- Email: [email protected]
Analyzing the Effects of Lipid Type on the α-Hemolysin Nanopore and 5HT3 Receptor Structure and Gating using Molecular Dynamics Simulations
Monday, February 13, 2017 1345-Pos Board B413 Proton Sensitivity of NCX: Modulation by Na, Ca and a Distinct ProtonSensing Domain Scott John1, Brian Kim2, Riccardo Olcese3, Joshua I. Goldhaber4, Michela Ottolia3. 1 Cardiology, UCLA, Los Angeles, CA, USA, 2Heart Institute, Cedars Sinai, Los Angeles, CA, USA, 3Anesthesiology, UCLA, Los Angeles, CA, USA, 4 Heart Institute, Cedars Sinai, Los Angeles, CA, USA. The Naþ-Ca2þ exchanger (NCX) is a plasma membrane transporter expressed in cardiac myocytes where it extrudes Ca2þ on a beat to beat basis, thereby influencing contractility. NCX is strongly inhibited by cytosolic protons potentially leading to contractile dysfunction and cellular injury under pathological conditions associated with a decrease in intracellular pH (e.g. ischemia/reperfusion). The molecular mechanisms underlying this regulation remain elusive. Previous studies suggested that NCX pH inhibition is enhanced by intracellular Na and that may originate from displacement of Ca from two regulatory Ca2þbinding domains. Using giant patch voltage clamp and site-directed mutagenesis, we found that NCX pH sensitivity can develop independently from both Naþ and Ca2þ regulation. In the absence of Ca2þ regulation (E516L) pH sensitivity increased, while removal of Naþ-dependent inactivation (K229Q) caused a decrease in NCX apparent proton affinity. The data indicate that NCX pH regulation originates from a separate intrinsic pH sensor whose affinity for protons can be modulated by cytoplasmic Naþ and Ca2þ. Substitution of Histidines 124 and 165 and Lysine 229 drastically decreased both the apparent affinity of NCX for intracellular protons (K1/2 values are, mM: 0.0435 0.004 for WT and 0.2850.08 for mutant) and the Hill coefficient (1.8150.10 for WT and1.0150.01 for mutant). These results suggest that these residues are key components of the NCX pH sensor. 1346-Pos Board B414 Structure of a CLC-Family Fluoride/Proton Antiporter Nicholas B. Last1, Randy B. Stockbridge1, Ashley E. Brammer1, Tania Shane1, Ludmila Kolmakova-Partensky1, Akiko Koide2, Shohei Koide2, Christopher Miller1. 1 Brandeis Univ./HHMI, Waltham, MA, USA, 2NYU School of Medicine, New York, NY, USA. The CLC superfamily of anion transporters and channels has been studied for decades, but large swaths of the superfamily remain unexamined. One of those unstudied branches was recently found to be a family of fluoride/proton antiporters (CLCF), evolved to protect bacteria against intracellular fluoride accumulation. In addition to the clearly altered selectivity, these transporters also have a different transport stoichiometry from the chloride transporters (1 F- / 1 proton, vs 2 Cl- / 1 proton), suggesting a change in the transport mechanism. We have crystallized and solved the structure of CLCF-Eca, the fluoride CLC from Enterococcus casseliflavus, in complex with monobody crystalliza˚ . To our knowledge this is the first structure of a fluotion chaperones at 3.28 A ride CLC, and also the first structure of a non-chloride CLC-transporter. While some critical functional elements from the chloride CLCs remain, such as the external glutamate gate, other regions involved in both selectivity and ion coupling have been significantly altered. 1347-Pos Board B415 Repeat-Swap Homology Modeling of the Anion Exchanger AE1 Reveals an Elevator-Like Antiport Mechanism Emel Ficici1, Jose D. Faraldo-Gomez1, Lucy R. Forrest2, Michael L. Jennings3. 1 National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA, 2National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 3Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. Anion Exchanger 1 (AE1), or band 3, is a membrane transporter found in erythrocytes and the kidney collecting duct. As part of the process of carbon dioxide clearance from these tissues, AE1 exchanges the accumulated bicarbonate for chloride in the blood plasma. After years of extensive biochemical and functional studies, the structure of the AE1 C-terminal domain (ACTD), which is the integral membrane domain catalyzing the anion exchange, has been finally ˚ . The structure comprises two structural repeats of solved at a resolution of 3.5 A inverted transmembrane topology, organized into what has been referred as ‘core’ and ‘gate’ domains. The two repeats are, however, structurally asymmetric and the protein adopts an outward-facing conformation. Here, we gain insights into the nature of the alternating-access mechanism of this transporter, by constructing a model of the inward-facing state using the so-called repeatswap homology modeling method. Comparison of the resulting model of the
