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Kenton J. Swartz - One of the best experts on this subject based on the ideXlab platform.
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Structural interactions of a Voltage Sensor toxin with lipid membranes.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Mihaela Mihailescu, Mirela Milescu, Kenton J. Swartz, Dmitriy Krepkiy, Klaus Gawrisch, Stephen H WhiteAbstract:Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including Voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a Voltage Sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to Voltage Sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the Voltage Sensor.
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Defining the Voltage Sensor Properties and Pharmacology of Nav1.9
Biophysical Journal, 2010Co-Authors: Frank Bosmans, Michelino Puopolo, Marie-france Martin-eauclaire, Bruce P. Bean, Kenton J. SwartzAbstract:The Voltage-activated sodium channel Nav1.9 is preferentially expressed in DRG neurons where it is believed to play an important role in pain perception. However, progress in revealing the gating characteristics and pharmacological sensitivities of Nav1.9 has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here we use a protein engineering approach to study the contributions of the four Nav1.9 Voltage Sensors to channel function. We define individual S3b-S4 paddle motifs within each Voltage Sensor and show that these structural motifs sense changes in membrane Voltage and determine the kinetics of Voltage Sensor activation. Toxins from tarantula and scorpion venom interact with each of these four motifs and can be used as pharmacological tools to alter Nav1.9 currents in native DRG neurons. Our results provide answers to fundamental questions on the functional role of the four Voltage Sensors in Nav1.9 and may be useful in developing new strategies to combat pain.
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Interactions between lipids and Voltage Sensor paddles detected with tarantula toxins
Nature Structural & Molecular Biology, 2009Co-Authors: Mirela Milescu, Frank Bosmans, Abdulrasheed A Alabi, Kenton J. SwartzAbstract:Increasing evidence indicates that membrane protein function can be affected by the surrounding membrane bilayer. A new study on Voltage-gated potassium channels using tarantula toxins suggests that lipid interaction with the Voltage Sensor can influence channel function. Voltage-activated ion channels open and close in response to changes in Voltage, a property that is essential for generating nerve impulses. Studies on Voltage-activated potassium (Kv) channels show that Voltage-Sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 Voltage-sensing domains and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters Voltage-Sensor activation in an S1–S4 Voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to Voltage-sensing domains and demonstrate that the pharmacological sensitivities of Voltage-activated ion channels are influenced by the surrounding lipid membrane.
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interactions between lipids and Voltage Sensor paddles detected with tarantula toxins
Nature Structural & Molecular Biology, 2009Co-Authors: Mirela Milescu, Jae Il Kim, Frank Bosmans, Abdulrasheed A Alabi, Seungkyu Lee, Kenton J. SwartzAbstract:Voltage-activated ion channels open and close in response to changes in Voltage, a property that is essential for generating nerve impulses. Studies on Voltage-activated potassium (Kv) channels show that Voltage-Sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 Voltage-sensing domains, and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters Voltage-Sensor activation in an S1–S4 Voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to Voltage-sensing domains and demonstrate that the pharmacological sensitivities of Voltage-activated ion channels are influenced by the surrounding lipid membrane.
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Deconstructing Voltage Sensor function and pharmacology in sodium channels.
Nature, 2008Co-Authors: Frank Bosmans, Marie-france Martin-eauclaire, Kenton J. SwartzAbstract:Voltage-activated sodium (Na(v)) channels are crucial for the generation and propagation of nerve impulses, and as such are widely targeted by toxins and drugs. The four Voltage Sensors in Na(v) channels have distinct amino acid sequences, raising fundamental questions about their relative contributions to the function and pharmacology of the channel. Here we use four-fold symmetric Voltage-activated potassium (K(v)) channels as reporters to examine the contributions of individual S3b-S4 paddle motifs within Na(v) channel Voltage Sensors to the kinetics of Voltage Sensor activation and to forming toxin receptors. Our results uncover binding sites for toxins from tarantula and scorpion venom on each of the four paddle motifs in Na(v) channels, and reveal how paddle-specific interactions can be used to reshape Na(v) channel activity. One paddle motif is unique in that it slows Voltage Sensor activation, and toxins selectively targeting this motif impede Na(v) channel inactivation. This reporter approach and the principles that emerge will be useful in developing new drugs for treating pain and Na(v) channelopathies.
