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Silica Nanoparticles Induce Calcium-Permeable Pores in Plasma Membranes
Tuesday, February 14, 2017 compartment, so that the plug and pore ring could again seal the channel. Here we prevented the intermediate from either backsliding or exiting through the lateral gate by (i) stalling a highly charged helix, the voltage sensor domain S4 of the potassium channel KvAP in the SecYEG pore and (ii) locking it between the ribosome on one side of the membrane and calmodulin on the other side. Dc was unable to gate this complex. We observed several conductivity levels, suggesting that the S4 helix was free to sample between the aqueous environment of the pore and the lipid interior. This observation (i) confirms the general view of how the translocon distinguishes membrane proteins from secretory proteins and (ii) disproves the hypothesis that the pore ring acts to maintain the membrane barrier in an active translocon. The work was supported by a grant from the Austrian science fund to Peter Pohl (P28213). 1. Saparov SM, et al. (2007) Determining the conductance of the SecY protein translocation channel for small molecules. Mol Cell 26(4):501-509. 2. Park E & Rapoport TA (2011) Preserving the membrane barrier for small molecules during bacterial protein translocation. Nature 473(7346):239-242. 3. Knyazev DG, Winter L, Bauer BW, Siligan C, & Pohl P (2014) Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation. J Biol Chem 289(35):24611-24616. 2042-Pos Board B362 A Designer Peptide Toxin Isolated by Phage Display that Inhibits the Human Voltage-Gated Proton Channel, hHv1 Kelleigh Kennedy1, Ruiming Zhao1, Qufei Li2, Eduardo Perozo2, Gerardo A. De Blas3, Martin A. Pavarotti3, Rodolfo Arias3, Luis S. Mayorga3, Steve A.N. Goldstein1. 1 Brandeis University, Waltham, MA, USA, 2University of Chicago, Chicago, IL, USA, 3National University of Cuyo, Mendoza, Argentina. hHv1 is critical to immune defense, maintaining pH homeostasis in white blood cells by extruding protons during respiratory burst to compensate for reactive oxygen species (ROS) production, and during sperm capacitation causing cytosolic alkalization. Hindering basic and clinical research, hHv1 is an orphan receptor without a potent and specific blocker. hHv1 is homodimeric; each subunit has a conduction pathway and is homologous to the four membrane-spanning segments that form the voltage sensing domains (VSD) in KV and NaV channels. To produce a toxin ligand for hHv1, we employed a phage display strategy whereby ~1 million novel peptides were fabricatedon an inhibitor cysteine knot (ICK) scaffold, a backbone stabilized by three disulfide bonds and found in nature to be rich in VSD-directed toxins. Designed by combinatorial permutation of 110 venom toxins, phage sorting was performed on purified recombinant hHv1 and specificity of binding validated by ELISA. Five novel peptides were identified (C2-C6), synthesized, and studied by external application to hHv1 channels expressed in HEK293T cells. Proton currents measured by whole cell patch clamp were inhibited by C6. Consistent with VSD trapping, C6 slows activation, accelerates deactivation, and shifts activation to more depolarized voltages. Inhibition by C6 is partial, showing at most 50% block at þ40 mV (1 mM) with a Ki of 150 nM. Partial block was not due to partial occupancy of the dimer: two C6 were seen per dimer by single particle photobleaching using C6 labeled with 5,6-TAMRA and hHv1 tagged with TFP. Moreover, a monomeric hHv1 channel was also blocked by only 50% with a Ki of 110 nM and bound one fluorescent C6 peptide. Of note, a point mutation in the channel epitope where C6 binds increased toxin affinity and produced complete blockade (accompanying abstract by Zhao, Kennedy et al). C6 was isolated on purified hHv1 protein but shown to inhibit two cellular responses proposed to depend on native hHv1 function. Human whole blood cells stimulated by PMA release ROS and this was suppressed in a dose dependent manner by C6 (IC50 100 nM). C6 at 20 mM also specifically suppressed the cytosolic calcium increase and acrosome reaction triggered by progesterone in human sperm. Both ROS generation and capacitation were insensitive to 10 or 20 mM of scorpion toxin blockers of Kv1.3 Kþ channels. 