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inward-facing state with the outward-facing crystal structure reveals that anion translocation takes place by way of an elevator-type mechanism in which the core domain moves vertically relative to the gate domain, which forms the dimerization interface. Taken together, the structures can account qualitatively for a wide range of biochemical and functional data, and suggest new avenues of experimentation. 1348-Pos Board B416 Functional Characterization of the SLC-Transporters PepT1 and Oct2 by Electrophysiological Real-Time Measurements using a High throughput System Maria Barthmes, Andre Bazzone, Stephan Holzhauser, Maximilian Kellner, Niels Fertig, Michael George, Andrea Br€uggemann. Nanion Technologies, Munich, Germany. The SLC transporters PepT1 and OCT2 are of high interest in the development process of new pharmacological agents, due to their involvement in drug uptake and secretion. Other membrane transporters, in particular members of the SLC family, are coming more and more into focus as potential drug targets. Due to this growing interest in membrane transporters, there is an increasing demand for reliable high throughput systems, enabling the measurement of membrane transporter activity. Electrophysiological methods generate detailed and high quality data. However, conventional electrophysiology is in most cases not applicable or inefficient for the investigation of membrane transporters, due to their usually low turnover rates compared to ion channels. Here we present for the first time electrophysiological data of the SLC transporters PepT1 and Oct2, generated with a fully automated high throughput platform, applying solid-supported membrane (SSM) based electrophysiology. This method, proven by about 100 peer-reviewed publications, allows the recording of a large number of individual transport proteins in parallel, ensuring a better signal to noise ratio compared to patch clamp. Using this system, we were able to compare the kinetics (Km, vmax) of three different OCT2 substrates. By a competitive inhibitor assay we determined the IC50 of a PepT1 inhibitor. Furthermore, we performed several case studies of various targets, such as NaK-ATPase, mitochondrial transporters, channel rhodopsin, sugar transporters, and others. 1349-Pos Board B417 High Throughput Analysis of Membrane Transport by using Arrayed Water-In-Oil Droplet Bilayers Rikiya Watanabe1,2, Naoki Soga1, Hiroyuki Noji1. 1 Department of Applied Chemistry, The University of Tokyo, Bunkyo-ku, Japan, 2Presto, JST, Bunkyo-ku, Japan. The maintenance of an appropriate intracellular environment is a constant challenge for all living organisms, from prokaryotes to multicellular eukaryotes. Intracellular homeostasis is maintained by transporting various compounds such as ions, sugars, amino acids, and drugs across the cell membrane. Therefore, the analysis of transmembrane transport is crucial to understanding cell physiology as well as for exploring the bioavailability of drugs. In this study, we developed a novel micro-device in which a large number of droplet interface bilayers (> 500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate. The droplet volume was controllable from 3.5 to 350 fL by changing the hydrophobic/hydrophilic pattern on the device, allowing high-throughput analysis of membrane transport mechanisms including membrane permeability to solutes (e.g., ions or small molecules) with or without the aid of transport proteins. Thus, this novel platform will pave the way for novel analytical and pharmacological applications such as drug screening. 1350-Pos Board B418 Analyzing the Effects of Lipid Type on the a-Hemolysin Nanopore and 5HT3 Receptor Structure and Gating using Molecular Dynamics Simulations Nicholas B. Guros, Jeffery B. Klauda. Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA. Alpha-hemolysin (a-HL) is a transmembrane protein used to measure the size and charge of macromolecules. In this process, a-HL is inserted into a tethered lipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (PHPC) lipids. However, all previous molecular dynamics (MD) simulations attempting to model this system have used a more common lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), to represent the PHPCtethered bilayer. PHPC possesses highly methylated hydrophobic chains which creates a more rigid structure. The result is that simulations using POPC lipids yield channel currents at least 20% less than experimental values. MD simulations conducted with both PHPC and POPC lipids for both the full and a
