Functional Heteromeric PIEZO1 Ion Channels

Sunday, February 28, 2016 potential. The photosensitization reactions are reflective of physiological oxidative stress induced by reactive oxygen species (ROS), suggesting such processes may occur naturally in cells. 467-Pos Board B247 Biased Signaling of GPCR Regulates Pancreatic Beta Cell Secretion and Survival Xiao Yu1, Shanglei Ning2, Jin-Peng Sun1. 1 Shandong University School of Medicine, Jinan, China, 2Shandong University Qilu Hospital, Jinan, China. Gastrointestinal hormones which are secreted by enteroendocrine cells play important role in the regulation of pancreatic b cell survival and insulin secretion. Here we demonstrated that cholecystokinin CCK-8s activates a G-proteincoupled receptor, CCKAR, and elevates both IP3 and cAMP levels. Under lowglucose condition, Gq-IP3 signaling contributes to the CCK-8s-induced insulin secretion, while Gs-cAMP signaling increases the CCK-8s-mediated insulin secretion greatly in high glucose conditions. CCK-8s also promotes the CCKAR/b-arrestin-1 formation in pancreatic b cells, and further activates ERK signaling. Using b-arrestin-1 siRNA-mediated knockdown cells and b-arrestin-1 knockout mice, we demonstrated that b-arrestin-1 mediates both CCK8s-induced insulin secretion and the protective function in pancreatic b cells. The association of b-arrestin-1 and CCKAR mediates cytoplasmic late-phase ERK phosphorylation, then activates the p90RSK-phospho-Bad pathway, which contributes to the anti-apoptotic effect of CCK-8s. Further studies on biased signaling of CCKAR in pancreatic b cells will help developing biased CCKAR ligands. 468-Pos Board B248 The Enigmatic Chloroplast Stt7 Kinase: Trans-Membrane Function with Cytochrome b6f Complex in situ; Kinase Activity in vitro Sandeep K. Singh1, Whitaker Cohn2, Saif S. Hasan1, Julian P. Whitelegge3, William A. Cramer1. 1 Biological Sciences, Purdue University, West Lafayette, IN, USA, 2 Department of Psychiatry and Biobehavioral Sciences, UCLA, Los Angeles, CA, USA, 3Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA, USA. The distribution of light energy between the two photosystems in oxygenic photosynthesis is regulated in the alga, C. reinhardtii, by a 754 residue State Transition Kinase, Stt7, which is unique in using a trans-membrane topology to carry out its signaling function (1). It is activated by oxidation of plastoquinol on the electrochemically positive, lumen side of the cytochrome b6f complex (2), and phosphorylates the major light-harvesting chlorophyll protein II (3) on the opposite side of the membrane (1). Stt7, which binds to cyt b6f (1), requires a disulfide bond on the p-side proximal to the quinol oxidation site for its activation (1, 3). In the present study, Stt7 has been cloned in E. coli and purified as a soluble protein whose monomer mass, determined by mass spectrometry is 79,501. On Clear Native PAGE, Stt7 runs close to a position expected for a heptamer. Purified Stt7 has significant redoxdependent kinase activity in vitro. The demonstration of in vitro activity, the absence of a documented membrane bound state, and the small stoichiometry (circa 1:20) of interaction with the b6f complex (1), suggest that reaction of the kinase with the LHCII occurs through a membrane-peripheral domain of the b6f complex. (1) Lemeille, S. et al. (2009) PLoS Biology 7, e1000045; (2) Vener, A. V. et al. (1997) PNAS, 94, 1585-1590. (3) Millner, P. et al., J. Biol. Chem., 257, 1736-1742, 1982. Support from NIHGMS038323.

Mechanosensation 469-Pos Board B249 Mechanosensetivity and Symmetry in K2P Channels Julian Tim Brennecke, Bert L. de Groot. Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany. We studied mechanosensetive two-pore domain (K2P) potassium channels (i.e. TRAAK and TREK-2) by Molecular Dynamics (MD) Simulations. Mechanosensetivity of these channels is mediated directly through the lipid bilayer. By applying a membrane pressure protocol combined with Computational Electrophysiology (CompEL) we aim to gain insight into the structural rearrangements underlying mechanosensing of the channels while simultaneously measuring the permeability. In the study we focus particularly on the question if the observed structural changes follow the pseudo-fourfold symmetry of the crystal structures or if asymmetric changes dominate. To this aim we quantify symmetric and asymmetric contributions to the observed conformational changes in the MD simulations.

