K+ Occupancy in the Cavity Determines the Ion Permeation Rate through the KV1.2 Channel

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Wednesday, February 15, 2017

by Iso application, contrary to WT cells. Together, our results suggest that ablation of the RyR2-S2030 site may result in a blunted increase of RyR2 Ca2þ sensitivity upon ß-adrenergic stimulation, and that the site represents a link between the adrenergic pathway and modulation of RyR2 channel activity. 2671-Pos Board B278 Molecular Cloning and Expression of cDNA Encoding the Ryanodine Receptor Type 2 from Rattus Norvegicus Cerebral Artery Smooth Muscle Jianxi Liu1, Guruprasad Kuntamallappanavar1, Venkatasushma Kalava2, Alex Dopico1. 1 Pharmacology, The University of Tennessee Hlth. Sci. Ctr., Memphis, TN, USA, 2Christian Brothers University, Memphis, TN, USA. Ryanodine receptors (RyRs) are a family of Ca2þ release channels found in intracellular organelles. RyR channels are ubiquitously expressed in many cell types and participate in a wide variety of physiological processes (e.g., neurotransmission, secretion, regulation of myogenic tone, cardiac contractility). We have cloned and sequenced cDNA encoding the Ca2þ release channel isoform 2 of the ryanodine receptor (RyR2) from smooth muscle cells of Rattus norvegicus cerebral arteries and determined RyR2 protein expression. The RyR2 cDNA is 14,850 bp in length, resulting in a protein product of 4,950 amino acids with a theoretical pI/MW of 5.81/561,699.41 Da. We used total RNA from endothelium free cerebral arteries and conducted reverse transcription. We divided the RyR2 cDNA in two equal parts because of its huge size; N-terminal part: 1-7,486 bp and C-terminal part: 7,487-14,862 bp. We used gene specific primers to get both N and C-terminal parts of the cDNA sequence. Primers for the N-terminal part: forward 5’ - CG ACG CGT ATG GCT GAT GCG GGC GAA G - 3’; reverse 5’ - GA CGC GTC GAC CAG CCG TGT CTA GAG AG - 3’; for C-terminal part: forward 5’ - GGA CGC GTC GAC CAC TAA GTG CTA CAG AC - 3’; reverse 5’ - GAT AAG AAT GCG GCC GCT TAA TTT AAC TGG TCC - 3’. After cloning the cDNAs into the mammalian vector pIRESneo, we characterized the clones by restriction analysis and confirmed the resulting cDNA sequence by automated analysis. Using immunohistochemistry, Western blotting and surface biotinylation assays, we evaluated the cytosolic and membrane surface expression of RyR2 in HEK cells. Our results demonstrate that RyR2 protein effectively expresses in both plasma and internal membranes. Our study demonstrates protein expression from the newly cloned RyR2 from cerebral artery smooth muscle. Functional characterization of this RyR2 is underway. Support: HL104631, R37AA11560 (AMD). 2672-Pos Board B279 MCU and EMRE Binding is Mediated through Intermembrane HelixHelix Interactions Charles Phillips. Brandeis University, Waltham, MA, USA. Mitochondrial calcium uptake is critical for physiological processes such as calcium signaling, energy production, and apoptosis, and is mediated by a calcium activated calcium channel known as the mitochondrial calcium uniporter. The uniporter is a protein complex, composed of the pore-forming MCU protein, and the important regulatory proteins EMRE, MICU1, and MICU2. It has been well established that EMRE, a small 10kDa single-pass transmembrane (TM) protein, is absolutely required for MCU activity. However, little is known about how it interacts with MCU. Here, using a Tryptophan scanning assay, we identify key residues absolutely required for MCU-EMRE interaction and MCU activation by EMRE. Specifically, we show that EMRE contains a GXXXG in the TM helix to pack against the TM1 of MCU. Residue swapping along with co-immunoprecipitation experiments establish that EMRE and MCU directly bind each other without the need of a bridging protein. This work is an important first step toward understanding how EMRE regulates MCU, and helps explain why animals have evolved a requirement for EMRE to facilitate mitochondrial calcium uptake.

Voltage-gated K Channels and Mechanisms of Voltage Sensing and Gating IV 2673-Pos Board B280 State-Dependent Structural Modeling and Atomistic Simulations of the hERG Potassium Channel Kevin R. DeMarco1, Phuong T. Nguyen1, Toby W. Allen2,3, Vladimir Yarov-Yarovoy4, Colleen E. Clancy5, Igor Vorobyov5. 1 Biophysics Graduate Group, University of California Davis, Davis, CA, USA, 2School of Applied Sciences, RMIT University, Melbourne, Australia, 3 Department of Chemistry, University of California Davis, Davis, CA, USA, 4 Department of Physiology and Membrane Biology, University of California Davis, Davis, CA, USA, 5Department of Pharmacology, University of California Davis, Davis, CA, USA.