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reduced form of the protein containing just the b-barrel indicate that PHPC lipids could more accurately describe the experimentally observed parameters. The 5-hydroxytryptamine receptor (5HT3) is the principle receptor responsible for modulating a wide range of neurological and digestive functions. Here we aim to better understand the response of the 5HT3 receptor to 5HT (serotonin) in an environment that mimics the native lipid composition. This work supports the experimental design of a field effect transistor (FET) device we are developing to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC)/cholesterol membrane, and 5HT to determine if ligand binding causes experimentally observed protein reassembly and channel opening. This work is unique in that these simulations possess a more realistic membrane composition and that they compare the conformation of the glycosylated protein to the non-glycosylated form providing structural details of the effects of glycosylation on the binding pocket and channel pore radius. 1351-Pos Board B419 Identification of the Ion Conduction Pathway in a TMEM16 Scramblase Tao Jiang1,2, Criss Hartzell3, Emad Tajkhorshid1,2. 1 Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2Beckman Institute for Advanced Science and Technology, Urbana, IL, USA, 3Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA. The TMEM16 family constitutes a class of widespread membrane proteins that is functionally split into ion channels and phospholipid scramblases. The recent crystallization of the fungal TMEM16 (nhTMEM16) has provided the first glimpse into the structural properties of TMEM16 proteins. This structure revealed a membrane-spanning hydrophilic crevice on the protein surface which is shown by our atomistic simulations to serve as the pathway for lipid movement. However, it is still puzzling how the same structure can also transport ions, because the structure does not reveal an obvious alternative pathway for ion permeation through the bilayer other than the path taken by phospholipids. In order to investigate the pathway and mechanism of ion permeation, and the weak ionic selectivity, we have performed multiple molecular dynamic (MD) simulations with nhTMEM16 embedded in asymmetric lipid bilayers under different levels of transmembrane electric potentials. The simulations reveal the formation of a ‘‘proteolipidic’’ pore along the hydrophilic crevice, where lipids play a structural role by lining the hydrophilic ion conduction pathway with their headgroups. Externalization of the anionic phospholipid PS along the pore wall was observed as water penetrates into the bilayer and extends across the membrane. Notably, ion permeation events through the aqueous pore formed between the protein and the lipid headgroups were also captured frequently for both cations (Naþ) and anions (Cl-). Moreover, the inclusion of various lipid compositions in the simulated systems allowed us to characterize the interaction between the ions and the pore-lining headgroups as they transverse the membrane. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s. 1352-Pos Board B420 Effect of Camp on the Human Erythrocyte KD/CA2D Exchanger Activity Daniel Landi-Conde, Alejandro Mata, Jesus G. Romero. Escuela de Biologı´a/ Lab. de Fisiologı´a Molecular y Biofisica IBE, Universidad Central Venezuela, Caracas, Venezuela, Bolivarian Republic of. Human erythrocyte has a half-life span of 120 days. The erythrocyte is a cell without nucleus so, this is not a process of classic apoptosis. The intracellular calcium concentration increases with the age of these cells. It has been suggested that the process of senescence of erythrocytes is related to the passage through the capillary bed. In our laboratory we have proposed the ‘‘hypothesis of Kþ’’, which is based on two mechanisms: the Human Erythrocyte Mechanoactivated Kþ Channel A and the Kþ/Ca2þ exchanger also proposed in our laboratory. The activation of this exchanger has a non-linear voltage dependence and a permeability sequence of Kþ> Rbþ >> Csþ and Ca2þ> Ba2þ >> Mg2þ. The cyclic adenosine monophosphate (cAMP) is a cyclic nucleotide generated from ATP by the action of the membrane protein adenylate cyclase, in response to various extracellular signals. This molecule acts as an important second messenger in most cell types. Its normal concentration in the cytosol is around 0.1 uM, but an extracellular signal can significantly increase its concentration in seconds. Here we present the effect of cAMP on the activity of the Kþ/Ca2þ exchanger of the Human Erythrocyte. cAMP produces a dose-dependent inhi-
bition on the exchanger activity (with a maximum of 45.33 5 5.12% in the direct mode and 58.64 5 15.68% in the reverse mode) presenting saturation at higher concentration than 1.0 uM. This inhibition has no dependence on membrane potential and this effect is exerted by cAMP on the permeation pathways (with a maximum of 42.66% inhibition in direct mode and a maximum of 54.29% inhibition in the reverse mode) and indeed it has no effect on the activation mechanism. Additionally, cAMP slow down the deactivation process of the exchanger by increasing Ʈd up to 215.22% in the direct mode and up to 229.25% for Ʈi in the reverse mode, in a dose-dependent manner, showing saturation at concentrations above 1.0 uM, this effect is also independent on the membrane potential. The existence of a binding site for cAMP in the Kþ/ Ca2þ exchanger intracellular face, showing a Ki in the submicromolar order, is proposed.