Francisco Bezanilla - One of the best experts on this subject based on the ideXlab platform.
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A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor
Biophysical journal, 2016Co-Authors: Ofra Barchad-avitzur, Francisco Bezanilla, Michael F. Priest, Noa Dekel, Hanna Parnas, Yair Ben-chaimAbstract:G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are Voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a Voltage Sensor. However, the typical domain that serves as a Voltage Sensor in Voltage-gated channels is not present in GPCRs, making the search for the Voltage Sensor in the latter challenging. Here, we examine the M2R and describe a Voltage Sensor that is comprised of tyrosine residues. This Voltage Sensor is crucial for the Voltage dependence of agonist binding to the receptor. The tyrosine-based Voltage Sensor discovered here constitutes a noncanonical by which membrane proteins may sense Voltage.
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The Influence of Voltage Sensor Activity on Arclight Dynamics
Biophysical Journal, 2015Co-Authors: Jeremy S. Treger, Michael F. Priest, Francisco BezanillaAbstract:Many genetically-encoded Voltage indicators have been developed based on Voltage-sensitive phosphatases. One of the most promising of these indicators is Arclight, largely due to its substantial signal in response to Voltage changes. Although it is evident that the fluorophore is fundamentally following the Voltage Sensor movement, the fluorescence change is comprised of multiple components; how these fluorescence components reflect Voltage Sensor movement remains poorly understood. Using simultaneous electrophysiological and optical recordings of oocytes expressing wild-type and mutant Arclight constructs, we investigated the influence of altered Voltage Sensor behavior on Arclight fluorescence. We found that the latency before fluorescence change onset correlated with gating charge movement, and that the fluorescence change following prolonged depolarizations showed alterations in kinetics and thermodynamics consistent with Voltage Sensor relaxation. Other features of fluorophore movement, however, were less obviously coupled to Voltage Sensor dynamics. These results may help guide future attempts to optimize the properties of Arclight and related fluorescent proteins. Support: NIH GM030376.
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Single Molecule Fluorescence of an S4-Based Voltage Sensor
Biophysical Journal, 2014Co-Authors: Jeremy S. Treger, Michael F. Priest, Tomoya Kubota, Ludivine Frezza, Francisco BezanillaAbstract:Single-channel recordings revolutionized our understanding of Voltage-gated ion channels by allowing observation of behaviors that are obscured by large ensemble averages. Yet despite its power, single-channel recordings only allow indirect inference about the motions of the Voltage Sensor since only transitions between "open" and "closed" states of the channel can be seen; most transitions between states remain hidden. For this reason, direct measurement of the motion of a single Voltage Sensor has long been a goal to understand the details of Voltage sensing; unfortunately, at present the elementary charge transition is below experimental resolution. We report here observation of fluorescence from single Voltage Sensors conjugated to fluorescent proteins. These recordings respond to Voltage and are able to recapitulate macroscopic recordings when averaged together. The protein we used is the "ArcLight" Voltage Sensor (Jin, L. et al. Neuron, 2012.), along with derivatives thereof. This Sensor consists of the Voltage sensing domain from Ci-VSP coupled to a GFP derivative, and it shows robust changes in fluorescence in response to Voltage. Our recordings are taken from oocyte membranes using total internal reflection microscopy at a frame rate of 500 hertz and at a temperature of approximately 13 degrees Celsius. This combination of low temperature and fast acquisition allows detection of residencies of the protein at distinct fluorescence levels with stochastic movement between these levels being biased by Voltage. Presumably these distinct fluorescence levels correspond to distinct states of the Voltage Sensor. The transitions between these states can be analyzed and modeled, producing a novel picture of how the Voltage Sensor moves and how these movements are influenced by membrane potential. Support: NIH GM030376.