Voltage gated ion channels are integral membrane proteins responsible for the propagation of electric signals in excitable cells such as cardiac myocytes. The voltage gated potassium channel, Kv11.1, encoded by the human ether-a-go-go related gene (hERG), mediates the rapid repolarization phase of the cardiac action potential. Inherited mutations in this gene, as well as channel interactions with various organic molecules, including pharmaceuticals, are associated with long QT syndrome (LQTS), a standard clinical indicator for increased risk of ventricular arrhythmias and sudden cardiac death. In the absence of published experimentally resolved structures of hERG channel, we used ROSETTA structural modeling software and a recent cryo-EM structure of a homologous EAG1 channel as a template to generate a closed-state hERG channel model. Furthermore, open conducting and open inactivated models of hERG were modeled using ROSETTA and the crystal structures of KvAP, Kv1.2, Kv1.2-Kv2.1 chimera and KcsA channels as templates, and incorporating structural constraints from mutagenesis studies. Model stability was assessed via microsecond-long all-atom molecular dynamics (MD) simulations of the channels in an explicitly hydrated lipid membrane environment. Additionally, hERG modulation by lipid membrane composition was probed by MD simulations of the channel models in lipid membranes with varied compositions. In particular, omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), is known to possess antiarrhythmic properties, exhibiting time-, voltage- and use-dependent blockade of hERG, preferentially binding to the open state of the channel. Conversely, some pharmaceuticals block hERG in a state-dependent manner, and exhibit pro-arrhythmogenic properties. These interactions were probed by MD simulations as well. This study will help elucidate such molecular mechanisms of hERG modulation potentially leading to decreased risks of LQTS and deadly arrhythmias. 2674-Pos Board B281 Effect of Membrane Composition on Ion Conduction in a Voltage-Gated Potassium Channel Niklaus B. Johner, Simon Berneche. Biozentrum, Basel Universit€at, Basel, Switzerland. Potassium channels constitute a super-family of membrane proteins playing a key role in the function of neurons. Of particular importance are the voltagegated potassium channels (Kv), of which there are around 40 sub-types. These channels have been studied extensively for several decades and great advances in the understanding of the mechanisms governing conduction and gating at the atomic scale have been made since the first crystal structure of a potassium channel was solved almost 20 years ago. Nevertheless, as molecular dynamics (MD) simulations have suggested that the conformational states captured in the crystal structures of voltage-gated potassium channels sustained permeation rates much lower compared to experimental measurements, the details of the ion conduction mechanism and whether the crystal structures truly represent the conducting state are still debated. We are using MD simulations to study the impact of membrane composition and voltage on the conformation of a Kv channel and its ion conductivity. Our results notably suggest that membrane thickness affects the function of the channel. These simulations illustrate how the microenvironment can impact the function of ion channels. 2675-Pos Board B282 KD Occupancy in the Cavity Determines the Ion Permeation Rate through the KV1.2 Channel Takashi Sumikama, Shigetoshi Oiki. Faculty of Medical Sciences, University of Fukui, Eiheiji-cho, Yoshida-gun, Fukui, Japan. The ion permeation mechanism through potassium channels has been examined extensively through experimental and simulation studies. Molecular dynamics (MD) simulations have demonstrated that rapidly permeating ions collide near the selectivity filter (SF) (the ‘‘knock-on’’ mechanism), but this oversimplified view is insufficient to account for the experimentally observed single-channel current amplitudes. Here, we examined the MD-simulated ion trajectories through the Kv1.2 potassium channel by developing an event-oriented analysis. In the potassium channels, a cavity stands between the intracellular bulk solution and the SF. The analysis showed that two ions in the SF were immobilized when an ion was empty in the cavity. We found surprisingly that the queueing ions in the SF became mobile and initiated outward motion when an ion entered the water-filled cavity. This cavity ion subsequently filled the space left in the SF and the cavity is ion-empty again. After an expulsion of the outermost ion in the SF and a following short relaxation, the ions in the SF became immobilized. Accordingly, outward ion movements were not continuous but exhibited alternating mobile and immobile (or queueing) phases, and the permeation process can be described as a cyclic phase diagram having two phases. The period spent