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470-Pos Board B250 ATP Release via GAP Junction Hemichannels in Rat Atrial Myocytes under Shear Stress Joon-Chul Kim, Sun-Hee Woo. College of Pharmacy, Chungnam National University, Daejeon, Korea, Republic of. We have previously shown that shear stress (~16 dyn/cm2) induces longitudinal Ca2þ propagation wave with a 0.2- to 3-s delay in atrial myocytes through the activation of P2Y1 purinergic signaling, and that the shear-mediated wave generation is inhibited by the blockade of gap junction hemichannels (J Physiol 2015 http://dx.doi.org/10.1113/JP271016). In the present study, we further examined whether atrial cells release ATP via the gap junction hemichannels. ATP releases of cells were directly measured with 2-s intervals as chemiluminescence emitted (at 562 nm) during ATP-driven luciferin-luciferase reaction. Shear stress was applied to fields of cells in a laminar flow chamber attached to the flow regulator. ATP was released from atrial myocytes by the application of shear stress in the range of 1-16 dyn/cm2 in a strength-dependent manner. The shear-induced ATP release occurred transiently with a maximal release (~1.3x1016 moles/mm2) at about 2 s and 90% decay at 4-6 s after the shear application (~16 dyn/cm2 for 10 s). Removal of external Ca2þ to enhance the activity of the gap junction hemichannels increased the shearmediated ATP release to ~320% of control. In cells pre-treated with carbenoxolone (50 mM for 10 min) or La3þ (2 mM for 5 min), the hemichannel blockers, ATP release by shear application was suppressed by ~50% and ~90%, respectively. The shear-induced ATP release was not affected by the pre-treatment of 50 mM Gd3þ (5 min) or 1 mM 9-anthracenecarboxylic acid (10 min), suggesting no role of maxi anion channel and Cl channels in shear-induced ATP release in these myocytes. Our data suggest that shear stress induces ATP release through the gap junction hemichannels in atrial myocytes. 471-Pos Board B251 Investigating the Role of Nav1.5 in Somatosensory Mechanosensation Evan O. Anderson, Eve R. Schneider, Jon D. Matson, Elena O. Gracheva, Slav N. Bagriantsev. Cellular & Molecular Physiology, Yale University, New Haven, CT, USA. Low-threshold mechanoreceptors (LTMR) are a diverse subpopulation of primary afferent neurons responsible for sensing the variety of mechanical stimuli experienced on a day-to-day basis. However, molecular details of touch sensation in LTMRs remain elusive. To investigate the neurons and molecules underlying touch sensation, we study the mechanosensation in a novel model system - trigeminal neurons of tactile foraging ducks (Anas peking). Ducks rely on the abundance of mechanoreceptors in the glabrous skin of their bill in order to identify food in the absence of olfactory or visual cues. To understand the molecular basis of mechanosensation in trigeminal LTMRs, we compared the transcriptome of duck trigeminal (TG) and dorsal root ganglia (DRG) (Schneider et al., PNAS 2014). The analysis revealed elevated expression of the mechanosensitive voltage-gated sodium channel Nav1.5 (SCN5a) in duck TG compared to DRG. Nav1.5 is known for its contribution to the cardiac action potential, but its role in the sense of touch is unexplored. Considering the high level of Nav1.5 expression in duck TG by RNA-seq and the mechanosensitivity of Nav1.5, we hypothesize a role of Nav1.5 in transduction or transmission of touch. Our work demonstrates a wide distribution of the SCN5a transcript in duck TG neurons, providing further rationale for a role of Nav1.5 in touch sensation. Here, we performed biophysical characterization of duck Nav1.5 in vitro and in trigeminal neurons. Our data reveals key functional differences between the human and duck orthologs of Nav1.5 and suggest a novel role for this channel in the sense of touch. 472-Pos Board B252 Functional Heteromeric PIEZO1 Ion Channels Philip Gottlieb, Chilman Bae, Radakrishnan Gnanasambandam, Frederick Sachs. Physiology and Bophysics, SUNY at BUffalo, Buffalo, NY, USA. Eukaryotic PIEZO1 ion channels are cation selective and open with membrane tension. The channel is further characterized by voltage dependent inactivation and a conductance of ~45 pS. We mutated the glutamic acid at position 2117 to an aspartic acid and this increased channel conductance to 72 pS. The mutations E2117A or E2117K lowered channel conductance. These mutations did not alter the current-voltage relationship or the ion selectivity, suggesting that the site is not in the selectivity filter but possibly near the pore. This is consistent with the cryo EM structure of mPiezo1 where the homologous residue is near, but not in the pore located toward the C-terminus. These mutations also affected the rates of activation and inactivation implying