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The Unique Role of the Domain IV Voltage-Sensor in Fast Inactivation
Biophysical Journal, 2013Co-Authors: Deborah L. Capes, Francisco Bezanilla, Manoel Arcisio-miranda, Marcel P. Goldschen-ohm, ChandaAbstract:Unlike the potassium channel, the Voltage-Sensors of the sodium channel are homologous but not identical. Prior studies have suggested that each Voltage-Sensor may play a different role in the processes of activation and inactivation. In order to characterize the role of the Voltage-Sensors in the process of fast inactivation, we neutralized the first three charges in specific Voltage-Sensors to glutamine. We reasoned that simultaneous neutralization of critical gating charges in a Voltage-Sensor would be sufficient to functionally impede the affected Voltage-Sensor and thus allow us to determine how the properties of fast inactivation are altered by removing that particular source of Voltage-dependence. Our experiments reveal that activation of the domain (D) IV Voltage-Sensor allows fast inactivation to occur. Our results provide solid evidence for the unique importance of the DIV Voltage-Sensor in the process of fast inactivation.
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A single charged Voltage Sensor is capable of gating the Shaker K+ channel
The Journal of general physiology, 2009Co-Authors: Dominique G. Gagnon, Francisco BezanillaAbstract:We sought to determine the contribution of an individual Voltage Sensor to Shaker's function. Concatenated heterotetramers of Shaker zH4 Δ(6–46) wild type (wt) in combination with a neutral S4 segment Shaker mutant (mut) with stoichiometries 2wt/2mut and 1wt/3mut were studied and compared with the 4wt concatenated homotetramer. A single charged Voltage Sensor is sufficient to open Shaker conductance with reduced delay (
Mirela Milescu - One of the best experts on this subject based on the ideXlab platform.
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Voltage-Sensor Pharmacology of Calcium Channels
Biophysical Journal, 2016Co-Authors: Autoosa Salari, Brooklynn R. White, Timothee Pale, Vincent L. Baggett, Mirela MilescuAbstract:Voltage-gated calcium (Cav) channels contain four homologous domains, presenting the possibility of multiple toxin binding sites. Studies of related Voltage-gated potassium (Kv) channels identified the S3b-S4 structural motif, which moves at the protein-lipid interface to drive Voltage-Sensor activation, as the pharmacological target for amphipathic neurotoxins. Here we show that the homologous S3b-S4 sequences within the Cav2 and Cav3 families can be transplanted into the homotetrameric Kv channel to individually examine their contributions to Voltage Sensor activation and pharmacology. We found that the four Cav motifs display distinct toxin binding properties. We also identified the molecular surfaces involved in the toxin-channel interaction. Furthermore, as gating-modifiers of Kv channels have been shown to favor binding to the lipid bilayer prior to binding to the channel, we probed the lipid partitioning capabilities of Cav toxins, using intrinsic tryptophan fluorescence. Our results indicate a strong correlation between the strength of lipid partitioning and channel inhibition.
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Structural interactions of a Voltage Sensor toxin with lipid membranes.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Mihaela Mihailescu, Mirela Milescu, Kenton J. Swartz, Dmitriy Krepkiy, Klaus Gawrisch, Stephen H WhiteAbstract:Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including Voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a Voltage Sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to Voltage Sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the Voltage Sensor.
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Interactions between lipids and Voltage Sensor paddles detected with tarantula toxins
Nature Structural & Molecular Biology, 2009Co-Authors: Mirela Milescu, Frank Bosmans, Abdulrasheed A Alabi, Kenton J. SwartzAbstract:Increasing evidence indicates that membrane protein function can be affected by the surrounding membrane bilayer. A new study on Voltage-gated potassium channels using tarantula toxins suggests that lipid interaction with the Voltage Sensor can influence channel function. Voltage-activated ion channels open and close in response to changes in Voltage, a property that is essential for generating nerve impulses. Studies on Voltage-activated potassium (Kv) channels show that Voltage-Sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 Voltage-sensing domains and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters Voltage-Sensor activation in an S1–S4 Voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to Voltage-sensing domains and demonstrate that the pharmacological sensitivities of Voltage-activated ion channels are influenced by the surrounding lipid membrane.
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interactions between lipids and Voltage Sensor paddles detected with tarantula toxins
Nature Structural & Molecular Biology, 2009Co-Authors: Mirela Milescu, Jae Il Kim, Frank Bosmans, Abdulrasheed A Alabi, Seungkyu Lee, Kenton J. SwartzAbstract:Voltage-activated ion channels open and close in response to changes in Voltage, a property that is essential for generating nerve impulses. Studies on Voltage-activated potassium (Kv) channels show that Voltage-Sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 Voltage-sensing domains, and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters Voltage-Sensor activation in an S1–S4 Voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to Voltage-sensing domains and demonstrate that the pharmacological sensitivities of Voltage-activated ion channels are influenced by the surrounding lipid membrane.
