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• W2047858693 abstract "The crystal structure of the sensorless pore module of a voltage-gated K+ (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules. The crystal structure of the sensorless pore module of a voltage-gated K+ (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules. Voltage-gated channels responsible for cellular excitability are assemblies of modular membrane protein subunits consisting of two distinct, tandemly arranged, functional modules. An N-terminal voltage-sensor module encompassing four transmembrane segments S1–S4 and a C-terminal pore module (PM) 3The abbreviations used are:PMpore moduleDPhPC1,2-diphytanoyl-sn-glycero-3-phosphocholineDOPC1,2-dioleoyl-sn-glycero-3-phosphocholineDPhPE1,2-diphytanoyl-sn-glycero-3-phosphoethanolamineDOPA1,2-dioleoyl-sn-glycero-3-phosphatidic acidDOPG1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)DPhPG1,2-diphytanoyl-sn-glycero-3-phospho-(1′-rac-glycerol)Lyso-PG1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol)DOPI1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol)lysyl-DOPG1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))]monoolein1-oleoyl-rac-glycerolDOPE-PEG3501,2-dioleoyl-sn-glycero-3-phosphoethanolamine N-(methoxy(polyethylene glycol)-350)Poopen probabilityγsingle channel conductanceτopenmean open timeτcritτ-criticaltdreceiver dead timePGphosphatidylglycerol. consisting of transmembrane segments S5 and S6 (1.Montal M. 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Cherezov V. Baker K.A. Stevens R.C. Montal M. Crystal structure of a voltage-gated K+ channel pore module in a closed state in lipid membranes.J. Biol. Chem. 2012; 287: 43063-43070Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 6.Chakrapani S. Cuello L.G. Cortes D.M. Perozo E. Structural dynamics of an isolated voltage-sensor domain in a lipid bilayer.Structure. 2008; 16: 398-409Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7.Butterwick J.A. MacKinnon R. Solution structure and phospholipid interactions of the isolated voltage-sensor domain from KvAP.J. Mol. Biol. 2010; 403: 591-606Crossref PubMed Scopus (80) Google Scholar) and function (8.Santos J.S. Grigoriev S.M. Montal M. Molecular template for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel.J. Gen. Physiol. 2008; 132: 651-666Crossref PubMed Scopus (22) Google Scholar, 9.Alabi A.A. Bahamonde M.I. Jung H.J. Kim J.I. Swartz K.J. Portability of paddle motif function and pharmacology in voltage sensors.Nature. 2007; 450: 370-375Crossref PubMed Scopus (188) Google Scholar, 10.Syeda R. Santos J.S. Montal M. Bayley H. Tetrameric assembly of KvLm K+ channels with defined numbers of voltage sensors.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 16917-16922Crossref PubMed Scopus (14) Google Scholar). The discovery of the voltage-gated K+ (Kv) channel from Listeria monocytogenes (KvLm) (11.Santos J.S. Lundby A. Zazueta C. Montal M. Molecular template for a voltage sensor in a novel K+ channel. I. Identification and functional characterization of KvLm, a voltage-gated K+ channel from Listeria monocytogenes.J. Gen. Physiol. 2006; 128: 283-292Crossref PubMed Scopus (23) Google Scholar), which embodies an incipient sensor (12.Lundby A. Santos J.S. Zazueta C. Montal M. Molecular template for a voltage sensor in a novel K+ channel. II. Conservation of a eukaryotic sensor fold in a prokaryotic K+ channel.J. Gen. Physiol. 2006; 128: 293-300Crossref PubMed Scopus (8) Google Scholar), allowed us to dissect the pore module from the intact subunit, to demonstrate that it recapitulates the full complement of functional features after reconstitution in lipid bilayers (8.Santos J.S. Grigoriev S.M. Montal M. Molecular template for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel.J. Gen. Physiol. 2008; 132: 651-666Crossref PubMed Scopus (22) Google Scholar), and to determine its crystal structure in a membrane (5.Santos J.S. Asmar-Rovira G.A. Han G.W. Liu W. Syeda R. Cherezov V. Baker K.A. Stevens R.C. Montal M. Crystal structure of a voltage-gated K+ channel pore module in a closed state in lipid membranes.J. Biol. Chem. 2012; 287: 43063-43070Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). These advances afford an opportunity to focus on this pore-only entity, extending from the N-terminal end of the S4–S5 linker to the C terminus of the protein, to investigate the role of the individual lipid monolayer components of the bilayer in modulating the channel properties. pore module 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-dioleoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine 1,2-dioleoyl-sn-glycero-3-phosphatidic acid 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) 1,2-diphytanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] 1-oleoyl-rac-glycerol 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine N-(methoxy(polyethylene glycol)-350) open probability single channel conductance mean open time τ-critical receiver dead time phosphatidylglycerol. It is widely recognized that the lipid bilayer components modulate the function of K+ channels (13.Forte M. Satow Y. Nelson D. Kung C. Mutational alteration of membrane phospholipid composition and voltage-sensitive ion channel function in paramecium.Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 7195-7199Crossref PubMed Scopus (39) Google Scholar, 14.Bell J.E. Miller C. Effects of phospholipid surface charge on ion conduction in the K+ channel of sarcoplasmic reticulum.Biophys. J. 1984; 45: 279-287Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 15.Moczydlowski E. Alvarez O. Vergara C. Latorre R. Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers.J. Membr. Biol. 1985; 83: 273-282Crossref PubMed Scopus (121) Google Scholar, 16.Bian J. Cui J. McDonald T.V. HERG K+ channel activity is regulated by changes in phosphatidylinositol 4,5-bisphosphate.Circ. 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Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids.Science. 2004; 304: 265-270Crossref PubMed Scopus (278) Google Scholar, 21.Lee S.Y. Lee A. Chen J. MacKinnon R. Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15441-15446Crossref PubMed Scopus (273) Google Scholar, 22.Schmidt D. Jiang Q.X. MacKinnon R. Phospholipids and the origin of cationic gating charges in voltage sensors.Nature. 2006; 444: 775-779Crossref PubMed Scopus (341) Google Scholar, 23.Marius P. Zagnoni M. Sandison M.E. East J.M. Morgan H. Lee A.G. Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening.Biophys. J. 2008; 94: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 24.Schmidt D. MacKinnon R. Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 19276-19281Crossref PubMed Scopus (132) Google Scholar, 25.Xu Y. Ramu Y. Lu Z. Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels.Nature. 2008; 451: 826-829Crossref PubMed Scopus (150) Google Scholar, 26.Whorton M.R. MacKinnon R. Crystal structure of the mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium.Cell. 2011; 147: 199-208Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 27.Iwamoto M. Oiki S. Amphipathic antenna of an inward rectifier K+ channel responds to changes in the inner membrane leaflet.Proc. Natl. Acad. Sci. 2013; 110: 749-754Crossref PubMed Scopus (60) Google Scholar, 28.Rodriguez-Menchaca A.A. Adney S.K. Tang Q.-Y. Meng X.-Y. Rosenhouse-Dantsker A. Cui M. Logothetis D.E. PIP2 controls voltage-sensor movement and pore opening of Kv channels through the S4-S5 linker.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E2399-E2408Crossref PubMed Scopus (72) Google Scholar, 29.Zaydman M.A. Silva J.R. Delaloye K. Li Y. Liang H. Larsson H.P. Shi J. Cui J. Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 13180-13185Crossref PubMed Scopus (132) Google Scholar, 30.Weingarth M. Prokofyev A. van der Cruijsen E.A. Nand D. Bonvin A.M. Pongs O. Baldus M. Structural determinants of specific lipid binding to potassium channels.J. Am. Chem. Soc. 2013; 135: 3983-3988Crossref PubMed Scopus (66) Google Scholar, 31.van der Cruijsen E.A. Nand D. Weingarth M. Prokofyev A. Hornig S. Cukkemane A.A. Bonvin A.M. Becker S. Hulse R.E. Perozo E. Pongs O. Baldus M. Importance of lipid-pore loop interface for potassium channel structure and function.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 13008-13013Crossref PubMed Scopus (39) Google Scholar, 32.van Dalen A. de Kruijff B. The role of lipids in membrane insertion and translocation of bacterial proteins.Biochim. Biophys. Acta. 2004; 1694: 97-109Crossref PubMed Scopus (57) Google Scholar). A role for lipids on the inactivation of Kv channels, namely the ability of the channel to close and remain closed for prolonged periods while voltage-clamped at an activating potential, has been previously demonstrated (24.Schmidt D. MacKinnon R. Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 19276-19281Crossref PubMed Scopus (132) Google Scholar, 33.Schmidt D. Cross S.R. MacKinnon R. A gating model for the archeal voltage-dependent K+ channel KvAP in DPhPC and POPE:POPG decane lipid bilayers.J. Mol. Biol. 2009; 390: 902-912Crossref PubMed Scopus (58) Google Scholar). In recent years this modulatory effect has been attributed solely to an interaction of the lipid bilayer with the voltage-sensor module. The mechanical properties of the membrane are dictated by its lipid composition and the interaction of the basic charges on the sensor with lipid headgroup phosphates as a means to facilitate sensor movement and therefore pore opening (22.Schmidt D. Jiang Q.X. MacKinnon R. Phospholipids and the origin of cationic gating charges in voltage sensors.Nature. 