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that the C-terminus is involved in channel gating. The mutation E2117D slowed the inactivation rate 8-fold. We made heteromeric channels in HEK cells by co-transfecting with high and low conductance mutants. This created active channels with two new unitary conductances indicating that pore formation occurs at the subunits’ interface. The number of new conducting states supports the trimer structure as revealed by Cryo EM. This is the first demonstration of functional heteromers of Piezo channels. Heteromeric formation is likely to be important for understanding the physiological activity of these channels. 473-Pos Board B253 Pore Determinants of Mechanosensitive Piezo Channels Qiancheng Zhao. Tsinghua University, Beijing, China. Piezo proteins have been proposed as the long-sought-after mechanosensitive (MS) cation channels in mammals that play critical roles in various mechanotransduction processes, such as touch sensation and vascular development. However, their ion-conducting pore and ion permeation mechanisms have remained undefined. Here we identify domains and specific residues in Piezo1 that control the essential pore properties, including unitary conductance, ion selectivity and pore blockage, pinpointing the ion-conducting pathway. By uncovering the bona fide ion-conducting pore, these findings not only provide definitive proof that Piezo proteins are genuine pore-forming subunits of MS cation channels, but also shed light on elucidating the ion permeation and gating mechanisms of this prototypic class of mammalian MS cation channels. 474-Pos Board B254 Characterization and Physiological Role of a Bacterial-Like Mechanosensitive Channel in Trypanosoma Cruzi Osmoregulation Noopur Dave, Christopher Skorka, Heather Lynch, Veronica Jimenez. Biological Science, California State University Fullerton, Fullerton, CA, USA. In order to complete its life cycle, Trypanosoma cruzi-the protozoan parasite that causes Chagas disease- faces various environmental changes as it propagates from an insect vector to a mammalian host. Previous studies have shown that T. cruzi has a robust osmoregulatory response, however the osmosensors involved in the detection and compensation pathways have not been identified. Mechanosensitive channels, which are activated by a stretch of the plasma membrane, have been associated with sensing of environmental changes in other organisms, but the function of these channels in T. cruzi is still unknown. In silico analysis of T. cruzi genome reveals the presence of mechanosensitive channels similar to the ones described in bacteria. We hypothesize that a bacterial-like mechanosensitive channel, TcMcS, is involved in osmoregulatory processes in T. cruzi. Overexpressing mutants using a tetracycline inducible system were developed to investigate the role of TcMcS in T. cruzi osmoregulation. Knockout mutants mediated by CRISPR/Cas9 were generated to test the essentiality of the protein. The localization and expression pattern of TcMcS varied in the three main life stages of T. cruzi. TcMcS seems to be localized in the contractile vacuole of epimastigote and trypomastigote forms, and in the plasma membrane of intracellular amastigote forms. Under hyposmotic stress, cells overexpressing TcMcS swell significantly less than wild- type parasites. Under EGTA treatment, this advantage was eliminated, suggesting that calcium plays a role in the osmoregulatory response. Known mechanosensitive channel blockers, including gadolinium and streptomycin, were found to elicit significant differences in the parasite’s ability to detect and compensate for the osmotic stress. Overall, our results support the idea that TcMcS is involved in sensing and compensation of osmotic stress in T. cruzi. Funding: NIH Grant R00AI101167, CSUF MHIRT Program, CSUF HHMI. 475-Pos Board B255 Systematic Discovery of the ‘Force-From-Lipid’ Principles Pietro Ridone1, Amrutha Patkunarajah2, Andrew Battle3, Boris Martinac4. 1 Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Chippendale, Australia, 2School of Medical Sciences, University of New South Wales, Sydney, Australia, 3School of Pharmacy, Griffith University, Gold Coast, Australia, 4Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Sydney, Australia. The functioning of bacterial mechanosensitive channels is governed by the lipid environment in which they are embedded. Several physical parameters such as lipid charge, saturation and topography, work in concert during transmission of tension from the bilayer onto the ion channel, thus making it a difficult task to dissect the contribution of each parameter to the channel’s mechanosensitivity.