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Voltage-Sensor activation with a tarantula toxin as cargo
Nature, 2005Co-Authors: L. Revell Phillips, Mirela Milescu, Yingying Li-smerin, Jae Il Kim, Joseph A. Mindell, Kenton J. SwartzAbstract:The opening and closing of Voltage-activated Na+, Ca2+ and K+ (Kv) channels underlies electrical and chemical signalling throughout biology, yet the structural basis of Voltage sensing is unknown. Hanatoxin is a tarantula toxin that inhibits Kv channels by binding to Voltage-Sensor paddles, crucial helix-turn-helix motifs within the Voltage-sensing domains that are composed of S3b and S4 helices. The active surface of the toxin is amphipathic, and related toxins have been shown to partition into membranes, raising the possibility that the toxin is concentrated in the membrane and interacts only weakly and transiently with the Voltage Sensors. Here we examine the kinetics and state dependence of the toxin-channel interaction and the physical location of the toxin in the membrane. We find that hanatoxin forms a strong and stable complex with the Voltage Sensors, far outlasting fluctuations of the Voltage Sensors between resting (closed) conformations at negative Voltages and activated (open) conformations at positive Voltages. Toxin affinity is reduced by Voltage-Sensor activation, explaining why the toxin stabilizes the resting conformation. We also find that when hanatoxin partitions into membranes it is localized to an interfacial region, with Trp 30 positioned about 8.5 A from the centre of the bilayer. These results demonstrate that Voltage-Sensor paddles activate with a toxin as cargo, and suggest that the paddles traverse no more than the outer half of the bilayer during activation.
Roderick Mackinnon - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidic acid modulation of Kv channel Voltage Sensor function
eLife, 2014Co-Authors: Richard K. Hite, Joel A. Butterwick, Roderick MackinnonAbstract:Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the Voltage Sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the Voltage Sensor's closed conformation. These findings support a novel mechanism of Voltage Sensor regulation by the signaling lipid phosphatidic acid.
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two separate interfaces between the Voltage Sensor and pore are required for the function of Voltage dependent k channels
PLOS Biology, 2009Co-Authors: Roderick Mackinnon, Seokyong Lee, Anirban BanerjeeAbstract:Voltage-dependent K+ (Kv) channels gate open in response to the membrane Voltage. To further our understanding of how cell membrane Voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the Voltage-Sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between Voltage Sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four Voltage Sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert Voltage-Sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the Voltage Sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the Voltage Sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate.
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Voltage Sensor Meets Lipid Membrane
Science (New York N.Y.), 2004Co-Authors: Roderick MackinnonAbstract:Several crystal structures of the bacterial Voltage-dependent Kchannel have provided intriguing insights into how Voltage-dependent ion channels work. In his Perspective, MacKinnon provides a primer on how the Voltage Sensor of Voltage-dependent Kchannels works, and updates the primer with new data provided by the latest spectroscopic study from Perozo9s group.
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Localization of the Voltage-Sensor toxin receptor on KvAP.
Biochemistry, 2004Co-Authors: Vanessa Ruta, Roderick MackinnonAbstract:A variety of venomous animals produce small protein toxins that impair the function of Voltage-dependent cation channels by affecting the motions of the Voltage-Sensor domains and altering the ener...
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a membrane access mechanism of ion channel inhibition by Voltage Sensor toxins from spider venom
Nature, 2004Co-Authors: Roderick MackinnonAbstract:Venomous animals produce small protein toxins that inhibit ion channels with high affinity. In several well-studied cases the inhibitory proteins are water-soluble and bind at a channel's aqueous-exposed extracellular surface1,2,3,4. Here we show that a Voltage-Sensor toxin (VSTX1) from the Chilean Rose Tarantula (Grammostola spatulata) reaches its target by partitioning into the lipid membrane. Lipid membrane partitioning serves two purposes: to localize the toxin in the membrane where the Voltage Sensor resides and to exploit the free energy of partitioning to achieve apparent high-affinity inhibition. VSTX1, small hydrophobic poisons and anaesthetic molecules reveal a common theme of Voltage Sensor inhibition through lipid membrane access. The apparent requirement for such access is consistent with the recent proposal that the Sensor in Voltage-dependent K+ channels is located at the membrane–protein interface5,6.