Wednesday, February 15, 2017 in the immobile (or queueing) phase was longest, indicating that the period determines the ion permeation rate or the single-channel current amplitude. The concentration dependency of the period was also investigated. We found that the period having an ion in the cavity is independent of the concentration, but the queueing period is inversely proportional to the concentration. Thus, the ion-bearing state in the cavity serves as a catalytic intermediate in the Michaelis-Menten-type kinetics, accounting for the current-concentration relationship. 2676-Pos Board B283 On Resolution of the Selectivity/Conductivity Paradox for the Potassium Ion Channel Dmitry G. Luchinsky1,2, Will A.T. Gibby1, Igor Kh Kaufman1, Dogan A. Timucin3, Peter V.E. McClintock1. 1 Physics, Lancaster University, Lancaster, United Kingdom, 2SGT Inc., Greenbelt, MD, USA, 3Intelligent Systems Division, NASA Ames Research Center, Moffett Field, CA, USA. The ability of the potassium channel to conduct Kþ at almost the rate of free diffusion, while discriminating strongly against the (smaller) Naþ ion, is of enormous biological importance [1]. Yet its function remains at the center of a ‘‘many-voiced debate’’ [2,3]. In this presentation, a first-principles explanation is provided for the seemingly paradoxical coexistence of high conductivity with high selectivity between monovalent ions within the channel. It is shown that the conductivity of the selectivity filter is described by the generalized Einstein relation. A novel analytic approach to the analysis of the conductivity is proposed, based on the derivation of an effective grand canonical ensemble for ions within the filter. The conditions for barrier-less diffusion-limited conduction through the KcsA filter are introduced, and the relationships between system parameters required to satisfy these conditions are derived. It is shown that the Eisenman selectivity equation is one of these, and that it follows directly from the condition for barrier-less conduction. The proposed theory provides analytical insight into the ‘‘knock-on’’ [1] and Coulomb blockade [4] mechanisms of Kþ conduction through the KcsA filter. It confirms and illuminates an earlier argument [3] that the ‘‘snug-fit’’ model cannot describe the fast diffusion-limited conduction seen in experiments. Numerical examples are provided illustrating agreement of the theory with experimentally-measured I-V curves. The results are not restricted to biological systems, but also carry implications for the design of artificial nanopores. [1] Morais-Cabral, J. H. H., Zhou, Y. & MacKinnon, R., Energetic optimization of ion conduction rate by the Kþ selectivity filter, Nature 414, 37-42 (2001). [2] Piasta, K. N., Theobald, D. L. & Miller, C., Potassium- selective block of barium permeation through single KcsA channels., J. Gen. Physiol. 138, 42136 (2011). ˚ qvist, J., Permeation Redux: Thermodynamics and [3] Horn, R., Roux, B. & A Kinetics of Ion Movement through Potassium Channels, Biophys. J. 106, 18591863 (2014). Kaufman, I. K., Luchinsky, D. G., Tindjong, R., McClintock, P. V. E. & Eisenberg, R. S., Multi-ion conduction bands in a simple model of calcium ion channels, Phys. Biol. 10, 026007 (2013). 2677-Pos Board B284 Weighted Ensemble Approach to In Silico Measure the I-V Relationship in a KD Ion Channel Sara Capponi1, Joshua Adelman2, John Rosenberg2, Michael Grabe1. 1 Cardiovascular Research Institute, UCSF, San Francisco, CA, USA, 2 Department of Biological Sciences, U Pitt, Pittsburgh, PA, USA. Weighted Ensemble (WE) sampling is a rigorous multi-replica method for full-atom simulations of equilibrium and not-equilibrium processes [1,2]. The strategy this method uses can be summarized in three steps: i) initiation of independent simulations from a starting state of the system; ii) progress coordinate definition and replication of the trajectories advancing along it; iii) splitting/replicating or merging/culling simulations in undersampled or oversampled bins, in which the conformational space has been divided. WE method has been demonstrated to accurately reproduce the results obtained by using full-atom molecular dynamics simulations, herein called brute-force dynamics [3]. The ionic current through a membrane ion channel model was calculated by means of WE simulations. The results show that the I-V relationship accurately reproduce that obtained by using bruteforce dynamics. Moreover, in presence of low ionic current, the efficiency of the WE approach increases greatly. Here, we aim to calculate the I-V relationship of the Kþ TRAAK ion channel embedded in a POPC bilayer by applying the WE method. Research supported by NIH grants R21 NS091941-01. [1] G. A. Huber and S. Kim, Biophys J, 70, 97 (1996). [2] B. W. Zhang et al., J Chem Phys, 132, 052107 (2010). [3] J. L. Adelman and M. Grabe, JCTC, 11(4), 1907 (2015)