The elasticity modulus, level of saturation, fatty acid length and the electrical charge of the lipids ultimately modify the lateral pressure profile of the bilayer at the hydrophobic interface with the ion channels. Even if we are currently unable to measure the lateral pressure profile directly, these changes can be indirectly measured in patch-clamp experiments by using MscS and MscL as probes. Our previous experimental results have shown how the lipid Cardiolipin differentially affects MscS and MscL in artificial bilayers, and our current research shows how lipid saturation plays an essential role in mechanosensitivity and gating frequency of these channels. By focusing on the change of the channel-activity parameters such as activation threshold, frequency of gating, open probability and hysteresis, we aim to generate a model of the ‘force-from-lipid’ principles through the systematic patch-clamping of liposomes of controlled composition. 476-Pos Board B256 Expression and Biophysical Characterization of Bacterial MechanoSensitive Ion Channel of Large Conductance into Mammalian Cells Alessandro Soloperto1, A. Bartolozzi1, G. Palazzolo1, M. Basso2, A. Contestabile1, M. Vassalli3, F. Difato1. 1 Neuroscience and Brain Technologies, Fondazione Istituto Italiano di Tecnologia, Genova, Italy, 2Dipartimento di Sistemi e Informatica, Universita` di Firenze, Firenze, Italy, 3Institute of Biophysics, National Research Council of Italy, Genova, Italy. Mechanobiology is an emerging field of science focused on the role of physical parameters in determining cell morphology and physiology. Mechanosensistive ion channels represent one of the more important cellular elements sensing and transducing mechanical forces in chemical signaling activating biological pathways. The mechanosensitive channel of large conductance (MscL) was the first mechano-sensitive ion channel to be isolated, cloned and sequenced, and therefore is the more characterized. In Escherichia Coli, MscL acts as an emergency release valve when the tension in the membrane lipid bilayer is getting near to the lytic limit. In the past, the MscL has been used as a comparative model for newly discovered mechano-sensitive channels, and recently, it has been recognized as a tool for potential application in nanotechnology. For example, the MscL have been successfully expressed in mammalian cells to achieve controlled delivery of bioactive molecules through application of mechanical forces. In the present work, we report the expression of the MscL channel into mammalian cells, in order to develop an experimental model of cells highly sensitive to mechanical forces. In order to fully characterize the biophysical features of this transmembrane protein, we developed an optical tweezers setup integrated with a patch-clamp recording system to apply calibrated forces on single cells and simultaneously record the electrophysiological response of the genetically modified cells. We strongly believe that heterologous cellular expression of the MscL may become a useful tool to achieve targeted modulation of cellular activity, as well as to understand the role of mechanical properties of the materials currently used for the development of cellurized scaffolds. 477-Pos Board B257 The Role of the C-Terminal Domain on the Gating Properties of Corynebacterium Glutamicum Mechanosensitive Channel MscCG Yoshitaka Nakayama1, Michael Becker2, Haleh Ebrahimian3, Tomoyuki Konishi4, Hisashi Kawasaki4, Reinhard Kramer5, Boris Martinac1. 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2University of Cologne, Koln, Germany, 3Wollongong University, Sydney, Australia, 4 Tokyo Denki University, Tokyo, Japan, 5University of Cologne, Koln, Japan. The gram-positive soil bacterium Corynebacterium glutamicum exports a large amount of glutamate through the mechanosensitive channel MscCG. This process is utilized for the world industrial glutamate production. MscCG belongs to a subfamily of bacterial MscS-like channels, which function in osmoregulation, however this channel shows structural and functional differences compared to MscS. To understand the role of the carboxyl terminal domain of MscCG, the chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in E. coli spheroplasts characterized by a lower activation threshold and slow channel closing compared to MscS. The chimeric channel MscS-(C-MscCG) was also successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at