Feb 15, 2017 - A salient feature of lysenin channels is the history-dependent gating in ... Medicine, Stanford, CA, USA, 2Mechanical Engineering, Stanford.
Wednesday, February 15, 2017
Other Channels II 2704-Pos Board B311 Lysenin Gating Implies Strong Interaction between the Voltage-Gating Sensor and the Bilayer Membrane: An Ionic Screening Study Samuel R. Kosydar1, Kaitlyn S. Ware1, Sheenah Bryant2, Nisha Shrestha2, Charles Hanna2, Daniel Fologea2. 1 Department of Physics, Boise State University, Boise, ID, USA, 2 Department of Physics/Biomolecular Sciences Graduate Program, Boise State University, Boise, ID, USA. Lysenin, a pore-forming toxin extracted from the earthworm E. foetida, inserts large conductance channels into membranes containing sphingomyelin. A salient feature of lysenin channels is the history-dependent gating in response to periodic transmembrane voltages for which the period greatly exceeds the relaxation time of the voltage-induced gating. Our work focuses on understanding the molecular mechanisms by which lysenin channels gain history-dependent functionality. We hypothesized that lysenin channels, upon interaction with electric fields, undergo conformational changes that induce gating by pushing the voltage-domain sensor deep inside the bilayer membrane. Our experiments aim at testing this hypothesis by analyzing the effects of ionic screening induced by addition of mono and multivalent ions to the support electrolyte. Results demonstrate that ionic screening weakens the electrostatic interactions between the external electric field and the voltage domain sensor, which manifests as a significant rightward shift of the channels’ open probability in response to ascending voltage ramps that elicit channel closure. The shift is both concentration and electrovalence dependent, characteristic of ionic screening. However, once channels close, descending voltage ramps, which elicit channel reopening, show no major effects of ionic strength on the open probability. Moreover, the reactivation open probability is invariant irrespective of ionic conditions. Consequently, we propose a model of hysteresis based on gating that is accompanied by significant movement of the voltage domain sensor. While the voltage domain sensor is exposed to external aqueous solutions it is subject to screening, but gating pushes the domain deep within the membrane, from where the ions are excluded owing to the high energy cost. Therefore, gating induces major changes in the energy landscape, which may explain the molecular memory of lysenin channels and the invariance of the reactivation pathway. 2705-Pos Board B312 Subunits that form Trimeric DEG/ENaC Mechano-Electrical Transduction Channels in Touch Receptor Neurons Sylvia Fechner1, Frederic Loizeau2, Adam L. Nekimken2, Beth L. Pruitt2, Miriam B. Goodman1. 1 Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA, 2Mechanical Engineering, Stanford University, Stanford, CA, USA. In C. elegans, the behavioral response to touch and the underlying molecular actors are thoroughly studied. However, it is still debated which and how many of several co-expressed pore-forming subunits assemble into a functional, multimeric mechano-electrical transduction (MeT) channel in vivo. Two members of the DEG/ENaC channel family, MEC-4 and MEC-10, are important to convert touch into behavioral responses. MEC-4 is required to form native MeT channels, whereas MEC-10 plays a regulatory role. Recently, a third homologous protein and potential subunit, DEGT-1, was identified; its role in touch sensation remains incompletely understood. Given that DEG/ENaC proteins are thought to assemble as trimers, the presence of a third, homologous protein opens new questions regarding the composition of native MeT channels. A key outstanding question is which of these homologous proteins co-assemble to form the channels responsible for touch sensation. With the gentle touch assay, we detected a more severe defect in touch sensation if both MEC-10 and DEGT-1 were removed simultaneously than we did by removing them individually. This genetic enhancement suggests that DEGT-1 is part of the native MeT channel complex. To investigate this further, we are analyzing the contribution of DEGT-1 to native MeT channels and to channel activity in Xenopus oocytes. Using in vivo patch clamp recordings of wild type and mutant touch receptor neurons lacking individual subunits or expressing proteins with altered pore domains, we seek to delineate how DEGT-1 contributes to the mechanosensitivity, voltagedependence and adaptation of the native MeT current. In Xenopus oocytes, we find that DEGT-1 is unable to form homomeric channels on its own, but can assemble into functional channels in the presence of MEC-4. This finding suggests that, like MEC-10, DEGT-1 plays a regulatory role in channel formation.
