(PC) membranes with varying hydropho- bic thicknesses. While the nAChR in each PC membrane adopts an uncoupled conformation, long acyl chain lipids promote very slow agonist-induced con- formational transitions to the desensi- tized state. Hydrophobic thickness likely influences the ability of the nAChR to undergo agonist-induced conformational transitions by promoting a-helix/a-helix interactions, which appears to lower the activation energy barriers between un- coupled and coupled conformations. 3261-Pos Board B416 Alpha7 Nicotinic Receptors: Intrinsic Kinetics and Modulation by PNU 120596 Arpad Mike, Krisztina Pesti, Anett K. Szabo, E.S. Vizi. Inst. of Experimental Medicine, Budapest, Hungary. We investigated the kinetics of choline evoked currents, and the effect of the positive modulator PNU 120596 on alpha7 nicotinic acetylcholine receptors expressed by GH4C1 cells (obtained from Siena Biotech SpA) in whole-cell and outside-out patch-clamp experiments. using a theta-tube system for rapid application of agonists, we measured the dependence of current kinetics on the solution exchange rate. By extrapolation and by kinetic modeling we es- timated the intrinsic kinetics of the receptor: what would be the amplitude and kinetics of choline-evoked currents at instantaneous agonist application. In the presence of PNU 120596 the single channel mean open time is dras- tically prolonged, this allowed us to determine the ratio of simultaneously open channels using nonstationary fluctuation analysis. By determining the approximate number of channels in a patch, we could determine the peak open probability of 10 mM choline-evoked currents in the absence of the modulator (0.0333 5 0.0056), as well as the open probability in the pres- ence of 10 mM choline and 10 mM PNU 120596 (0.632 5 0.065). We per- formed kinetic experiments to determine the affinity of PNU 120596 to three different conformational states of the receptor: resting state, desensitized state and a slowly developing second desensitized state. We found that PNU 120596 was ineffective at both the resting and the slow desensitized states, while it bound to the desensitised state and re-activated the receptors. We investigated the nature of cooperativity between the agonist and the modulator. We found that while the agonist increases the apparent affinity of the modulator only by inducing desensitized conformation (which is pre- ferred by the modulator), the modulator induces a true increase of agonist affinity probably by allosterically affecting the conformation of the agonist binding site itself. 3262-Pos Board B417 Anesthetic Binding Sites within the Transmembrane Domain of the a7 Nicotinic Acetylcholine Receptor Vasyl Bondarenko, David Mowrey, Yan Xu, Pei Tang. University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. General anesthetics inhibit the a7 neuronal nicotinic acetylcholine receptor (nAChR) with different sensitivities, but the location of anesthetic binding sites remains unclear. Here we used high-resolution solution NMR to study the interaction of general anesthetics ketamine and halothane with the trans- membrane domain (TMD) of the human a7 nAChR. The TMD was expressed in E. coli and purified into detergent LDAO micelles. The specific residues involved in anesthetic binding were identified by anesthetics-induced chemi- cal shift changes in 1 H- 15 N HSQC NMR spectra. Ketamine binds to an intra- subunit site involving residues of all four helices at the intracellular end of the TMD. Two halothane interaction sites were detected. The first site was located near the intracellular end of the TMD, similar to the ketamine site, but in- volved only residues in TM1 and TM2. The second halothane site was formed by residues in the middle of TM1 and TM3. This site was not observed for ketamine binding, probably because ketamine was too large to penetrate deep into the TMD. Interestingly, unlike the previous finding in the a4b2 nAChR, anesthetic binding to the extracellular end of the TMD was not ob- served in the a7 nAChR. This difference may be responsible for their distinct sensitivity to halothane. In addition, halothane modulated motion less in a7 than in a4b2 on the micro- to milli-second (ms-ms) timescale, as demonstrated by changes in peak intensity, line width, and peak splitting for some residues. This work was supported by NIH grants: R01GM066358, R01GM056257, and R37GM049202. 