2006; 444: 775-779Crossref PubMed Scopus (341) Google Scholar, 25.Xu Y. Ramu Y. Lu Z. Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels.Nature. 2008; 451: 826-829Crossref PubMed Scopus (150) Google Scholar). The crystal structure of the sensorless PM of a Kv channel showed four immobilized lipids filling and surrounding a crevice between subunits at the extracellular surface of the channel, suggesting an affinity for lipids in that region (5.Santos J.S. Asmar-Rovira G.A. Han G.W. Liu W. Syeda R. Cherezov V. Baker K.A. Stevens R.C. Montal M. Crystal structure of a voltage-gated K+ channel pore module in a closed state in lipid membranes.J. Biol. Chem. 2012; 287: 43063-43070Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Therefore, we asked if different lipids could act as selective modulators of ion channel function by acting as co-factors in the ion translocation process. Functional studies using single channel current recordings of a sensorless PM reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of the lipid headgroup on gating the two channel gates located at the selectivity filter (34.Hoshi T. Zagotta W.N. Aldrich R.W. Two types of inactivation in Shaker K+ channels. Effects of alterations in the carboxy-terminal region.Neuron. 1991; 7: 547-556Abstract Full Text PDF PubMed Scopus (569) Google Scholar, 35.Choi K.L. Aldrich R.W. Yellen G. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 5092-5095Crossref PubMed Scopus (394) Google Scholar, 36.Liu Y. Jurman M.E. Yellen G. Dynamic rearrangement of the outer mouth of a K+ channel during gating.Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar), facing the extracellular medium, and at the bundle crossing (37.Liu Y. Holmgren M. Jurman M.E. Yellen G. Gated access to the pore of a voltage-dependent K+ channel.Neuron. 1997; 19: 175-184Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar), in direct contact with the cytoplasmic facing leaflet of the membrane. Taking advantage of the uniqueness of asymmetric bilayers (38.Montal M. Asymmetric lipid bilayers response to multivalent ions.Biochim. Biophys. Acta. 1973; 298: 750-754Crossref PubMed Scopus (30) Google Scholar, 39.Rothman J.E. Kennedy E.P. Asymmetrical distribution of phospholipids in the membrane of Bacillus megaterium.J. Mol. Biol. 1977; 110: 603-618Crossref PubMed Scopus (116) Google Scholar, 40.Op den Kamp J.A. Lipid asymmetry in membranes.Annu. Rev. Biochem. 1979; 48: 47-71Crossref PubMed Scopus (976) Google Scholar, 41.Devaux P.F. Morris R. Transmembrane asymmetry and lateral domains in biological membranes.Traffic. 2004; 5: 241-246Crossref PubMed Scopus (216) Google Scholar, 42.van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids. Where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4480) Google Scholar) allowed us to uncover two mechanisms for the stabilization of the open channel conformation. First, we discovered that the inclusion of lipids with headgroups that coat the extracellular membrane-solution interface with hydroxyl groups such as those found in glycerol, phosphoglycerol, phosphoinositol, and lysyl-phosphoglycerol increased drastically the probability of finding the channel open. This effect was observed to be independent of headgroup charge, number of acyl chains, and whether the chains were methyl branched or not. We propose that the stabilizing effect of these surface-coating hydroxyls is indirectly mediated by an interaction with water at the membrane-solution interface. We further validate this proposal by demonstrating that the addition of 1% (0.17 m) ethylene glycol or 0.2 m mannitol to the extracellular bathing solution of lipid bilayers lacking exposed hydroxyls emulates the gain in open conformation stability observed when lipids with exposed hydroxyls are present at the extracellular membrane interface. Second, we unveiled a π-cation interaction (43.Ma J.C. Dougherty D.A. The cation-π interaction.Chem. Rev. 1997; 97: 1303-1324Crossref PubMed Scopus (3314) Google Scholar) between a lysine containing lipid headgroup and a conserved aromatic residue in Kv channels at the protein cytoplasmic surface as an additional stabilizer to generate an open, rarely interrupted, K+ permeation path. The new findings provide evidence that the PM of Kv channels may be transformed into an open conductor through interactions with lipid modulators that target either the filter gate, through a destabilization of water structure, or the bundle gate, via a direct interaction, and thereby stabilize the conducting conformation of the channel. The implication is that lipids are crucial not only for proper protein folding (44.Valiyaveetil F.I. Zhou Y. MacKinnon R. Lipids in the structure, folding, and function of the KcsA K+ channel.Biochemistry. 