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2678-Pos Board B285 Quantum Calculations of Large Segments of a Voltage Sensing Domain of a Voltage Gated Channel Alisher M. Kariev, Michael E. Green. Chemistry, City College of the City University of New York, New York, NY, USA. Several large scale calculations of sections of the voltage sensing domain (VSD) of the Kv1.2 channel (pdb: 3Lut), including structure optimizations at HF/6-31G* level, were done with applied electric fields of both polarities, or without field. Atoms at the ends of the S1, S2, and S3 segments were frozen. With the S4 segment free to move, the backbone displacement of S4 relative ˚ in calculations that to the S1,S2,S3 block remained below approximately 2 A included the side chains. Thus gating current would not come from the motion of S4 perpendicular to the membrane surface. Optimizations with protons in several possible positions are being carried out, and a possible response of protons to field, in which they move through exchange of partners, is suggested by the energy of the systems in which the protons shift, e.g., from arginine to glutamate. This leaves an unionized salt bridge with energy less than that of the ionized salt bridge (see accompanying Kariev/Green abstract). Proton shifts are required: e.g., calculated open state, salt bridge R300 nitrogen to E226 oxygen distance, with Y266 very close, do not agree with the X-ray structure with the salt bridge ionized. Second, the E183-R297 salt bridge distance corresponds to the X-ray structure, ionized or not, but the neutral case is appreciably lower in energy. Thus this salt bridge is likely to be neutral in the open state. Based on related results, we suggest a hypothesis for a proton pathway that creates the gating current in Kv1.2. It also appears that the Hv1 channel deviates from Kv1.2 near R201/D108/R204(Hv1):R300/E226/R303(Kv1.2), leading to a hypothesis for a path for the protons in Hv1. Water molecules also are an essential part of the path in the upper and lower sections of the VSD. 2679-Pos Board B286 Quantum Calculations of Salt Bridges, their Ionization State in the Interior of Voltage Sensing Domains of Voltage Gated Channels, and Some Consequences Alisher M. Kariev, Michael E. Green. Chemistry, City College of the City Univ of NY, New York, NY, USA. Quantum calculations on salt bridges with varying amounts of water show that ionization of the salt bridge requires 3 molecules of water to be certain, and a minimum of 2 molecules to have reasonable probability, depending on other parts of the surroundings. Calculations relevant to VSDs show essentially the same results. There is a hydrophobic region at the VSD center, where the salt bridge appears unionized; i.e., the energy is lower when E183 of the Kv1.2 potassium channel (3Lut numbering) is not ionized. However, there are complications in the calculations, as protons, hence charge, may transfer to or from other neighboring residues (e.g. tyrosine) that do not belong to what is normally considered part of a salt bridge; these triangular arrangements may be ionized in ways that are not expected. Work is now in progress to determine whether it is the expected arginine from the S4 transmembrane segment, which becomes neutral, or rather a neighboring tyrosine, which would become negatively charged, that provides the proton to neutralize the glutamate. A possibly related topic: the standard gating models depend in part on R to C mutations followed by reaction with MTS reagents, which are assumed to require the cysteine to migrate to a water accessible region in order to ionize, the interior of the VSD being assumed to be too hydrophobic to allow ionization. The assumptions of ionized salt bridges and the impossibility of ionization of the cysteine may be incompatible. Also the R to C mutation leaves a large cavity, which may be filled by water or the MTS reactive headgroup, so it is not certain that the cysteine would have to migrate to ionize. 2680-Pos Board B287 True Or False? ‘‘The Arginines and Lysines of the S4 Segment of a Voltage-Sensitive Ion Channel Repel One Another Electrostatically’’ H. Richard Leuchtag. Retired, Kerrville, TX, USA. The Channel Activation by Electrostatic Repulsion (CAbER) hypothesis1— that activation of a voltage-sensitive ion channel on depolarization is powered by an increase in the electrostatic repulsions between the positively charged arginine and lysine residues of S4 segments—has been challenged: Criticisms2: The S4 positive charges are not necessarily real. Almost as many acid groups with putatively negative charges are on S2 and S3; they should be salt bridged, making the combination neutral. The charges are not obviously there; quantum calculations suggest that with no or only one water molecule in a voltage sensor domain, salt bridges don’t ionize. When they do ionize, the charges are less than one because of charge transfer. Rebuttal: The claim that the positive charges are not real contradicts the accepted concept that