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2706-Pos Board B313 Tracking Pore Hydration within the Red-Activatable Channelrhodopsin ReaChR by Site-Directed Labeling with Infrared-Active Azido Probes Benjamin S. Krause1, Joel C.D. Kaufmann2, Jens Kuhne3, Johannes Vierock1, Thomas Huber4, Thomas P. Sakmar4, Klaus Gerwert3, Franz J. Bartl2, Peter Hegemann1. 1 Institute of Biology, Experimental Biophysics, Humboldt Universit€at zu Berlin, Berlin, Germany, 2Institute for Medical Physics and Biophysics, Charite´ Berlin, Berlin, Germany, 3Department of Biophysics, Ruhr-University Bochum, Bochum, Germany, 4Laboratory of Chemical Biology and Signal Transduction, Rockefeller University, New York, NY, USA. Ion channels like Channelrhodopsins (ChRs) constitute at least two distinct states, one is permeable (conducting) for charges and one is not (non-conducting). Mostly, ion transport across a membrane requires a continuous water channel throughout the entire protein, which has to build up upon activation by a certain stimulus, e.g. ligand, substrate or for ChRs with light, and collapses succeeding cessation of the activating trigger. Within the last years, researchers have intensively studied the mechanism how poreformation is achieved in ChRs but still a profound understanding, especially on the closing event, is lacking. In the current study, we introduced the artificial IR-active probe (p-azido phenyl alanine) in ChR at certain sites aligning the ion pathway by amber stop codon suppression. By exploiting its unique spectral properties (nas ~ 2100 cm1) and its sensitivity towards polarity changes, we tracked hydration dynamics within the pore region and the inner gate in the red-activatable ChR (ReaChR) by infrared techniques. Our data imply that channel closure coincides a late dehydration event happening within the interface of the central and the inner gate. Furthermore, site-directed mutagenesis of inner gate residues suggest that this spatial constriction is essential for preventing intracellular water influx within the closed configuration of the ion channel. Thus, alteration of the inner gate structure by mutations leads to water invasion inside the intracellular part of the pore already in the dark state, establishing a prehydrated closed state. 2707-Pos Board B314 Mechanism of Water and Solute Cotransport by the Sodium Glucose Cotransporter SGLT1 Christine Siligan1, Andreas Horner1, Sergey Akimov2, Peter Pohl1. 1 Institute of Biophysics, JKU Linz, Linz, Austria, 2A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russian Federation. The molecular mechanism of carrier mediated water and solute cotransport is unresolved. It has long been believed that water may be engulfed into proteinaceous cavities and thus be moved along with the solute in a fixed stoichiometric ratio from one side of the membrane to the other. Since solute and solvent flux measurements through the potassium-chloride-cotransporter or the glucose sodium cotransporter (SGLT1) in epithelial monolayers and/or reconstituted lipid bilayers did not confirm stoichiometric coupling, we tested the hypothesis that water and glucose share part of their pathway through SGLT1. To this end we reconstituted the purified SGLT1 into lipid vesicles and exposed the proteoliposomes to osmotic gradients of different origin. SGLT1 facilitates passive water flow. Its unitary water permeability pf was higher in the presence of sucrose than in the presence of glucose indicating a reflection coefficient s of glucose smaller than one. The decrease pf was due to occlusion of the water pathway by bound glucose molecules. Mutation of the glucose binding pocket released the block. The glucose concentration at which pf dropped to 50 % of its maximal value characterizes the glucose affinity of SGLT1’s inwardly open conformation. It was two orders of magnitude larger than the affinity in the outwardly open conformation, i.e. the previously reported concentration at which SGLT1 achieves half of its maximum rate. Thus glucose transport requires conformational transitions while water is channeled as long as the glucose binding site is unoccupied explaining as to why both substrates are transported at despairingly different rates. 2708-Pos Board B315 Chimeric Innexins Reveal Complexities of Electrical Rectification Jamal B. Williams, Martha Skerrett. Biology, SUNY Buffalo State, Buffalo, NY, USA. The innexins ShakingB Lethal (SBL) and ShakingB Neuralþ16 (SBN16) form heterotypic gap junctions in the Giant Fiber System of Drosophilia melanogaster. The junctions favor arthodromic transmission of action potentials and exhibit properties of rectification when expressed in vivo. Sequence
