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Leslie M Loew - One of the best experts on this subject based on the ideXlab platform.
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Integration of modeling with experimental and clinical findings synthesizes and refines the central role of inositol 1,4,5-trisphosphate receptor 1 in spinocerebellar ataxia
Frontiers in neuroscience, 2015Co-Authors: Sherry-ann Brown, Leslie M LoewAbstract:A suite of models was developed to study the role of inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in spinocerebellar ataxias (SCAs). Several SCAs are linked to reduced abundance of IP3R1 or to supranormal sensitivity of the receptor to activation by its ligand inositol 1,4,5-trisphosphate (IP3). Detailed multidimensional models have been created to simulate biochemical calcium signaling and Membrane Electrophysiology in cerebellar Purkinje neurons. In these models, IP3R1-mediated calcium release is allowed to interact with ion channel response on the cell Membrane. Experimental findings in mice and clinical observations in humans provide data input for the models. The SCA modeling suite helps interpret experimental results and provides suggestions to guide experiments. The models predict IP3R1 supersensitivity in SCA1 and compensatory mechanisms in SCA1, SCA2, and SCA3. Simulations explain the impact of calcium buffers. Results show that IP3R1-mediated calcium release activates voltage-gated calcium-activated potassium channels in the plasma Membrane. The SCA modeling suite unifies observations from experiments in a number of SCAs. The cadre of simulations demonstrates the central role of IP3R1.
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Monitoring Membrane Potential with Second-Harmonic Generation
Cold Spring Harbor protocols, 2014Co-Authors: Stacy A. Wilson, Aaron Lewis, Andrew C. Millard, Leslie M LoewAbstract:This protocol describes the nonlinear optical phenomenon known as second-harmonic generation (SHG) and discusses its special attributes for imaging Membrane-potential changes in single cells and multicellular preparations. Undifferentiated N1E-115 mouse neuroblastoma cells are used as a model cellular system for Membrane Electrophysiology. Styryl and naphthylstyryl dyes, also known as hemicyanines, are a class of electrochromic Membrane-staining probes that have been used to monitor Membrane potential by fluorescence; they also produce SHG images of cell Membranes with SHG intensities that are sensitive to voltage. These experiments allow for the precise characterization of the voltage sensitivity of SHG and identification of the optimal wavelength for the incident laser fundamental light. This protocol presents the steps for the culture, staining, patching, and imaging of cells. The details of the imaging system and the measurements obtained are discussed, as are the prospects of this technology for imaging Membrane potential changes in neuronal preparations.
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Computational analysis of calcium signaling and Membrane Electrophysiology in cerebellar Purkinje neurons associated with ataxia
BMC Systems Biology, 2012Co-Authors: Sherry-ann Brown, Leslie M LoewAbstract:Background Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1^delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins. Results We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and Membrane potential in Purkinje cell function. Conclusion Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1.
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Computational analysis of calcium signaling and Membrane Electrophysiology in cerebellar Purkinje neurons associated with ataxia
BMC systems biology, 2012Co-Authors: Sherry-ann Brown, Leslie M LoewAbstract:Background Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins.
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nonlinear optical measurement of Membrane potential around single molecules at selected cellular sites
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Gadi Peleg, Aaron Lewis, Michal Linial, Leslie M LoewAbstract:Membrane potential around single molecules has been measured by using the nonlinear optical phenomenon of second harmonic generation. This advance results from the interaction between a highly dipolar molecule with a selectively directed highly polarizable 1-nm gold particle. With this approach, a second harmonic signal, which is enhanced by the nanoparticle, is detected from a volume of nanometric dimensions. This present work clearly shows that functional cellular imaging around single molecules is possible by selectively directing an antibody with a 1-nm gold label to a specific Membrane protein. The results of this work open the way for three-dimensional, high resolution functional imaging of Membrane Electrophysiology in cells and cellular networks.
Nikolaos M. Tsoukias - One of the best experts on this subject based on the ideXlab platform.
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Can endothelial hemoglobin-α regulate nitric oxide vasodilatory signaling?
