it decreased the peak initial response by ∼15%. Comparison of the kinetics of recovery of ACh responses after exposure to ACh and azietomidate or to ACh alone indicated that at subsecond times, azietomidate inhibited nAChRs without enhancing the kinetics of agonist-induced desensitization. In nAChRs frozen after 50-ms exposure to agonist and [3H]azietomidate, amino acids were photolabeled in the ion channel [position M2-20 (αGlu-262, βAsp-268, δGln-276)], in δM1 (δCys-236), and in αMA/αM4 (αGlu-390, αCys-412) that were also photolabeled in nAChRs in the equilibrium desensitized state at approximately half the efficiency. These results identify azietomidate binding sites at the extracellular end of the ion channel, in the δ subunit helix bundle, and in the nAChR cytoplasmic domain that seem similar in structure and accessibility in the open and desensitized states of the nAChR. The American Society for Pharmacology and Experimental Therapeutics ER -