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(pHEXT). Sensing currents of Ci-VSP recorded during activation slowed down Two-pore domain (K2P) channels are important regulators of as pHEXT was decreased. Noteworthy, the kinetic of deactivating sensing cur- cellular electrical excitability integrating a wide range of stimuli including rents was less affected, suggesting that the initial activation of the VSD phosphorylation, various lipids, temperature, intracellular/extracellular pH involved a pHEXT-sensitive, rate-limiting step. In spite of these observations, and membrane voltage. Here we investigated the mechanism of voltage the voltage dependence of activating sensing currents was almost unaltered sensing/gating in K2P channels. We show that TASK-3 and TRAAK are by pHEXT (6.5 to 9.0). In contrast, voltage dependence was strongly modulated mostly closed at the resting membrane potential but become strongly activated by pHEXT after VSD relaxation. In fact, the shift in voltage dependence for by depolarisation similar to classical Kv channels. Moreover, strong voltage sensing current was 30 mV larger at pHEXT 6.5 with respect to 9.0. These ob- activation was also seen for other K2P channels such as TREK-1, TREK-2, servations indicated that the equilibrium between the resting and active states TRESK and TALK-2, however, only when intracellular Kþ was replaced was pHEXT insensitive. Conversely, the equilibrium between the relaxed and by other permeant ions such as NH4þ,Csþ or Rbþ. Strikingly, voltage acti- þ the relaxed-resting states was unambiguously pHEXT sensitive. Furthermore, vation was abolished upon intracellular Na or NMDGþ replacement sug- given that decreasing pHEXT slowed sensing currents, this observation suggests gesting that movements of permeant ions in the electric field focused in the that the energy barrier between the resting and active states, rather than the dif- selectivity filter power voltage gating and, thus, are likely to represent the ference in energy between these states, was modulated by pHEXT. In contrast, gating charge in K2P channels. Indeed, mutations in the selectivity filter, ex- for the relaxed VSD, pHEXT strongly modulated the difference in energy be- periments with high affinity pore blockers and cysteine modification establish tween the relaxed and the relaxed-resting states. the selectivity filter as the voltage gate. Furthermore, voltage activation in K2P channels exhibits distinct functional features compared to classical Kv 3771-Pos Board B499 channel gating as the V1/2 for voltage activation was strictly coupled to the Gating-Coupled Clustering-Dispersion Dynamics of the KcsA Potassium K2P channel reversal potential and the kinetics of voltage gating were largely Channel in a Lipid Membrane voltage-independent. The latter finding suggests a voltage-dependent fast 1,2 3 2 Ayumi Sumino , Daisuke Yamamoto , Masayuki Iwamoto , multi ion binding step to a nonconductive but ion accessible selectivity filter 4 2 Takehisa Dewa , Shigetoshi Oiki . and a second slower voltage-independent gating step leading to the conductive 1JST/PRESTO, Saitama, Japan, 2University of Fukui, Yoshida-gun, Japan, 3 4 state of the selectivity filter. These findings demonstrate a novel concept of Fukuoka University, Fukuoka, Japan, Nagoya Institute of Technology, voltage sensing in Kþ channels and point to a new role of K2P channels in Nagoya, Japan. cell excitability as the strong voltage dependence would enable K2P channel Using atomic force microscopy (AFM), we previously uncovered the open-gate to contribute significantly to the repolarisation phase of neural (e.g. TRAAK) structure of membrane-embedded KcsA potassium channels by removing the or cardiac (e.g. TASK-3) actions potentials. potentially sight-obstructing cytoplasmic domain (CPD). Here, we demonstrate the clustering and dispersion of CPD-truncated KcsA channels in the mem- 3774-Pos Board B502 brane and show that these processes are associated with pH-dependent gating. Shifting the Gating Equilibrium of a via Hydrophobic At neutral pH, AFM revealed self-assembled nanoclusters of closed channels Mismatch on a mica-attached membrane, and SDS-PAGE demonstrated supra- Dylan O. Burdette, Adrian Gross. molecular assembly on a liposomal membrane. At acidic pH, the open-gated Biochemistry and Molecular Biology, Rosalind Franklin University of channels were dispersed as single channels with a separation well beyond the Medicine and Science, North Chicago, IL, USA. contact distance. The clustering-dispersion dynamics were captured using When the potassium channel KcsA transitions from the closed to the open state, high-speed AFM. These results suggest that the channels undergo conforma- the inner helices transition outward and away from the axis of symmetry. This tional changes within the cluster but do not open until the channels are substantial conformational