American journal of physiology. Heart and circulatory physiology, 2017Co-Authors: Jaimit Parikh, Adam Kapela, Nikolaos M. TsoukiasAbstract:We used mathematical modeling to investigate nitric oxide (NO)-dependent vasodilatory signaling in the arteriolar wall. Detailed continuum cellular models of calcium (Ca2+) dynamics and Membrane Electrophysiology in smooth muscle and endothelial cells (EC) were coupled with models of NO signaling and biotransport in an arteriole. We used this theoretical approach to examine the role of endothelial hemoglobin-α (Hbα) as a modulator of NO-mediated myoendothelial feedback, as previously suggested in Straub et al. (Nature 491: 473-477, 2012). The model considers enriched expression of inositol 1,4,5-triphosphate receptors (IP3Rs), endothelial nitric oxide synthase (eNOS) enzyme, Ca2+-activated potassium (KCa) channels and Hbα in myoendothelial projections (MPs) between the two cell layers. The model suggests that NO-mediated myoendothelial feedback is plausible if a significant percentage of eNOS is localized within or near the myoendothelial projection. Model results show that the ability of Hbα to regulate the myoendothelial feedback is conditional to its colocalization with eNOS near MPs at concentrations in the high nanomolar range (>0.2 μM or 24,000 molecules). Simulations also show that the effect of Hbα observed in in vitro experimental studies may overestimate its contribution in vivo, in the presence of blood perfusion. Thus, additional experimentation is required to quantify the presence and spatial distribution of Hbα in the EC, as well as to test that the strong effect of Hbα on NO signaling seen in vitro, translates also into a physiologically relevant response in vivo.NEW & NOTEWORTHY Mathematical modeling shows that although regulation of nitric oxide signaling by hemoglobin-α (Hbα) is plausible, it is conditional to its presence in significant concentrations colocalized with endothelial nitric oxide synthase in myoendothelial projections. Additional experimentation is required to test that the strong effect of Hbα seen in vitro translates into a physiologically relevant response in vivo.
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A mathematical model of vasoreactivity in rat mesenteric arterioles: I. Myoendothelial communication.
Microcirculation (New York N.Y. : 1994), 2009Co-Authors: Adam Kapela, Anastasios Bezerianos, Nikolaos M. TsoukiasAbstract:ABSTRACTTo study the effect of myoendothelial communication on vascular reactivity, we integrated detailed mathematical models of Ca2+ dynamics and Membrane Electrophysiology in arteriolar smooth muscle (SMC) and endothelial (EC) cells. Cells are coupled through the exchange of Ca2+, Cl−, K+, and Na+ ions, inositol 1,4,5-triphosphate (IP3), and the paracrine diffusion of nitric oxide (NO). EC stimulation reduces intracellular Ca2+ ([Ca2+]i) in the SMC by transmitting a hyperpolarizing current carried primarily by K+. The NO-independent endothelium-derived hyperpolarization was abolished in a synergistic-like manner by inhibition of EC SKCa and IKCa channels. During NE stimulation, IP3 diffusing from the SMC induces EC Ca2+ release, which, in turn, moderates SMC depolarization and [Ca2+]i elevation. On the contrary, SMC [Ca2+]i was not affected by EC-derived IP3. Myoendothelial Ca2+ fluxes had no effect in either cell. The EC exerts a stabilizing effect on calcium-induced calcium release–dependent SMC Ca2+...