2002; 41: 10771-10777Crossref PubMed Scopus (297) Google Scholar) but also to modulate, in a deterministic manner, the properties of the channel. KvLm PM, WT, and mutants were expressed in Escherichia coli XL1-Blue with a C-terminal His tag and purified by Ni2+-affinity and size exclusion chromatography as previously described (45.Santos J.S. Syeda R. Montal M. Stabilization of the conductive conformation of a Kv channel. The lid mechanism.J. Biol. Chem. 2013; 288: 16619-16628Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Single channel mutations were introduced using the QuikChange site-directed mutagenesis kit (Agilent) according to the manufacturer's instructions. Liposomes were composed of 100 mol % of 1,2-diphytanoyl-sn-glycero-3 phosphocholine (DPhPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), or 90 mol % of DPhPC and 10 mol % of the indicated test lipids: DOPC, DPhPE, 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-diphytanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPhPG), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (lyso-PG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) (DOPI), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (lysyl-DOPG), 1-oleoyl-rac-glycerol (monoolein) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine N-(methoxy(polyethylene glycol))-350 (DOPE-PEG350) (Avanti Polar Lipids, Alabaster, AL) (Fig. 1). Liposomes were prepared as previously described (45.Santos J.S. Syeda R. Montal M. Stabilization of the conductive conformation of a Kv channel. The lid mechanism.J. Biol. Chem. 2013; 288: 16619-16628Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). When indicated, ethylene glycol (0.17 m) or mannitol (0.2 m) were supplemented to the rehydrated liposome suspension prior to extrusion. For reconstitution, the protein was diluted ∼100–300-fold into preformed liposomes to give final protein concentrations 2–5 μg/ml. The proteoliposomes were incubated on ice for 15 min prior to bilayer recording experiments. Fresh aliquots of liposomes and protein were used for every experiment. Single channel currents were recorded from droplet interface bilayers as described (10.Syeda R. Santos J.S. Montal M. Bayley H. Tetrameric assembly of KvLm K+ channels with defined numbers of voltage sensors.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 16917-16922Crossref PubMed Scopus (14) Google Scholar, 46.Bayley H. Cronin B. Heron A. Holden M.A. Hwang W.L. Syeda R. Thompson J. Wallace M. Droplet interface bilayers.Mol. Biosyst. 2008; 4: 1191-1208Crossref PubMed Scopus (388) Google Scholar). Briefly, a 10 × 10 × 4-mm Plexiglas chamber was filled with hexadecane (Sigma) containing 1 mol % of DPhPC. A 0.2-mm diameter Ag/AgCl wire electrode (40 mm in length) was attached to each of two micromanipulators (NMN-21, Narishige, Japan). Droplets (∼200 nl) were placed with a pipette on each of the electrodes, which had been coated with 3% (w/v) low-melt agarose (65 °C). The electrode carrying the droplet with the proteoliposomes in 0.5 m KCl, 10 mm HEPES, pH 7.4, was connected to the grounded end (cis side) of the amplifier head-stage. The second electrode, in a droplet containing exclusively liposomes in the same buffer, was connected to the working end of the head-stage (trans side). The chamber, electrodes, and the amplifier head-stage were enclosed in a Faraday cage. The droplets were incubated in hexadecane containing 1 mol % of DPhPC until a monolayer of lipid had formed around them (∼5 min). A bilayer spontaneously formed when the two droplets were brought into contact. Regardless of bilayer composition, and symmetric or asymmetric configuration, the proteoliposome droplet was always placed on the cis compartment to ensure that the filter gate of the channel protein remained on the cis side and was exposed to only one class of lipids. To confirm the orientation of the protein in the reconstituted bilayers with the consequent outward flow of current (from bundle to filter), the open channel blocker tetrabutyl ammonium was used (45.Santos J.S. Syeda R. Montal M. Stabilization of the conductive conformation of a Kv channel. The lid mechanism.J. Biol. Chem. 2013; 288: 16619-16628Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Tetrabutyl ammonium stocks (5 mm) were made in droplet solution (0.5 m KCl, 10 mm HEPES, pH 7.4) and injected into the bundle-facing droplet (trans side) at the end of each experiment to a final concentration of 50 μm using a Nano injector (VWR instrument). In all configurations, channel orientation was verified by sided tetrabutyl ammonium block. The test lipids were always supplemented at 10 mol % in 90 mol % of