Energy Transducing Complexes and Electron and Proton Transfer 1353-Pos Board B421 Intramolecular Friedel-Crafts Acylation Promoted by Hexafluoroisopropanol Alexander Y.Z. Li. Chemistry (College of Arts and Sciences) and CICBDD (UNC Eshelman School of Pharmacy), University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. The Friedel-Crafts (FC) acylation, discovered by Charles Friedel and James Crafts, is utilized to synthesize aryl ketones. The traditional intermolecular FC acylation requires a stoichiometric amount of Lewis acid (i.e., AlCl3) catalysts to drive the reaction to completion due to complex formation between product ketones and catalysts. We have discovered a modified FC acylation where hexafluoroisopropanol (HFIP) was used as a promoter and solvent. Due to the strong hydrogenbond donor ability of HFIP, it was shown that multiple arylalkyl acyl chlorides are able to undergo intramolecular FC acylation in HFIP at room temperature. In this work, we seek to refine this procedure for the optimized synthesis of 6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one. The procedure involves (1) the conversion of 4-(3,4-dimethoxyphenyl)butanoic acid to 4-(3,4-dimethoxyphenyl)butanoyl chloride with thionyl chloride, followed by (2) the FC acylation of 4-(3,4-dimethoxyphenyl)butanoyl chloride to form 6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one in HFIP. This methodology illustrates an improvement over traditional FC acylation as it requires no additional reagents or catalysts and generates no byproducts. In addition, it is operationally simple, requiring only solvent evaporation and product purification after reaction completion. 1354-Pos Board B422 Tautomeric Equilibria of Cytosine and Isocytosine in Gas Phase and in Solution Avichai Fagan, Shuhua Ma. Chemistry, Towson University, Towson, MD, USA. Tautomerism is very important in computer-aided drug design because numerous compounds in small molecule databases could exist in different tautomeric structures. Structure determines the functionality of a small molecule when it is docked to the binding site of a target protein. Thus, if the structure of the ligand is not the most stable tautomer in the protein environment, the properties of the resulting protein-ligand complex will not be accurately calculated. Tautomeric equilibrium of a compound is highly influenced by the medium. For a specific ligand and its derivatives, it is therefore essential to understand potential links of tautomer stability in different media, as this information helps determine the tautomeric forms of a ligand used in docking studies. Cytosine and isocytosine are of pharmaceutical importance. It has been reported that cytosine and its derivatives can bind to the active site of E. Coli IspE protein, which is one of the attractive targets for the development of novel drugs against malaria and tuberculosis. Isocytosine and its derivatives are inhibitors of b-secretase (BACE-1) which is a key target for the treatment of Alzheimer’s disease. Both cytosine and isocytosine can exist in a number of tautomers. Understanding the relative stability of these tautomers in the gas phase and aqueous solution will help design inhibitors of IspE protein and BACE-1. We determined the two most stable tautomers of cytosine and isocytosine in gas phase by calculating the relative free energies of these tautomers. The tautomers with the lowest free energies in gas phase were then modeled in aqueous solution, free energy of solvation of these molecules have been calculated. The solvent effect on tautomeric equilibrium of cytosine and isocytosine will be discussed in detail.