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Multiscale Mathematical Modeling of Microvascular Tone Regulation
IFMBE Proceedings, 2009Co-Authors: Adam Kapela, Tushar Gadkari, Sridevi Nagaraja, Nikolaos M. TsoukiasAbstract:Microvascular tone and blood flow are determined by passive biomechanical properties of the vessel wall and by active constrictions of the vascular smooth muscle cells (SMC). The intracellular calcium concentration in SMC is the major determinant of the active tone and is controlled by a complex system of intracellular and intercellular signaling pathways. We present here a methodology for integrating mathematical descriptions at the cellular level into computational multicellular/whole-vessel models. Detailed descriptions of calcium dynamics and Membrane Electrophysiology in isolated endothelial and smooth muscle cells are formulated and validated based on subcellular- and cellular-level data. An endothelial/smooth muscle model and multicellular vessel model are formulated by integrating individual cells and accounting for intercellular communication. The biomechanical properties of the vessel wall are integrated into the model by the calcium-dependent active force development and stretch-sensitive Membrane channels. The model can simulate agonist-induced vasorelaxation and vasoconstriction, conducted vasoreactivity, myogenic tone and pressure-induced diameter changes. The model can also be used to test hypotheses about tone regulation, such as the role of myoendothelial projections and extracellular potassium in endothelium derived hyperpolarizing factor. Disease-specific changes at the subcellular level can be incorporated into the model to predict their effect on responses at the vessel level. Thus, the integration of subcellular data into a computational model can allow the analysis of complex microcirculatory function in health and disease, hypothesis testing, and guide new experimentation.
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A mathematical model of Ca2+ dynamics in rat mesenteric smooth muscle cell: Agonist and NO stimulation
Journal of theoretical biology, 2008Co-Authors: Adam Kapela, Anastasios Bezerianos, Nikolaos M. TsoukiasAbstract:A mathematical model of calcium dynamics in vascular smooth muscle cell (SMC) was developed based on data mostly from rat mesenteric arterioles. The model focuses on (a) the plasma Membrane Electrophysiology; (b) Ca2+ uptake and release from the sarcoplasmic reticulum (SR); (c) cytosolic balance of Ca2+, Na+, K+, and Cl ions; and (d) IP3 and cGMP formation in response to norepinephrine(NE) and nitric oxide (NO) stimulation. Stimulation with NE induced Membrane depolarization and an intracellular Ca2+ ([Ca2+]i) transient followed by a plateau. The plateau concentrations were mostly determined by the activation of voltage-operated Ca2+ channels. NE causes a greater increase in [Ca2+]i than stimulation with KCl to equivalent depolarization. Model simulations suggest that the effect of[Na+]i accumulation on the Na+/Ca2+ exchanger (NCX) can potentially account for this difference.Elevation of [Ca2+]i within a concentration window (150-300 nM) by NE or KCl initiated [Ca2+]i oscillations with a concentration-dependent period. The oscillations were generated by the nonlinear dynamics of Ca2+ release and refilling in the SR. NO repolarized the NE-stimulated SMC and restored low [Ca2+]i mainly through its effect on Ca2+-activated K+ channels. Under certain conditions, Na+-K+-ATPase inhibition can result in the elevation of [Na+]i and the reversal of NCX, increasing resting cytosolic and SR Ca2+ content, as well as reactivity to NE. Blockade of the NCX's reverse mode could eliminate these effects. We conclude that the integration of the selected cellular components yields a mathematical model that reproduces, satisfactorily, some of the established features of SMC physiology. Simulations suggest a potential role of intracellular Na+ in modulating Ca2+ dynamics and provide insights into the mechanisms of SMC constriction, relaxation, and the phenomenon of vasomotion. The model will provide the basis for the development of multi-cellular mathematical models that will investigate microcirculatory function in health and disease.
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A mathematical model of plasma Membrane Electrophysiology and calcium dynamics in vascular endothelial cells
American journal of physiology. Cell physiology, 2007Co-Authors: Haroldo S. Silva, Adam Kapela, Nikolaos M. TsoukiasAbstract:Vascular endothelial cells (ECs) modulate smooth muscle cell (SMC) contractility, assisting in vascular tone regulation. Cytosolic Ca2+ concentration ([Ca2+]i) and Membrane potential (Vm) play impo...
Raimund Dutzler - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of a ClC transporter by solid-supported Membrane Electrophysiology
The Journal of general physiology, 2013Co-Authors: Juan J. Garcia-celma, Adrian Szydelko, Raimund DutzlerAbstract:EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported Membrane Electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.