Frank Bosmans - One of the best experts on this subject based on the ideXlab platform.
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Defining the Voltage Sensor Properties and Pharmacology of Nav1.9
Biophysical Journal, 2010Co-Authors: Frank Bosmans, Michelino Puopolo, Marie-france Martin-eauclaire, Bruce P. Bean, Kenton J. SwartzAbstract:The Voltage-activated sodium channel Nav1.9 is preferentially expressed in DRG neurons where it is believed to play an important role in pain perception. However, progress in revealing the gating characteristics and pharmacological sensitivities of Nav1.9 has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here we use a protein engineering approach to study the contributions of the four Nav1.9 Voltage Sensors to channel function. We define individual S3b-S4 paddle motifs within each Voltage Sensor and show that these structural motifs sense changes in membrane Voltage and determine the kinetics of Voltage Sensor activation. Toxins from tarantula and scorpion venom interact with each of these four motifs and can be used as pharmacological tools to alter Nav1.9 currents in native DRG neurons. Our results provide answers to fundamental questions on the functional role of the four Voltage Sensors in Nav1.9 and may be useful in developing new strategies to combat pain.
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Interactions between lipids and Voltage Sensor paddles detected with tarantula toxins
Nature Structural & Molecular Biology, 2009Co-Authors: Mirela Milescu, Frank Bosmans, Abdulrasheed A Alabi, Kenton J. SwartzAbstract:Increasing evidence indicates that membrane protein function can be affected by the surrounding membrane bilayer. A new study on Voltage-gated potassium channels using tarantula toxins suggests that lipid interaction with the Voltage Sensor can influence channel function. Voltage-activated ion channels open and close in response to changes in Voltage, a property that is essential for generating nerve impulses. Studies on Voltage-activated potassium (Kv) channels show that Voltage-Sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 Voltage-sensing domains and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters Voltage-Sensor activation in an S1–S4 Voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to Voltage-sensing domains and demonstrate that the pharmacological sensitivities of Voltage-activated ion channels are influenced by the surrounding lipid membrane.
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interactions between lipids and Voltage Sensor paddles detected with tarantula toxins
Nature Structural & Molecular Biology, 2009Co-Authors: Mirela Milescu, Jae Il Kim, Frank Bosmans, Abdulrasheed A Alabi, Seungkyu Lee, Kenton J. SwartzAbstract:Voltage-activated ion channels open and close in response to changes in Voltage, a property that is essential for generating nerve impulses. Studies on Voltage-activated potassium (Kv) channels show that Voltage-Sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 Voltage-sensing domains, and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters Voltage-Sensor activation in an S1–S4 Voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to Voltage-sensing domains and demonstrate that the pharmacological sensitivities of Voltage-activated ion channels are influenced by the surrounding lipid membrane.
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Deconstructing Voltage Sensor function and pharmacology in sodium channels.
Nature, 2008Co-Authors: Frank Bosmans, Marie-france Martin-eauclaire, Kenton J. SwartzAbstract:Voltage-activated sodium (Na(v)) channels are crucial for the generation and propagation of nerve impulses, and as such are widely targeted by toxins and drugs. The four Voltage Sensors in Na(v) channels have distinct amino acid sequences, raising fundamental questions about their relative contributions to the function and pharmacology of the channel. Here we use four-fold symmetric Voltage-activated potassium (K(v)) channels as reporters to examine the contributions of individual S3b-S4 paddle motifs within Na(v) channel Voltage Sensors to the kinetics of Voltage Sensor activation and to forming toxin receptors. Our results uncover binding sites for toxins from tarantula and scorpion venom on each of the four paddle motifs in Na(v) channels, and reveal how paddle-specific interactions can be used to reshape Na(v) channel activity. One paddle motif is unique in that it slows Voltage Sensor activation, and toxins selectively targeting this motif impede Na(v) channel inactivation. This reporter approach and the principles that emerge will be useful in developing new drugs for treating pain and Na(v) channelopathies.