2043-Pos Board B363 Identification of Activating and Inhibiting Gating Modifier Toxins that Target the S3-S4 Loop of the Human Proton Channel, hHv1 Ruiming Zhao1, Kelleigh Kennedy1, Qufei Li2, Eduardo Perozo2, Steve A.N. Goldstein1. 1 Brandeis University, Waltham, MA, USA, 2University of Chicago, Chicago, IL, USA. Human Hv1 channels (hHv1) are implicated in a wide variety of cellular functions including reactive oxygen species production, sperm capacitation and cell proliferation in cancer. Each channel has two hHv1 subunits that are homologous to the S1-S4 voltage sensing domains (VSD) in voltage-gated Kþ and Naþ channels. The S3-S4 loop of VSD is a hot spot for interaction of known gating modifier toxins, which either activate or inhibit by stabilizing the
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VSD in different conformations. In the accompanying abstract (Kennedy, Zhao, et al.), we describe isolation of C5 and C6 from ~1 million member phage display library constructed on an inhibitor cysteine knot (ICK) scaffold, a backbone found in many gating modifier neurotoxins. As expected, the S3-S4 loop of hHv1 was found to be the major binding epitope for C6 toxin. Whereas, ci-Hv1 channels from sea squirt were insensitive to 1 mM C6, transplanting the hHv1 S3b-S4a motif to ci-Hv1 conferred the chimera with sensitivity (maximal blockade 70%; half maximal inhibition at 35 nM); the hHv1 motif was also able to confer C6 blockade to otherwise insensitive Kv2.1. Scanning the motif in hHv1 by Cys scanning across 12 residues revealed four sites that decreased affinity of C6 over 3-fold, one site that increased inhibition 75-fold (and permits 100% inhibition), and one site that allows C6 binding to activate the channel by 30% (half maximal activation at 250 nM). Consistent with a shared VSD trapping mechanism, C6 inhibits whereas C5 activates, yet both modify the kinetics of activation and deactivation as well as the voltage-dependence of gating, and while they are 39 and 41 residues, respectively, the C-terminal 31 residues are identical. A tethered-toxin strategy has been employed to map the C6 interaction surface with hHv1 as a prelude to designing more potent inhibitors and activators; ten important residues that are charged or polar have been identified. 2044-Pos Board B364 HCN3 Channel Expression in Human Leukocytes Chiara Piantoni1, Angelica Gualdoni1, Claudia Bazzini1, Raffaella Milanesi1, Miryam Adelfio1, Annalisa Bucchi1, Andrea Barbuti1, Matteo Pecchiari2, Mirko Baruscotti1, Dario DiFrancesco1. 1 Department of Biosciences, Universita` degli Studi di Milano, Milan, Italy, 2 Department of Pathophysiology and Transplantation, Universita` degli Studi di Milano, Milan, Italy. Ivabradine is a heart rate-reducing agent, which specifically and selectively blocks pacemaker HCN channels. Strong evidence shows that Ivabradine also reduces inflammatory processes and the suggested mechanism of action is the modification of the vascular shear stress caused by the primary bradycardic effect of the drug (Li et al., 2008 Plos one 11(2): e0149694.doi:10.1371). Leukocytes, the key players of the immune response, express several ion channels (Kv1.3, KCa3.1, TRPM4, ORAI1 and others) which control the functional state of the cell and are therefore involved in cell activation and inflammatory processes. Since HCN channels are important determinants of membrane potential in both excitable and non-excitable cell types, we investigated the presence of these channels in leukocytes. Although qualitative PCR experiments revealed a positive amplification of all HCN mRNA isoforms (HCN1-2-3-4), preliminary quantitative PCR carried out in total human leukocytes showed a reliable signal only for HCN3. We therefore proceeded with the investigation of HCN3 channel expression in human leukocytes. Immunolabelling with commercial anti-HCN3 antibody confirmed the presence of this isoform in human total leukocytes. Flow cytometry analysis revealed that the major sources of HCN3 signal appear to be granulocytes and lymphocytes. We finally tested the effect of Ivabradine (1 mM) on the activation process of human total leukocytes. The results show a 29% (n=5) reduction in the activation readout parameters in the presence of Ivabradine. These results raise the possibility that the mechanism by which Ivabradine reduces inflammation is a direct binding to HCN3 channels in human leukocytes and we therefore suggest that HCN3 channels may play a functional role in specific human leukocytes subpopulations. Future electrophysiological experiments will further investigate this possibility. 