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Transport Properties of a Prokaryotic CLC Transporter Assayed by Solid-Supported Membrane Electrophysiology
Biophysical Journal, 2012Co-Authors: Juan J. Garcia-celma, Adrian Szydelko, Raimund DutzlerAbstract:EcClC (or ClC-Ec1), a prokaryotic member of the ClC family of channels and transporters of known structure, acts as a coupled chloride/proton antiporter. To investigate its electrogenic activity, proteoliposomes containing reconstituted EcClC were adsorbed on a solid-supported Membrane (SSM) electrode. The application of concentration jumps of different anions results in robust transient currents with a selectivity sequence that agrees with previously reported measurements on planar lipid bilayers. The transient currents increase at acidic pH with an apparent pKa of 4.6.The pH dependences of three mutations that are known to impair proton translocation while preserving chloride transport (mutations E148A, Y445A, and E203Q) have also been investigated. In agreement with previous results, E148A shows weak pH dependence. The transport impaired Y445A and the uncoupled E203Q present a shift in the apparent pKa towards less acidic values. Mutation E203Q, in addition, presents residual electrogenic activity at neutral pH values. Taken together, our results support the idea that chloride transport in the wild-type varies with pH mainly in response to titration of the external glutamate (E148).
Randy B. Stockbridge - One of the best experts on this subject based on the ideXlab platform.
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The structural basis of promiscuity in small multidrug resistance transporters
Nature Communications, 2020Co-Authors: Ali A. Kermani, Christian B. Macdonald, Olive E. Burata, B. Koff, Akiko Koide, Eric Denbaum, Shohei Koide, Randy B. StockbridgeAbstract:By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported Membrane Electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the Membrane for the hydrophobic substituents of transported drug-like cations. Gdx-Clo is a bacterial transporter from the small multidrug resistance (SMR) family. Here, the authors use solid supported Membrane Electrophysiology to characterize Gdx-Clo functionally and report crystal structures of Gdx-Clo which confirm the dual topology architecture and offer insight into substrate binding and transport mechanism.
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The structural basis of promiscuity in small multidrug resistance transporters.
Nature communications, 2020Co-Authors: Ali A. Kermani, Christian B. Macdonald, Olive E. Burata, Akiko Koide, Eric Denbaum, Shohei Koide, B Ben Koff, Randy B. StockbridgeAbstract:By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported Membrane Electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family's extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the Membrane for the hydrophobic substituents of transported drug-like cations.
Juan J. Garcia-celma - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of a ClC transporter by solid-supported Membrane Electrophysiology
The Journal of general physiology, 2013Co-Authors: Juan J. Garcia-celma, Adrian Szydelko, Raimund DutzlerAbstract:EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported Membrane Electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.
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Transport Properties of a Prokaryotic CLC Transporter Assayed by Solid-Supported Membrane Electrophysiology
Biophysical Journal, 2012Co-Authors: Juan J. Garcia-celma, Adrian Szydelko, Raimund DutzlerAbstract:EcClC (or ClC-Ec1), a prokaryotic member of the ClC family of channels and transporters of known structure, acts as a coupled chloride/proton antiporter. To investigate its electrogenic activity, proteoliposomes containing reconstituted EcClC were adsorbed on a solid-supported Membrane (SSM) electrode. The application of concentration jumps of different anions results in robust transient currents with a selectivity sequence that agrees with previously reported measurements on planar lipid bilayers. The transient currents increase at acidic pH with an apparent pKa of 4.6.The pH dependences of three mutations that are known to impair proton translocation while preserving chloride transport (mutations E148A, Y445A, and E203Q) have also been investigated. In agreement with previous results, E148A shows weak pH dependence. The transport impaired Y445A and the uncoupled E203Q present a shift in the apparent pKa towards less acidic values. Mutation E203Q, in addition, presents residual electrogenic activity at neutral pH values. Taken together, our results support the idea that chloride transport in the wild-type varies with pH mainly in response to titration of the external glutamate (E148).