The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Michael E. Green - One of the best experts on this subject based on the ideXlab platform.
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The Role of Proton Transport in Gating Current in a Voltage Gated Ion Channel, as Shown by Quantum Calculations
MDPI AG, 2018Co-Authors: Alisher M. Kariev, Michael E. GreenAbstract:Over two-thirds of a century ago, Hodgkin and Huxley proposed the existence of voltage gated ion channels (VGICs) to carry Na+ and K+ ions across the cell membrane to create the nerve impulse, in response to depolarization of the membrane. The channels have multiple physiological roles, and play a central role in a wide variety of diseases when they malfunction. The first channel structure was found by MacKinnon and coworkers in 1998. Subsequently, the structure of a number of VGICs was determined in the open (ion conducting) state. This type of channel consists of four voltage sensing domains (VSDs), each formed from four transmembrane (TM) segments, plus a pore domain through which ions move. Understanding the gating mechanism (how the channel opens and closes) requires structures. One TM segment (S4) has an Arginine in every third position, with one such segment per domain. It is usually assumed that these Arginines are all ionized, and in the resting state are held toward the intracellular side of the membrane by voltage across the membrane. They are assumed to move outward (extracellular direction) when released by depolarization of this voltage, producing a capacitive gating current and opening the channel. We suggest alternate interpretations of the evidence that led to these models. Measured gating current is the total charge displacement of all atoms in the VSD; we propose that the prime, but not sole, contributor is proton motion, not displacement of the charges on the Arginines of S4. It is known that the VSD can conduct protons. Quantum calculations on the Kv1.2 potassium channel VSD show how; the key is the amphoteric nature of the Arginine side chain, which allows it to transfer a proton. This appears to be the first time the Arginine side chain has had its amphoteric character considered. We have calculated one such proton transfer in detail: this proton starts from a tyrosine that can ionize, transferring to the NE of the third Arginine on S4; that Arginine’s NH then transfers a proton to a glutamate. The backbone remains static. A mutation predicted to affect the proton transfer has been qualitatively confirmed experimentally, from the change in the gating current-voltage curve. The total charge displacement in going from a normal closed potential of −70 mV across the membrane to 0 mV (open), is calculated to be approximately consistent with measured values, although the error limits on the calculation require caution in interpretation
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Cys Mutation + MTS Caution is Needed in Interpretation of Arg Reaction in the S4 Transmembrane Segment of a Voltage Sensing Domain (VSD) of a Voltage Gated Channel: Results of Quantum Calculations
Biophysical Journal, 2014Co-Authors: Alisher M. Kariev, Michael E. GreenAbstract:In all previous work on voltage gated channels, mutation of the Arginines on the S4 segment of the VSD, then reaction with an MTS reagent, followed by channel shutdown, has been taken to mean that the Arginine was exposed on the surface from which the reagent was applied. This may require more care in interpretation. Cysteine is smaller than the Arginine by about the size of the reactive sulfonate on the MTS; The mutation leaves a large cavity where the Arginine side chain had been, so the MTS can reach the cysteine, possibly via the omega pore. The backbone atoms need not move. The distance between S4 and S2 or S3 remains largely unchanged. Salt bridges, (e.g., R297-E183) however, are disturbed; when cysteine is in the reactive (negative) form, it constitutes a charge reversal mutation, as the Arginine was (presumably) positive. Quantum calculations on configurations of this region for R300C of the VSD of Kv1.2 show that the cys anion can fold away from the cavity where it could react, in a manner dependent on the water and protons present. See the preprint posted at http://arxiv.org/abs/1309.1373. Optimizations have been done at BLYP/6-31G∗∗ level. Acknowledgement: Computations were done at the Brookhaven National Laboratory CFN cluster, and the CUNY hpc facility.
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A possible role for phosphate in complexing the Arginines of S4 in voltage gated channels.
Journal of Theoretical Biology, 2004Co-Authors: Michael E. GreenAbstract:Abstract Phosphate ions are known to complex guanidinium groups, which are the side chains of Arginine. Voltage gated channels that allow passage of ions through cell membranes, producing, for example the nerve impulse, are in many cases composed of four domains, each with six transmembrane segments. The S4 transmembrane segments of these channels have Arginines placed in such a way that they would be expected to complex phosphate. Known phosphate–Arginine complexes are reasonably strong. Here, an ab initio calculation reinforces the expectation that a strong complex could form. As a consequence, if the S4 moved, it would carry either no charge, or at most half of what is expected from fully charged Arginines. This suggests that it may be necessary to rethink voltage gating models in which the gating current is produced by physical motion of the S4 transmembrane segments.
Ehud Y Isacoff - One of the best experts on this subject based on the ideXlab platform.
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voltage sensing Arginines in a potassium channel permeate and occlude cation selective pores
Neuron, 2005Co-Authors: Francesco Tombola, Medha M Pathak, Ehud Y IsacoffAbstract:Summary Voltage-gated ion channels sense voltage by shuttling Arginine residues located in the S4 segment across the membrane electric field. The molecular pathway for this Arginine permeation is not understood, nor is the filtering mechanism that permits passage of charged Arginines but excludes solution ions. We find that substituting the first S4 Arginine with smaller amino acids opens a high-conductance pathway for solution cations in the Shaker K + channel at rest. The cationic current does not flow through the central K + pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain. The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane Arginine permeation. We propose that when S4 moves it ratchets between conformations in which one Arginine after another occupies and occludes to ions the narrowest part of this pathway.
