The Location and Nature of General Anesthetic Binding Sites on the Active Conformation of Firefly Luciferase; A Time Resolved Photolabeling Study

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP–driven production of light. We have used time–resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC–MSMS revealed a similar conformation–dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies

Time-Resolved Photolabeling of the Nicotinic Acetylcholine Receptor by [3H]Azietomidate, an Open-State Inhibitor

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 <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

Identification of photolabeled residues in the peptide Gln-340 – Lys-366.

<p>Conditions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g004" target="_blank">Fig. 4</a>. <b>A.</b> 3-azibutanol (1 mM) photolabels Asp-358. <b>B.</b> TFD-benzyl alcohol (100 µM) photolabels Ser-349.</p