change can be expected to significantly alter the dispersed. The interplay between the gating conformational change of individ- both the shape and volume of the protein within the membrane. In this study ual channels and the collective behaviour of the clustering-dispersion dynamics we explored the possibility of exploiting this shape and volume change to provides insight into understanding membrane-mediated protein-protein inter- gate the channel using hydrophobic mismatch to alter the membrane induced actions and functional cooperativity. lateral forces on the channel. We spin-labeled residues of the inner helices 3772-Pos Board B500 known to exhibit strong spin-spin interaction (residues 103, 108, 109, 112, Towards the Incorporation of Functional Proteins in Teth- 116 and 118) and monitored spin-spin interactions by cw-EPR as we altered ered Membranes both pH and membrane composition. As previously observed, the spin-spin in- David P. Hoogerheide1,2, Amit Vaish1, Tatiana Rostovtseva2, teractions weakened when the open state was favored by lowering pH, due to Adam Kuszak3, Sergey Bezrukov2, Susan Krueger1, Hirsh Nanda1,4. the outward movement of the inner helices. We then altered the length of the 1NIST Center for Neutron Research, National Institute of Standards and acyl chains from 18 to 22 carbons at neutral pH. Over this range of chain Technology, Gaithersburg, MD, USA, 2National Institute of Child Health and lengths the strength of the spin-spin interactions changed to approximately Human Development, National Institutes of Health, Bethesda, MD, USA, the same degree as observed when lowering pH. We interpret these results to 3National Institute of Diabetes and Digestive and Kidney Diseases, National mean that the state distribution of the KcsA channel can be shifted by altering Institutes of Health, Bethesda, MD, USA, 4Department of Physics, Carnegie the membrane induced lateral forces on the channel. Mellon University, Pittsburgh, PA, USA. Biomimetic platforms play an important role in the study of a variety of biolog- 3775-Pos Board B503 ical processes. Tethered bilayer membranes (tBLMs) constitute platforms that Correlated Single-Molecule Spectroscopy and Patch-Clamp Studies of are useful in the study of membrane properties and a variety of integral and pe- Voltage Gated Ion Channel Activation Dynamics in Living Cells ripheral membrane proteins. The usual approach to forming tBLMs is to tether Dibyendu Sasmal, H. Peter Lu. the lipids directly to the substrate. For certain integral membrane proteins, the Bowling Green State University, Bowling Green, OH, USA. proteins can be tethered directly to the substrate and the lipid membrane recon- We have studied the NMDA receptor dynamics in HEK cells and colicin ion stituted around the tethered proteins. Here we report progress toward functional channel in , using our newly developed combining real-time sin- and structural characterization of bilayer membranes tethered using two gle-molecule FRET imaging with single-channel electric current recording. different ion channels, KcsA and the voltage-dependent anion channel We were able to probe single ion-channel open-close activation and associ- (VDAC). The bilayers are characterized using surface plasmon resonance, elec- ated protein conformational changes simultaneously, and thus providing a trochemical impedance spectroscopy, and neutron reflectivity. The one- molecular-level understanding of the dynamics and mechanism of ion- dimensional structural information derived from neutron reflectivity spectra channel activations. The kinetic behavior of ion channel proteins is regulated allows us to characterize the surface-bound proteins and describe the reconsti- by subtle conformational changes that are often difficult to characterize by tution of the lipid bilayer membrane. conventional ensemble-averaged static structure analysis and by interpreta- tions of ion-channel electrophysiological measurements. Subtle structural 3773-Pos Board B501 dynamics of ion channels play an important role in regulating channel func- A Novel Mechanism of Voltage Sensing and Gating in K2P Potassium tion and selectivity. Using our unique approaches, we have identified Channels the conformational regulated NMDA ion-channel activities and the associ- Marcus Schewe1, Markus Rapedius1, Ehsan Nematian-Ardestani1, ated conformational change dynamics. We were able to probe fluctuating Thomas Linke2, Klaus Benndorf2, Stephen J. Tucker3, Thomas Baukrowitz1. polypeptide-domain diffusional motions of single-molecule colicin 1Institute of Physiology, Kiel, Germany, 2Institute of Physiology II, Jena, channels across a lipid bilayer. A new solvation-desolvation model for Germany, 3Biological Physics Group, Clarendon Laboratory, Department of voltage-gated ion-channel dynamics has been developed based on our exper- Physics, Oxford, United Kingdom. imental results.