Alisher M. Kariev - One of the best experts on this subject based on the ideXlab platform.
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The Role of Proton Transport in Gating Current in a Voltage Gated Ion Channel, as Shown by Quantum Calculations
MDPI AG, 2018Co-Authors: Alisher M. Kariev, Michael E. GreenAbstract:Over two-thirds of a century ago, Hodgkin and Huxley proposed the existence of voltage gated ion channels (VGICs) to carry Na+ and K+ ions across the cell membrane to create the nerve impulse, in response to depolarization of the membrane. The channels have multiple physiological roles, and play a central role in a wide variety of diseases when they malfunction. The first channel structure was found by MacKinnon and coworkers in 1998. Subsequently, the structure of a number of VGICs was determined in the open (ion conducting) state. This type of channel consists of four voltage sensing domains (VSDs), each formed from four transmembrane (TM) segments, plus a pore domain through which ions move. Understanding the gating mechanism (how the channel opens and closes) requires structures. One TM segment (S4) has an Arginine in every third position, with one such segment per domain. It is usually assumed that these Arginines are all ionized, and in the resting state are held toward the intracellular side of the membrane by voltage across the membrane. They are assumed to move outward (extracellular direction) when released by depolarization of this voltage, producing a capacitive gating current and opening the channel. We suggest alternate interpretations of the evidence that led to these models. Measured gating current is the total charge displacement of all atoms in the VSD; we propose that the prime, but not sole, contributor is proton motion, not displacement of the charges on the Arginines of S4. It is known that the VSD can conduct protons. Quantum calculations on the Kv1.2 potassium channel VSD show how; the key is the amphoteric nature of the Arginine side chain, which allows it to transfer a proton. This appears to be the first time the Arginine side chain has had its amphoteric character considered. We have calculated one such proton transfer in detail: this proton starts from a tyrosine that can ionize, transferring to the NE of the third Arginine on S4; that Arginine’s NH then transfers a proton to a glutamate. The backbone remains static. A mutation predicted to affect the proton transfer has been qualitatively confirmed experimentally, from the change in the gating current-voltage curve. The total charge displacement in going from a normal closed potential of −70 mV across the membrane to 0 mV (open), is calculated to be approximately consistent with measured values, although the error limits on the calculation require caution in interpretation
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Cys Mutation + MTS Caution is Needed in Interpretation of Arg Reaction in the S4 Transmembrane Segment of a Voltage Sensing Domain (VSD) of a Voltage Gated Channel: Results of Quantum Calculations
Biophysical Journal, 2014Co-Authors: Alisher M. Kariev, Michael E. GreenAbstract:In all previous work on voltage gated channels, mutation of the Arginines on the S4 segment of the VSD, then reaction with an MTS reagent, followed by channel shutdown, has been taken to mean that the Arginine was exposed on the surface from which the reagent was applied. This may require more care in interpretation. Cysteine is smaller than the Arginine by about the size of the reactive sulfonate on the MTS; The mutation leaves a large cavity where the Arginine side chain had been, so the MTS can reach the cysteine, possibly via the omega pore. The backbone atoms need not move. The distance between S4 and S2 or S3 remains largely unchanged. Salt bridges, (e.g., R297-E183) however, are disturbed; when cysteine is in the reactive (negative) form, it constitutes a charge reversal mutation, as the Arginine was (presumably) positive. Quantum calculations on configurations of this region for R300C of the VSD of Kv1.2 show that the cys anion can fold away from the cavity where it could react, in a manner dependent on the water and protons present. See the preprint posted at http://arxiv.org/abs/1309.1373. Optimizations have been done at BLYP/6-31G∗∗ level. Acknowledgement: Computations were done at the Brookhaven National Laboratory CFN cluster, and the CUNY hpc facility.
Francesco Tombola - One of the best experts on this subject based on the ideXlab platform.
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voltage sensing Arginines in a potassium channel permeate and occlude cation selective pores
Neuron, 2005Co-Authors: Francesco Tombola, Medha M Pathak, Ehud Y IsacoffAbstract:Summary Voltage-gated ion channels sense voltage by shuttling Arginine residues located in the S4 segment across the membrane electric field. The molecular pathway for this Arginine permeation is not understood, nor is the filtering mechanism that permits passage of charged Arginines but excludes solution ions. We find that substituting the first S4 Arginine with smaller amino acids opens a high-conductance pathway for solution cations in the Shaker K + channel at rest. The cationic current does not flow through the central K + pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain. The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane Arginine permeation. We propose that when S4 moves it ratchets between conformations in which one Arginine after another occupies and occludes to ions the narrowest part of this pathway.
Patrizia Alberti - One of the best experts on this subject based on the ideXlab platform.
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delivery of antisense peptide nucleic acids to cells by conjugation with small Arginine rich cell penetrating peptide r w 9
PLOS ONE, 2014Co-Authors: Celine Cordier, Fatima Boutimah, Mathilde Bourdeloux, Florian Dupuy, Patrizia AlbertiAbstract:Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the Arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of Arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the Arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of Arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of Arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that Arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.