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inward-facing state with the outward-facing crystal structure reveals that anion translocation takes place by way of an elevator-type mechanism in which the core domain moves vertically relative to the gate domain, which forms the dimerization interface. Taken together, the structures can account qualitatively for a wide range of biochemical and functional data, and suggest new avenues of experimentation. 1348-Pos Board B416 Functional Characterization of the SLC-Transporters PepT1 and Oct2 by Electrophysiological Real-Time Measurements using a High throughput System Maria Barthmes, Andre Bazzone, Stephan Holzhauser, Maximilian Kellner, Niels Fertig, Michael George, Andrea Br€uggemann. Nanion Technologies, Munich, Germany. The SLC transporters PepT1 and OCT2 are of high interest in the development process of new pharmacological agents, due to their involvement in drug uptake and secretion. Other membrane transporters, in particular members of the SLC family, are coming more and more into focus as potential drug targets. Due to this growing interest in membrane transporters, there is an increasing demand for reliable high throughput systems, enabling the measurement of membrane transporter activity. Electrophysiological methods generate detailed and high quality data. However, conventional electrophysiology is in most cases not applicable or inefficient for the investigation of membrane transporters, due to their usually low turnover rates compared to ion channels. Here we present for the first time electrophysiological data of the SLC transporters PepT1 and Oct2, generated with a fully automated high throughput platform, applying solid-supported membrane (SSM) based electrophysiology. This method, proven by about 100 peer-reviewed publications, allows the recording of a large number of individual transport proteins in parallel, ensuring a better signal to noise ratio compared to patch clamp. Using this system, we were able to compare the kinetics (Km, vmax) of three different OCT2 substrates. By a competitive inhibitor assay we determined the IC50 of a PepT1 inhibitor. Furthermore, we performed several case studies of various targets, such as NaK-ATPase, mitochondrial transporters, channel rhodopsin, sugar transporters, and others. 1349-Pos Board B417 High Throughput Analysis of Membrane Transport by using Arrayed Water-In-Oil Droplet Bilayers Rikiya Watanabe1,2, Naoki Soga1, Hiroyuki Noji1. 1 Department of Applied Chemistry, The University of Tokyo, Bunkyo-ku, Japan, 2Presto, JST, Bunkyo-ku, Japan. The maintenance of an appropriate intracellular environment is a constant challenge for all living organisms, from prokaryotes to multicellular eukaryotes. Intracellular homeostasis is maintained by transporting various compounds such as ions, sugars, amino acids, and drugs across the cell membrane. Therefore, the analysis of transmembrane transport is crucial to understanding cell physiology as well as for exploring the bioavailability of drugs. In this study, we developed a novel micro-device in which a large number of droplet interface bilayers (> 500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate. The droplet volume was controllable from 3.5 to 350 fL by changing the hydrophobic/hydrophilic pattern on the device, allowing high-throughput analysis of membrane transport mechanisms including membrane permeability to solutes (e.g., ions or small molecules) with or without the aid of transport proteins. Thus, this novel platform will pave the way for novel analytical and pharmacological applications such as drug screening. 1350-Pos Board B418 Analyzing the Effects of Lipid Type on the a-Hemolysin Nanopore and 5HT3 Receptor Structure and Gating using Molecular Dynamics Simulations Nicholas B. Guros, Jeffery B. Klauda. Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA. Alpha-hemolysin (a-HL) is a transmembrane protein used to measure the size and charge of macromolecules. In this process, a-HL is inserted into a tethered lipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (PHPC) lipids. However, all previous molecular dynamics (MD) simulations attempting to model this system have used a more common lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), to represent the PHPCtethered bilayer. PHPC possesses highly methylated hydrophobic chains which creates a more rigid structure. The result is that simulations using POPC lipids yield channel currents at least 20% less than experimental values. MD simulations conducted with both PHPC and POPC lipids for both the full and a
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Monday, February 13, 2017