2045-Pos Board B365 Silica Nanoparticles Induce Calcium-Permeable Pores in Plasma Membranes Alicia Sanchez, Kateryna Demydenko, Peter Hoet, Karel Talavera. KU Leuven, Leuven, Belgium. Amorphous silica nanoparticles (SiNPs) are extensively used for their beneficial properties in cosmetics and food industry as an additive and fining agent. However, the high levels of exposure have raised concerns about health hazards, and it has been shown that SiNPs can penetrate tissues and cells resulting in health problems. In this study we first evaluated the effects of SiNPs on intracellular Ca2þ concentration ([Ca2þ]i) in different cell types (HEK293, CHO and mouse airway epithelial cells). We found that extracellular application of 9 nm SiNPs (LudoxÒ) at 25 C produced a pronounced increase in [Ca2þ]i when the cells were co-stimulated with arachidonic acid or LPS. These effects were not observed when Ca2þ was omitted in the extracellular solution, indicating that SiNPs induce a Ca2þ entry pathway through the plasma membrane. Similar results were obtained when cells were exposed to SiNPs and heating from 25 to 35 C, suggesting that SiNPs are sufficient to induce the Ca2þ entry
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pathway at physiological temperatures. Whole-cell patch-clamp experiments revealed that SiNPs trigger large currents that could be blocked by the cation channel blocker ruthenium red in a voltage-dependent manner. Analysis of the selectivity properties of this current showed that they are mainly carried by cations. Taken together, our results demonstrate that SiNPs induce Ca2þ-permeable pores in the plasma membrane, and that this phenomenon is enhanced by factors that increase membrane fluidity. We propose that this Ca2þ entry pathway may be relevant for the toxicological properties of SiNPs.
2. Zhou, et al & Guo, P. (2016). Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity. Biomaterials, 105: 222. 3. Wendell, et al & Guo, P. (2009). Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature nanotechnology, 4:765. 4. Haque, et al & Guo, P. (2012). Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor. ACS nano, 6:3251.
2046-Pos Board B366 How to Get Large Drugs through Small Pores? Exploiting the Porins Pathway in Pseudomonas Aeruginosa Susruta Samanta1, Tommaso D’Agostino1, Ishan Ghai2, Monisha Pathania3, Silvia Acosta Gutierrez1, Mariano Andrea Scorciapino1, Igor Bodrenko1, Richard Wagner2, Bert van den Berg3, Mathias Winterhalter2, Matteo Ceccarelli1. 1 Physics, University of Cagliari, Monserrato, Italy, 2Jacobs University, Bremen, Germany, 3University of Newcastle, Newcastle upon Tyne, United Kingdom. The main focus of our study is to identify the structural features responsible for the transport of molecules through substrate-specific channels of Gram negative bacterium Pseudomonas aeruginosa. We present a precise molecular analysis of the structure and dynamics of OccK8 to explain the translocation of natural amino acid residues and antibiotics of two different chemical families through it. We used molecular dynamics simulations to obtain information on the molecule/channel interactions and proposed quantitative structure-function relationships based on them. We found some physical chemical properties of molecules playing an important role in modulating the translocation through OccK8. Molecules with zwitterionic structures have an affinity towards the polar eyelet region and presence of negative charge help interaction with the basic ladder facilitating translocation. A favorable chemical structure helps to widen the otherwise small constriction region helping translocation of relatively large antibiotics. Whenever possible, liposome swelling assays and electrophysiology at single-molecule level were used to confirm our data.
