State-Dependent Structural Modeling and Atomistic Simulations of the ...

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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