reduced form of the protein containing just the b-barrel indicate that PHPC lipids could more accurately describe the experimentally observed parameters. The 5-hydroxytryptamine receptor (5HT3) is the principle receptor responsible for modulating a wide range of neurological and digestive functions. Here we aim to better understand the response of the 5HT3 receptor to 5HT (serotonin) in an environment that mimics the native lipid composition. This work supports the experimental design of a field effect transistor (FET) device we are developing to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC)/cholesterol membrane, and 5HT to determine if ligand binding causes experimentally observed protein reassembly and channel opening. This work is unique in that these simulations possess a more realistic membrane composition and that they compare the conformation of the glycosylated protein to the non-glycosylated form providing structural details of the effects of glycosylation on the binding pocket and channel pore radius. 1351-Pos Board B419 Identification of the Ion Conduction Pathway in a TMEM16 Scramblase Tao Jiang1,2, Criss Hartzell3, Emad Tajkhorshid1,2. 1 Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2Beckman Institute for Advanced Science and Technology, Urbana, IL, USA, 3Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA. The TMEM16 family constitutes a class of widespread membrane proteins that is functionally split into ion channels and phospholipid scramblases. The recent crystallization of the fungal TMEM16 (nhTMEM16) has provided the first glimpse into the structural properties of TMEM16 proteins. This structure revealed a membrane-spanning hydrophilic crevice on the protein surface which is shown by our atomistic simulations to serve as the pathway for lipid movement. However, it is still puzzling how the same structure can also transport ions, because the structure does not reveal an obvious alternative pathway for ion permeation through the bilayer other than the path taken by phospholipids. In order to investigate the pathway and mechanism of ion permeation, and the weak ionic selectivity, we have performed multiple molecular dynamic (MD) simulations with nhTMEM16 embedded in asymmetric lipid bilayers under different levels of transmembrane electric potentials. The simulations reveal the formation of a ‘‘proteolipidic’’ pore along the hydrophilic crevice, where lipids play a structural role by lining the hydrophilic ion conduction pathway with their headgroups. Externalization of the anionic phospholipid PS along the pore wall was observed as water penetrates into the bilayer and extends across the membrane. Notably, ion permeation events through the aqueous pore formed between the protein and the lipid headgroups were also captured frequently for both cations (Naþ) and anions (Cl-). Moreover, the inclusion of various lipid compositions in the simulated systems allowed us to characterize the interaction between the ions and the pore-lining headgroups as they transverse the membrane. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s. 1352-Pos Board B420 Effect of Camp on the Human Erythrocyte KD/CA2D Exchanger Activity Daniel Landi-Conde, Alejandro Mata, Jesus G. Romero. Escuela de Biologı´a/ Lab. de Fisiologı´a Molecular y Biofisica IBE, Universidad Central Venezuela, Caracas, Venezuela, Bolivarian Republic of. Human erythrocyte has a half-life span of 120 days. The erythrocyte is a cell without nucleus so, this is not a process of classic apoptosis. The intracellular calcium concentration increases with the age of these cells. It has been suggested that the process of senescence of erythrocytes is related to the passage through the capillary bed. In our laboratory we have proposed the ‘‘hypothesis of Kþ’’, which is based on two mechanisms: the Human Erythrocyte Mechanoactivated Kþ Channel A and the Kþ/Ca2þ exchanger also proposed in our laboratory. The activation of this exchanger has a non-linear voltage dependence and a permeability sequence of Kþ> Rbþ >> Csþ and Ca2þ> Ba2þ >> Mg2þ. The cyclic adenosine monophosphate (cAMP) is a cyclic nucleotide generated from ATP by the action of the membrane protein adenylate cyclase, in response to various extracellular signals. This molecule acts as an important second messenger in most cell types. Its normal concentration in the cytosol is around 0.1 uM, but an extracellular signal can significantly increase its concentration in seconds. Here we present the effect of cAMP on the activity of the Kþ/Ca2þ exchanger of the Human Erythrocyte. cAMP produces a dose-dependent inhi-
bition on the exchanger activity (with a maximum of 45.33 5 5.12% in the direct mode and 58.64 5 15.68% in the reverse mode) presenting saturation at higher concentration than 1.0 uM. This inhibition has no dependence on membrane potential and this effect is exerted by cAMP on the permeation pathways (with a maximum of 42.66% inhibition in direct mode and a maximum of 54.29% inhibition in the reverse mode) and indeed it has no effect on the activation mechanism. Additionally, cAMP slow down the deactivation process of the exchanger by increasing Ʈd up to 215.22% in the direct mode and up to 229.25% for Ʈi in the reverse mode, in a dose-dependent manner, showing saturation at concentrations above 1.0 uM, this effect is also independent on the membrane potential. The existence of a binding site for cAMP in the Kþ/ Ca2þ exchanger intracellular face, showing a Ki in the submicromolar order, is proposed.