3263-Pos Board B418 Probing the Trans-Membrane Domain of GLIC, a Prokaryotic Ligand- Gated Ion Channel Mona A. Alqazzaz, Sarah CR Lummis. University of Cambridge, Cambridge, United Kingdom. The Gloeobacter ligand-gated ion channel, GLIC, has up to 28% sequence identity with eukaryotic Cys-loop receptors, and many key residues are con- served, especially in the 2nd trans-membrane pore lining region, M2. The M2 region is responsible for ion selectivity, ion flux, and binding a wide range of non-competitive inhibitors (Alqazzaz et al., 2011). The aim of this work was to investigate the GLIC M2 region, especially His235 (or 11’), which has been proposed as essential in proton sensing linked to channel opening (Wang et al., 2012), and residues that are conserved across the Cys-loop receptor family. To probe the roles of specific amino acids in GLIC M2 trans-membrane domain, we substituted M2 residues lining the ion pore and M2 residues facing M3 trans-membrane domains. We generated over 40 mutations at various positions including those at Glu 222(-2’), Thr 226 (2’), Ser 230 (6’), Leu 232 (8’), Ile 233 (9’), Ala 234 (10’), Ile 236 (12’), Ala 237 (13’) and Phe 238 (14’) using site- directed mutagenesis, and tested their function using two-electrode voltage clamp as previously described (Alqazzaz et al., 2011). Our results showed that Ser6’, Ile9’ and His11’ residues are very sensitive to subsitution with all substituents resulting in non-functional receptors. We conclude that His 11’ has a role in channel opening/closing, and the residues Thr226, Ser230, and Ile233 also play an important role in receptor function. Alqazzaz M, Thompson AJ, Price KL, Breitinger HG, Lummis SC (2011). Cys- loop receptor channel blockers also block GLIC. Biophysical journal 101(12): 2912-2918. Wang HL, Cheng X, Sine SM (2012). Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. The Journal of biolog- ical chemistry 287(9): 6482-6489. 3264-Pos Board B419 Functional Modulation of the ELIC by General Anesthetics and Alcohols Qiang Chen 1 , Tommy S. Tillman 2 , Yan Xu 3 , Pei Tang 4 . 1 Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA, 2 Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 3 Department of Anesthesiology, Pharmacology and Chemical Biology, and Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 4 Department of Anesthesiology, Pharmacology and Chemical Biology, and Computational and System Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. ELIC is a prokaryotic pentameric ligand gated ion channel (pLGIC) homolo- gous to Cys-loop receptors, which are molecular targets of general anesthetics and alcohols. Crystal structures of ELIC and its complex with the antagonist acetylcholine have been resolved recently, suggesting the possibility of using ELIC to understand the structural basis of anesthetic and alcohol modulation on pLGICs. However, the pharmacological profiles of ELIC for modulation by general anesthetics and alcohols have not been well defined. In this study, we characterized these profiles for ELIC expressed in Xenopus laevis oocytes with two-electrode voltage clamp techniques. We found that the ELIC current elicited by the agonist propylamine could be inhibited by both volatile and intravenous general anesthetics at clinically relevant concentrations. ELIC is also inhibited by ethanol and other n-alcohols with potency increasing with the number of carbons until n-nonanol, where inhibition is cut off. Alcohol modulation on ELIC is similar to that on GABAR-r1 but different from nAChRs, which are potentiated by ethanol. In addition, ELIC is inhibited by the benzodiazepine drug diazepam. In summary, ELIC shares some pharmaco- logical characteristics of cation-conducting eukaryotic pLGICs and is a suitable model for the structural study of the actions of general anesthetics and alcohols on pLGICs. Supported by NIH (R01GM066358, R01GM056257, and R37GM049202). 3265-Pos Board B420 Alcohol and Anesthetic Binding to Pentameric Ligand-Gated Ion Channels Revealed in a Prokaryotic Model System Rebecca J. Howard 1 , Ludovic Sauguet 2 , Torben Broemstrup 3 , Samuel Murail 2 , Ui S. Lee 4 , Suzzane Horani 4 , James R. Trudell 5 , Pierre-Jean Corringer 2 , Erik Lindahl 3 , Marc Delarue 2 , R Adron Harris 4 . 1 Skidmore College, Saratoga Springs, NY, USA, 2 Institut Pasteur, Paris, France, 3 Royal Institute of Technology, Stockholm, Sweden, 4 The University of Texas at Austin, Austin, TX, USA, 5 Stanford University School of Medicine, Stanford, CA, USA. Wednesday, February 6, 2012 635a