2049-Pos Board B369 Perturbation of Bilayer Surface Tension Differentially Modulates Mechanosensitive Ion Channels Navid Bavi1,2, Charles D. Cox1,2, Omid Bavi3, Boris Martinac1,2. 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2St. Vincent’s Clinical School, University of New South Wales, Sydney, Australia, 3 Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran, Islamic Republic of. The lipid composition of biological membranes modulates the activity of integral membrane proteins (1). This is particularly important for mechanosensitive (MS) channels, regardless of their gating paradigm. Here we use the surface active agent 2,2,2-trifluoroethanol (TFE) as a pharmacological tool to study the effect of surface tension perturbations on an array of bacterial and mammalian MS channels including MscL, Piezo1 and TREK-1. We chose TFE (a general anaesthetic) due to the fact that its effect on the bacterial channel MscS has already been studied. In particular TFE facilitates MscS activation from the periplasmic side, while it abolishes MscS current from the cytoplasmic side (2, 3). Here, we demonstrate that 2 % v/v TFE can also facilitate the activation of: (i) MscL if TFE is added to either bilayer leaflet, (ii) Piezo1 only if added to the cytoplasmic side and (iii) TREK-1 only from the extracellular side. Our molecular dynamics simulations revealed TFE increases the surface tension and the first moment of the transbilayer pressure profile markedly and hence facilitates activation of MscL. Using our molecular dynamics, energetic analysis and collective experimental data, we postulate there is a close relationship between MS channel shape and its activation mechanism by surface tension perturbations. The activation curve of MscL, which is a cylindrical protein, was shifted to the left (activated easier) upon addition of TFE from either side. MscS and TREK on the other hand, which are conical, were only facilitated from the extracellular side. The activation curve of Piezo1 was also shifted to left only when TFE was added to the cytoplasmic side. Given surface active drugs are adsorbed onto cell membranes, these findings provide a mechanistic understanding of their non-specific impact on the function of different membrane proteins, particularly MS ion channels. 1. Cantor RS (1998) The lateral pressure profile in membranes: a physical mechanism of general anesthesia. Toxicol Lett 100-101:451-458. 2. Akitake B, Spelbrink RE, Anishkin A, Killian JA, de Kruijff B, & Sukharev S (2007) 2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS. Biophys J 92(8):2771-2784. 3. Nomura T, Cox CD, Bavi N, Sokabe M, & Martinac B (2015) Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes. FASEB J 29(10):4334-4345.
2047-Pos Board B367 Molecular Transport through Large Diameter DNA Origami Channels Swati Krishnan, Friedrich Simmel. Physics, TU munich, Munich, Germany. We introduce a large DNA membrane channel with a z 4 nm diameter pore, which spontaneously inserts into flat lipid bilayer membranes using hydrophobic functionalizations or streptavidin linkages between biotinylated channels and lipids. Using a dye influx assay, we demonstrate formation of membrane pores in giant unilamellar vesicles. The versatility of DNA nanotechnology makes these channels promising molecular devices for synthetic biology and biosensing. 2048-Pos Board B368 Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor Zhouxiang Ji, Shaoying Wang, Zhengyi Zhao, Zhi Zhou, Farzin Haque, Peixuan Guo. The Ohio State University, Columbus, OH, USA. Insertion of biological nanopores to the lipid membranes has inspired single molecule sensing of peptides and proteins. Most biological membrane nanopores are around 1.2 nm, too small for the folded protein to pass through the nanopore. A well-studied bacteriophage phi29 DNA packaging motor with a large channel has been inserted into the lipid membrane, served as the biosensor of nucleic acid and chemical. Herein, we reported that the large channel of bacteriophage phi29 packaging motor can be used for fingerprinting of peptides or small proteins. Different peptides can be distinguished well with the blockage, dwell time or ionic signature, and the oligomeric states of peptides can be investigated in real time. The translocation was confirmed by single molecule florescence imaging. Further, Ni-NTA nanogold binding assay showed that peptides were translocated from the N-terminal to the C-terminal of the channel. Single direction insertion of the nanopore into the channel was controlled by polar hydrophobicity of the N or C terminal. Our results demonstrate the potential of this nanopore system for the detection of peptide biomarkers related to certain diseases. Reference: 1. Ji, Z. et al & Guo, P. (2016). Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor. Small, 12: 4572.