Energy Transducing Complexes and Electron and Proton Transfer 1353-Pos Board B421 Intramolecular Friedel-Crafts Acylation Promoted by Hexafluoroisopropanol Alexander Y.Z. Li. Chemistry (College of Arts and Sciences) and CICBDD (UNC Eshelman School of Pharmacy), University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. The Friedel-Crafts (FC) acylation, discovered by Charles Friedel and James Crafts, is utilized to synthesize aryl ketones. The traditional intermolecular FC acylation requires a stoichiometric amount of Lewis acid (i.e., AlCl3) catalysts to drive the reaction to completion due to complex formation between product ketones and catalysts. We have discovered a modified FC acylation where hexafluoroisopropanol (HFIP) was used as a promoter and solvent. Due to the strong hydrogenbond donor ability of HFIP, it was shown that multiple arylalkyl acyl chlorides are able to undergo intramolecular FC acylation in HFIP at room temperature. In this work, we seek to refine this procedure for the optimized synthesis of 6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one. The procedure involves (1) the conversion of 4-(3,4-dimethoxyphenyl)butanoic acid to 4-(3,4-dimethoxyphenyl)butanoyl chloride with thionyl chloride, followed by (2) the FC acylation of 4-(3,4-dimethoxyphenyl)butanoyl chloride to form 6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one in HFIP. This methodology illustrates an improvement over traditional FC acylation as it requires no additional reagents or catalysts and generates no byproducts. In addition, it is operationally simple, requiring only solvent evaporation and product purification after reaction completion. 1354-Pos Board B422 Tautomeric Equilibria of Cytosine and Isocytosine in Gas Phase and in Solution Avichai Fagan, Shuhua Ma. Chemistry, Towson University, Towson, MD, USA. Tautomerism is very important in computer-aided drug design because numerous compounds in small molecule databases could exist in different tautomeric structures. Structure determines the functionality of a small molecule when it is docked to the binding site of a target protein. Thus, if the structure of the ligand is not the most stable tautomer in the protein environment, the properties of the resulting protein-ligand complex will not be accurately calculated. Tautomeric equilibrium of a compound is highly influenced by the medium. For a specific ligand and its derivatives, it is therefore essential to understand potential links of tautomer stability in different media, as this information helps determine the tautomeric forms of a ligand used in docking studies. Cytosine and isocytosine are of pharmaceutical importance. It has been reported that cytosine and its derivatives can bind to the active site of E. Coli IspE protein, which is one of the attractive targets for the development of novel drugs against malaria and tuberculosis. Isocytosine and its derivatives are inhibitors of b-secretase (BACE-1) which is a key target for the treatment of Alzheimer’s disease. Both cytosine and isocytosine can exist in a number of tautomers. Understanding the relative stability of these tautomers in the gas phase and aqueous solution will help design inhibitors of IspE protein and BACE-1. We determined the two most stable tautomers of cytosine and isocytosine in gas phase by calculating the relative free energies of these tautomers. The tautomers with the lowest free energies in gas phase were then modeled in aqueous solution, free energy of solvation of these molecules have been calculated. The solvent effect on tautomeric equilibrium of cytosine and isocytosine will be discussed in detail.