Exploring Structural Interactions of Tarantula Toxins with Lipid Membranes using Rosetta and Molecular Dynamics Simulation

Tuesday, March 1, 2016 > meta-carborane derivative. Both ortho-boronicaine and dimethyl metaboronicaine had longer durations of analgesia than lidocaine. Differences in analgesic efficacies are rationalized by variations in chemical structure and protein binding characteristics. Preliminary studies using expressed voltage gated Na channels show that these compounds block channel currents in a state dependent and reversible manner. Financial support to G.R.K. from the University of Missouri Intellectual Property Fast Track Funding Program is acknowledged. 2207-Pos Board B351 Use-Dependent Block of Human Cardiac Sodium Channels by GS967 Franck Potet, Alfred L. George Jr. Northwestern University, Chicago, IL, USA. GS967 is a recently described selective late sodium current (INaL) blocker that has been shown to exert potent antiarrhythmic effects in rabbit ventricular myocytes exposed to ATX-II. The antiarrhythmic mechanism was attributed to preferential suppression of INaL with little effect on peak sodium current (INaP). We examined the effects of GS967 on INaP and INaL recorded in tsA201 cells expressing human NaV1.5. As previously described, we observed that 1 mM GS967 exhibited tonic block of INaL (63%) to a significantly greater extent than INaP (20%) without modifying the inactivation kinetics. To test the use-dependent block (UDB) of NaV1.5, we applied a series of 50 short depolarizing pulses (20 ms) at 20 mV with different frequencies (2, 10 and 20 Hz). GS967 caused a reduction of INaP in a frequency-dependent manner (block of 31%, 73% and 85% at 2, 10 and 20 Hz, respectively) consistent with UDB. This UDB was best explained by a significant slowing of recovery from fast and slow inactivation with a significant enhancement of slow inactivation in the presence of GS967. Using the SyncroPatch 384, we observed GS967 to be a more potent use-dependent blocker of INaP (IC50 = 0.07 mM) than ranolazine (16 mM) and lidocaine (17 mM). While, GS967 was found to exert these same effects on a prototypical long-QT syndrome mutation (delKPQ), an engineered mutation at an interaction site for local anesthetics (LA) (F1760A) attenuated the effect of GS967 on UDB. Lastly, an engineered mutation known to blunt slow inactivation (F1417C) also significantly reduced the effect of GS967 on UDB. We conclude that GS967 is a preferential inhibitor of INaL human NaV1.5, but it also exerts a previously unreported strong effect on slow inactivation and recovery from inactivation resulting in substantial UDB. 2208-Pos Board B352 Conformational Changes of the NMDA Receptors Associated with Ethanol-Induced Inhibition Hamid Reza Noori1,2, Christian Mu¨cksch2, Herbert Urbassek2. 1 Institute of Psychopharmacology, Central Institute of Mental Health, Mannheim, Germany, 2Department of Physics, Technical University of Kaiserslautern, Kaiserslautern, Germany. Alcohol addiction ranks among the leading global causes of preventable death and disabilities in human population. Understanding the sites of ethanol action that mediate its acute and chronic effects is critical to develop appropriate treatment options for this disorder. The N-methyl-D-Asparate (NMDA) receptors are ligand- and voltage-gated heterotetrameric ion channels, which not only play a key role in the development and function of the brain but are also known to directly interact with alcohol in a concentrationdependent manner. However, the exact molecular mechanisms and conformational dynamics of this interaction are not yet well understood. Computational studies may shed light at spatiotemporal dimensions that pose limitations to experimental investigations and thereby improve our understanding of the molecular interactions of alcohol with the NMDA receptors in a hypothesis-free manner. Here, we conducted a series of molecular dynamics simulations of the interaction of ethanol molecules at a moderate concentration (20 mM) with the wild-type ligand-free crystal structure GluN1-GluN2B NMDA ˚ resolution) under physiological conditions. The coReceptor of rat (4 A agonists glutamate and glycine were distributed randomly within the simulation box at concentrations reflecting the active zone of vesicular release. In total, eight configurations were simulated (100 ns/configuration) to investigate sites of action of ethanol as well the conformational dynamics of the NMDA receptor in absence/presence of each co-agonist or ethanol and any combination thereof. The simulations suggest that by a double hydrogen-bond ( 2.9 kcal/mol) with tryptophan 635 and phenylalanine 638 located at the transmembrane M3 helix of the GluN2B subunit, alcohol not only reduces the pore radius of the ion channel within the TMD but also decreases accessibility of glutamate to the ligand binding sites by altering the structure of the ligand binding domain and significantly widening the receptor in that area.