2050-Pos Board B370 Novel Properties of LRRC8-Mediated VRAC Currents Antonella Gradogna1, He´ctor Gaita´n-Pen˜as2, Lara Laparra-Cuervo3, Carles Solsona4, Vı´ctor Ferna´ndez-Duen˜as5, Alejandro Barrallo-Gimeno2, Francisco Ciruela6, Melike Lakadamyali3, Rau´l Este´vez2, Michael Pusch1. 1 Istituto di Biofisica, Genoa, Italy, 2Unitat de Fisiologı´a, Departament de Cie`ncies Fisiolo`giques II, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 3The Barcelona Institute of Science and Technology, ICFO-Institut de Cie`ncies Foto`niques, Barcelona, Spain, 4Laboratory of Neurobiology, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 5Unitat de Farmacologia, Departament Patologia i Terape`utica Experimenta, IDIBELLUniversitat de Barcelona, Barcelona, Spain, 6Unitat de Farmacologia, Departament Patologia i Terape`utica Experimental, IDIBELL-Universitat de Barcelona, Barcelona, Spain. Heteromers of the LRRC8A protein with other LRRC8 members (B, C, D, E) form the volume regulated anion channel (VRAC). We used the Xenopus oocyte system to study functional and structural properties of these channels. 8A/8C, 8A/8D, and 8A/8E heteromers gave rise to small volume stimulated currents. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity that was further
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VSD in different conformations. In the accompanying abstract (Kennedy, Zhao, et al.), we describe isolation of C5 and C6 from ~1 million member phage display library constructed on an inhibitor cysteine knot (ICK) scaffold, a backbone found in many gating modifier neurotoxins. As expected, the S3-S4 loop of hHv1 was found to be the major binding epitope for C6 toxin. Whereas, ci-Hv1 channels from sea squirt were insensitive to 1 mM C6, transplanting the hHv1 S3b-S4a motif to ci-Hv1 conferred the chimera with sensitivity (maximal blockade 70%; half maximal inhibition at 35 nM); the hHv1 motif was also able to confer C6 blockade to otherwise insensitive Kv2.1. Scanning the motif in hHv1 by Cys scanning across 12 residues revealed four sites that decreased affinity of C6 over 3-fold, one site that increased inhibition 75-fold (and permits 100% inhibition), and one site that allows C6 binding to activate the channel by 30% (half maximal activation at 250 nM). Consistent with a shared VSD trapping mechanism, C6 inhibits whereas C5 activates, yet both modify the kinetics of activation and deactivation as well as the voltage-dependence of gating, and while they are 39 and 41 residues, respectively, the C-terminal 31 residues are identical. A tethered-toxin strategy has been employed to map the C6 interaction surface with hHv1 as a prelude to designing more potent inhibitors and activators; ten important residues that are charged or polar have been identified. 2044-Pos Board B364 HCN3 Channel Expression in Human Leukocytes Chiara Piantoni1, Angelica Gualdoni1, Claudia Bazzini1, Raffaella Milanesi1, Miryam Adelfio1, Annalisa Bucchi1, Andrea Barbuti1, Matteo Pecchiari2, Mirko Baruscotti1, Dario DiFrancesco1. 1 Department of Biosciences, Universita` degli Studi di Milano, Milan, Italy, 2 Department of Pathophysiology and Transplantation, Universita` degli Studi di Milano, Milan, Italy. Ivabradine is a heart rate-reducing agent, which specifically and selectively blocks pacemaker HCN channels. Strong evidence shows that Ivabradine also reduces inflammatory processes and the suggested mechanism of action is the modification of the vascular shear stress caused by the primary bradycardic effect of the drug (Li et al., 2008 Plos one 11(2): e0149694.doi:10.1371). Leukocytes, the key players of the immune response, express several ion channels (Kv1.3, KCa3.1, TRPM4, ORAI1 and others) which control the functional state of the cell and are therefore involved in cell activation and inflammatory processes. Since HCN channels are important determinants of membrane potential in both excitable and non-excitable cell types, we investigated the presence of these channels in leukocytes. Although qualitative PCR experiments revealed a positive amplification of all HCN mRNA isoforms (HCN1-2-3-4), preliminary quantitative PCR carried out in total human leukocytes showed a reliable signal only for HCN3. We therefore proceeded with the investigation of HCN3 channel expression in human leukocytes. Immunolabelling with commercial anti-HCN3 antibody confirmed the presence of this isoform in human total leukocytes. Flow cytometry analysis revealed that the major sources of HCN3 signal appear to be granulocytes and lymphocytes. We finally tested the effect of Ivabradine (1 mM) on the activation process of human total leukocytes. The results show a 29% (n=5) reduction in the activation readout parameters in the presence of Ivabradine. These results raise the possibility that the mechanism by which Ivabradine reduces inflammation is a direct binding to HCN3 channels in human leukocytes and we therefore suggest that HCN3 channels may play a functional role in specific human leukocytes subpopulations. Future electrophysiological experiments will further investigate this possibility. 2045-Pos Board B365 Silica Nanoparticles Induce Calcium-Permeable Pores in Plasma Membranes Alicia Sanchez, Kateryna Demydenko, Peter Hoet, Karel Talavera. KU Leuven, Leuven, Belgium. Amorphous silica nanoparticles (SiNPs) are extensively used for their beneficial properties in cosmetics and food industry as an additive and fining agent. However, the high levels of exposure have raised concerns about health hazards, and it has been shown that SiNPs can penetrate tissues and cells resulting in health problems. In this study we first evaluated the effects of SiNPs on intracellular Ca2þ concentration ([Ca2þ]i) in different cell types (HEK293, CHO and mouse airway epithelial cells). We found that extracellular application of 9 nm SiNPs (LudoxÒ) at 25 C produced a pronounced increase in [Ca2þ]i when the cells were co-stimulated with arachidonic acid or LPS. These effects were not observed when Ca2þ was omitted in the extracellular solution, indicating that SiNPs induce a Ca2þ entry pathway through the plasma membrane. Similar results were obtained when cells were exposed to SiNPs and heating from 25 to 35 C, suggesting that SiNPs are sufficient to induce the Ca2þ entry
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pathway at physiological temperatures. Whole-cell patch-clamp experiments revealed that SiNPs trigger large currents that could be blocked by the cation channel blocker ruthenium red in a voltage-dependent manner. Analysis of the selectivity properties of this current showed that they are mainly carried by cations. Taken together, our results demonstrate that SiNPs induce Ca2þ-permeable pores in the plasma membrane, and that this phenomenon is enhanced by factors that increase membrane fluidity. We propose that this Ca2þ entry pathway may be relevant for the toxicological properties of SiNPs.