DPhPC. To increase the signal-to-noise ratio all electrical measurements were performed in 0.5 m KCl, 10 mm HEPES, pH 7.4, at 100 mV and 22 ± 2 °C, unless otherwise indicated. Single channel currents were sampled at 20 kHz using an Axon 200B patch clamp amplifier, filtered by using a low pass Bessel filter (80 dB/decade) with a corner frequency of 2 kHz, and then digitized with a DigiData 1320 A/D converter (Axon Instruments). All pre-processing and analysis of the single channel recordings was performed with QuB software. Currents in symmetric lysyl-DOPG were further filtered to 400–700 Hz, whereas additional off-line filtering of 2 kHz was used for all other recordings. Event detection was performed by time course fitting with the segmental k-means algorithm (SKM) (47.Qin F. Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling.Biophys. J. 2004; 86: 1488-1501Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Stretches of the recordings in which the channel was closed were included in the analysis; hence, the calculated Po is proportional to the ratio of open time/total time of the recording. Segments of continuous recordings in the range of 30 s ≤ t ≤ 400 s were used for analysis. To avoid the detection of erroneous events, the receiver dead time (td) was set at 300 μs for all recordings except for PM in symmetric lysyl-DOPG for which the td was set to 700–1000 μs. Accordingly, transitions shorter than the td were ignored; transitions longer than the td were accepted as “events.” To calculate the median burst length, bursts were defined as a group of three or more opening transitions with intraburst closures shorter than τ-critical (τcrit) and terminated by an interburst closure longer than τcrit. The value of τcrit was calculated from the closed dwell time histograms in QuB for each record (3 ms ≤ τcrit ≤ 20 ms). All the results reported were done at least in triplicate; the calculated values are reported as mean ± S.E. N and n denote the number of events and number of experiments, respectively. Statistical significance was assessed using the unpaired, two-tailed Student's t test. Wherever the statement “equal within error” appears, the p value was >0.05, indicating that the two groups in question are equal and there is no significant statistical difference. By contrast, wherever the open probability (or any other parameter) is stated to be “x-fold higher,” the p value was <0.05 indicating that the differences are statistically significant. To determine which residues in the PM are within 5 Å of a lipid phosphate or choline headgroup we used YASARA (Yasara Bioscience) to run a 10 ns molecular dynamics simulation (48.Krieger E. Darden T. Nabuurs S.B. Finkelstein A. Vriend G. Making optimal use of empirical energy functions. Force-field parameterization in crystal space.Proteins. 2004; 57: 678-683Crossref PubMed Scopus (700) Google Scholar) of KvLm PM (5.Santos J.S. Asmar-Rovira G.A. Han G.W. Liu W. Syeda R. Cherezov V. Baker K.A. Stevens R.C. Montal M. Crystal structure of a voltage-gated K+ channel pore module in a closed state in lipid membranes.J. Biol. Chem. 2012; 287: 43063-43070Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) in DOPC bilayers. At the end of the simulation, residues within 5 Å of a phosphate oxygen or a choline nitrogen were identified, given that the Debye screening at 0.5 m salt is 4 Å. The simulation was performed at 25 °C and pH 7.4 with the salt concentration fixed at 0.5 m KCl. The AMBER03 force-field was employed (49.Duan Y. Wu C. Chowdhury S. Lee M.C. Xiong G. Zhang W. Yang R. Cieplak P. Luo R. Lee T. Caldwell J. Wang J. Kollman P. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations.J. Comput. Chem. 2003; 24: 1999-2012Crossref PubMed Scopus (3730) Google Scholar). To investigate the functional consequence of an interaction between the PM of a Kv channel and the surrounding lipids, PM function was screened in lipid bilayers with varying headgroup chemistry and acyl chains (Fig. 1). The sensorless PM of KvLm was reconstituted in droplet interface bilayers in which both cytoplasmic and extracellular leaflets were of equal composition (symmetric bilayers, Fig. 1J); channel function was assayed by monitoring the single channel steady-state activity at a depolarizing potential of 100 mV while bathing both sides of the membrane in 0.5 m KCl. Channel function was first assayed in 100 mol % of DPhPC, DOPC, or DPhPE. In these bilayers of zwitterionic phospholipid" @default.
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• W2047858693 date "2014-02-01" @default.
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• W2047858693 title "Lipid Bilayer Modules as Determinants of K+ Channel Gating" @default.
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