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2209-Pos Board B353 Exploring Structural Interactions of Tarantula Toxins with Lipid Membranes using Rosetta and Molecular Dynamics Simulation Phuong T. Nguyen1,2, Jon T. Sack2, Toby W. Allen3, Vladimir Yarov-Yarovoy2. 1 Biophysics Graduate Group, UC Davis, Davis, CA, USA, 2Physiology and Membrane Biology, UC Davis, Davis, CA, USA, 3RMIT University, Melbourne, Australia. Tarantula venom toxins belong to a class of cystine knot peptide toxins that have three disulfide bridges stabilizing the inner core of the toxin structure. These toxins target a wide range of ion channels and modulate physiological activities of channels upon binding. Experimental data indicates that tarantula toxins which target voltage sensors of ion channels interact with cell membrane bilayers and potentially form interactions with channels at the membraneprotein interface. However, how these peptide toxins partition and orient in the membrane are not fully understood. We used Rosetta structural modeling and molecular dynamics simulation, to embed three tarantula toxins, VsTx1, GxTx-1E and SgTx1, into lipid membrane environments. Our results suggest that the three tarantula toxins prefer a similar orientation and depth in the membrane bilayer. We observe that hydrophobic residues on the toxins embed deeper in the hydrophobic lipid core region, basic residues prefer interactions with lipid head groups, and acidic residues are exposed to water environment. This unique orientation of peptide toxins exposes residues critical for interaction with the voltage sensors within the lipid environment, suggesting that toxins may utilize this embedding and orientation to interact favorably with voltage sensors of ion channels. 2210-Pos Board B354 Validation of KCa3.1 Channel Small Molecules Interaction Sites Predicted by Rosetta Hai M. Nguyen1, Latika Singh1, Heike Wulff1, Vladimir Yarov-Yarovoy2. 1 Pharmacology, University of California, Davis, CA, USA, 2Physiology and Membrane Biology, University of California, Davis, CA, USA. The calcium-activated intermediate-conductance KCa3.1 channel is an effective regulator of intracellular calcium and an attractive pharmacological target for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, the development of drugs for this medically relevant channel is hindered by the unavailability of a crystal structure useful for structure-guided drug design. Using the Rosetta modeling method we generated a homology model of the KCa3.1 channel transmembrane region using the Kv1.2-Kv2.1 channel structure (pdb id: 2R9R) as a template. Docking of known KCa3.1 small molecule blockers into the KCa3.1 model, identified, surprisingly, two independent sites for the dihydropyridine nifedipine and for its isoster methyl-5-acetyl-4-(4-chloro-3-(trifluoromethyl)phenyl)-2,6dimethyl-4H-pyran-3-carboxylate. While nifedipine is predicted to bind in the fenestration between the pore-lining S5 and S6 segments, the pyran isoster has its lowest energy binding configurations in the inner pore region. We validated these predictions via site-directed mutagenesis and patch-clamp recording. Blocking of KCa3.1 by nifedipine was significantly reduced by replacing the side chains at the fenestration positions L209, T212 and V272 with either alanine or valine. Replacement with bulkier phenylalanines confirmed T212 and V272 as the main interacting sites for nifedipine without compromising the affinity of inner pore blockers like TRAM-34 or the 4-phenyl pyran. In contrast, the inner pore T250S and V275A mutants, which are known to nullify TRAM-34 binding disrupted binding of the 4-phenyl pyran but did not interfere with nifedipine binding. We conclude that the Rosetta modeling approach can be useful to distinguish the molecular mechanisms of action of KCa channel modulators and has promising potential in guiding the development of clinically relevant drugs. 2211-Pos Board B355 Cardiac Small Conductance Calcium-Activated K Channels Maintain Repolarization Reserve in a Pharmacological Model of Type 3 Long QT Syndrome Jum Suk Ko, Dechun Yin, Thomas H. Everett, Zhenhui Chen, Michael Rubart, Peng-Sheng Chen. Medicine, Indiana University, Indianapolis, IN, USA. Drug-induced long QT syndrome (diLQT) is a major public health problem. Whether small conductance calcium-activated K (SK) channels are involved in modulating cardiac repolarization reserve in diLQT is unclear. To examine their role in diLQT, we determined the effects of SK current inhibition on optically measured action potential duration at 80% repolarization (APD80) in isolated perfused, immobilized (10 microM 5-blebbistatin) mouse hearts, in which ventricular repolarization was prolonged by the late Na current (INa-L) activator ATX-II (15 nM). ATX-II at this concentration had no significant