2. Zhou, et al & Guo, P. (2016). Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity. Biomaterials, 105: 222. 3. Wendell, et al & Guo, P. (2009). Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature nanotechnology, 4:765. 4. Haque, et al & Guo, P. (2012). Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor. ACS nano, 6:3251.
2046-Pos Board B366 How to Get Large Drugs through Small Pores? Exploiting the Porins Pathway in Pseudomonas Aeruginosa Susruta Samanta1, Tommaso D’Agostino1, Ishan Ghai2, Monisha Pathania3, Silvia Acosta Gutierrez1, Mariano Andrea Scorciapino1, Igor Bodrenko1, Richard Wagner2, Bert van den Berg3, Mathias Winterhalter2, Matteo Ceccarelli1. 1 Physics, University of Cagliari, Monserrato, Italy, 2Jacobs University, Bremen, Germany, 3University of Newcastle, Newcastle upon Tyne, United Kingdom. The main focus of our study is to identify the structural features responsible for the transport of molecules through substrate-specific channels of Gram negative bacterium Pseudomonas aeruginosa. We present a precise molecular analysis of the structure and dynamics of OccK8 to explain the translocation of natural amino acid residues and antibiotics of two different chemical families through it. We used molecular dynamics simulations to obtain information on the molecule/channel interactions and proposed quantitative structure-function relationships based on them. We found some physical chemical properties of molecules playing an important role in modulating the translocation through OccK8. Molecules with zwitterionic structures have an affinity towards the polar eyelet region and presence of negative charge help interaction with the basic ladder facilitating translocation. A favorable chemical structure helps to widen the otherwise small constriction region helping translocation of relatively large antibiotics. Whenever possible, liposome swelling assays and electrophysiology at single-molecule level were used to confirm our data.
2049-Pos Board B369 Perturbation of Bilayer Surface Tension Differentially Modulates Mechanosensitive Ion Channels Navid Bavi1,2, Charles D. Cox1,2, Omid Bavi3, Boris Martinac1,2. 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2St. Vincent’s Clinical School, University of New South Wales, Sydney, Australia, 3 Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran, Islamic Republic of. The lipid composition of biological membranes modulates the activity of integral membrane proteins (1). This is particularly important for mechanosensitive (MS) channels, regardless of their gating paradigm. Here we use the surface active agent 2,2,2-trifluoroethanol (TFE) as a pharmacological tool to study the effect of surface tension perturbations on an array of bacterial and mammalian MS channels including MscL, Piezo1 and TREK-1. We chose TFE (a general anaesthetic) due to the fact that its effect on the bacterial channel MscS has already been studied. In particular TFE facilitates MscS activation from the periplasmic side, while it abolishes MscS current from the cytoplasmic side (2, 3). Here, we demonstrate that 2 % v/v TFE can also facilitate the activation of: (i) MscL if TFE is added to either bilayer leaflet, (ii) Piezo1 only if added to the cytoplasmic side and (iii) TREK-1 only from the extracellular side. Our molecular dynamics simulations revealed TFE increases the surface tension and the first moment of the transbilayer pressure profile markedly and hence facilitates activation of MscL. Using our molecular dynamics, energetic analysis and collective experimental data, we postulate there is a close relationship between MS channel shape and its activation mechanism by surface tension perturbations. The activation curve of MscL, which is a cylindrical protein, was shifted to the left (activated easier) upon addition of TFE from either side. MscS and TREK on the other hand, which are conical, were only facilitated from the extracellular side. The activation curve of Piezo1 was also shifted to left only when TFE was added to the cytoplasmic side. Given surface active drugs are adsorbed onto cell membranes, these findings provide a mechanistic understanding of their non-specific impact on the function of different membrane proteins, particularly MS ion channels. 1. Cantor RS (1998) The lateral pressure profile in membranes: a physical mechanism of general anesthesia. Toxicol Lett 100-101:451-458. 2. Akitake B, Spelbrink RE, Anishkin A, Killian JA, de Kruijff B, & Sukharev S (2007) 2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS. Biophys J 92(8):2771-2784. 3. Nomura T, Cox CD, Bavi N, Sokabe M, & Martinac B (2015) Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes. FASEB J 29(10):4334-4345.
2047-Pos Board B367 Molecular Transport through Large Diameter DNA Origami Channels Swati Krishnan, Friedrich Simmel. Physics, TU munich, Munich, Germany. We introduce a large DNA membrane channel with a z 4 nm diameter pore, which spontaneously inserts into flat lipid bilayer membranes using hydrophobic functionalizations or streptavidin linkages between biotinylated channels and lipids. Using a dye influx assay, we demonstrate formation of membrane pores in giant unilamellar vesicles. The versatility of DNA nanotechnology makes these channels promising molecular devices for synthetic biology and biosensing. 2048-Pos Board B368 Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor Zhouxiang Ji, Shaoying Wang, Zhengyi Zhao, Zhi Zhou, Farzin Haque, Peixuan Guo. The Ohio State University, Columbus, OH, USA. Insertion of biological nanopores to the lipid membranes has inspired single molecule sensing of peptides and proteins. Most biological membrane nanopores are around 1.2 nm, too small for the folded protein to pass through the nanopore. A well-studied bacteriophage phi29 DNA packaging motor with a large channel has been inserted into the lipid membrane, served as the biosensor of nucleic acid and chemical. Herein, we reported that the large channel of bacteriophage phi29 packaging motor can be used for fingerprinting of peptides or small proteins. Different peptides can be distinguished well with the blockage, dwell time or ionic signature, and the oligomeric states of peptides can be investigated in real time. The translocation was confirmed by single molecule florescence imaging. Further, Ni-NTA nanogold binding assay showed that peptides were translocated from the N-terminal to the C-terminal of the channel. Single direction insertion of the nanopore into the channel was controlled by polar hydrophobicity of the N or C terminal. Our results demonstrate the potential of this nanopore system for the detection of peptide biomarkers related to certain diseases. Reference: 1. Ji, Z. et al & Guo, P. (2016). Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor. Small, 12: 4572.
2050-Pos Board B370 Novel Properties of LRRC8-Mediated VRAC Currents Antonella Gradogna1, He´ctor Gaita´n-Pen˜as2, Lara Laparra-Cuervo3, Carles Solsona4, Vı´ctor Ferna´ndez-Duen˜as5, Alejandro Barrallo-Gimeno2, Francisco Ciruela6, Melike Lakadamyali3, Rau´l Este´vez2, Michael Pusch1. 1 Istituto di Biofisica, Genoa, Italy, 2Unitat de Fisiologı´a, Departament de Cie`ncies Fisiolo`giques II, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 3The Barcelona Institute of Science and Technology, ICFO-Institut de Cie`ncies Foto`niques, Barcelona, Spain, 4Laboratory of Neurobiology, IDIBELL-Universitat de Barcelona, Barcelona, Spain, 5Unitat de Farmacologia, Departament Patologia i Terape`utica Experimenta, IDIBELLUniversitat de Barcelona, Barcelona, Spain, 6Unitat de Farmacologia, Departament Patologia i Terape`utica Experimental, IDIBELL-Universitat de Barcelona, Barcelona, Spain. Heteromers of the LRRC8A protein with other LRRC8 members (B, C, D, E) form the volume regulated anion channel (VRAC). We used the Xenopus oocyte system to study functional and structural properties of these channels. 8A/8C, 8A/8D, and 8A/8E heteromers gave rise to small volume stimulated currents. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity that was further