ELECTROPHYSIOLOGICAL STUDIES OF VOLTAGE GATED CHANNELS

Summary

Principal Investigator: FRANCISCO J BEZANILLA
Abstract: The long term objective of this project is the understanding of voltage-dependent gating of ion channels at the molecular level. In this proposal experiments are designed to describe structural aspects of Shaker and squid potassium channelsand human skeletal muscle sodium channels. Cloned, engineered channels will be expressed in Xenopus oocytes and the function will be assessed with electrophysiological techniques while the structure will be probed with optical and chemical modification techniques. There are four specific aims. 1) Correlation of structural changes with the function of the voltage sensor. This will be approached using the technique of histidine scanning mutagenesis on the charges of the S4 and S2 segments. This technique utilizes protons to probe the accessibility of engineered histidine residues, usually replacing basic residues of the protein. In addition, fluorescent probes attached to specific sites of the channels (mutated to cysteine) will be used to assess changes in environment and correlate them with gating currents. 2) Measurements of distances in the channel molecule. This aim will use the fluorescence resonance energy transfer and its variant, lanthanide-based resonance energy transfer, to measure distances between specific sites across subunits or between an specific site in the channel and an specific toxin sitting on the pore of the channel. The sites of attachment of the fluorophores and lanthanides are engineered cysteines in the channel molecule and Agitoxin II. Distance measurements will be done on sites in the S2, S3 and S4 segments using a newly developed optical setup that allows simultaneous voltage clamp and accurate measurements of gating currents. Distance measurements will be performed at different membrane potentials to assess possible distance during activation of the conductance. 3) Study of the activation and inactivation pathways. In this aim a study of the initial fast event of gating and a detailed characterization of the events leading to channel opening and slow inactivation will be studied with noise analysis of gating currents in the Shaker K. channel and with gating currents in the Sodium channel to correlate them with the structural information obtained in aims 1 and 2. 4) Modeling. Kinetic modeling will be done to account for the results in electrophysiologyical and optical experiments. Simulations of fluctuations produced by voltage ramps will be compared to the noise analysisexperiments of aim 3 to test models of activation and inactivation. In addition, molecular modeling will be done based on the results of distance measurements, including possible distance changes occurring during activation. These experiments are expected to give us insight on the molecular rearrangements concomitant with voltage-dependent gating, which is a basic property of many membranechannelsand it has critical importance in excitability and cell homeostasis.
Funding Period: 1981-08-01 - 2009-07-31
more information: NIH RePORT

Top Publications

  1. pmc Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction
    Rikard Blunck
    Département de Physique et Groupe d Etude des Protéines Membranaires GEPROM, Universite de Montreal, Montreal, Quebec, H3C 3J7, Canada
    J Gen Physiol 128:569-81. 2006
  2. pmc Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential
    Jérôme Lacroix
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA
    J Biol Chem 286:17945-53. 2011
  3. pmc Properties of deactivation gating currents in Shaker channels
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA
    Biophys J 100:L28-30. 2011
  4. pmc Depolarization induces a conformational change in the binding site region of the M2 muscarinic receptor
    Noa Dekel
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    Proc Natl Acad Sci U S A 109:285-90. 2012
  5. pmc Intermediate state trapping of a voltage sensor
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 140:635-52. 2012
  6. pmc Symmetry-constrained analysis of pulsed double electron-electron resonance (DEER) spectroscopy reveals the dynamic nature of the KcsA activation gate
    Olivier Dalmas
    Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA
    J Am Chem Soc 134:16360-9. 2012
  7. pmc Molecular mechanism for depolarization-induced modulation of Kv channel closure
    Alain J Labro
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 140:481-93. 2012
  8. pmc An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations
    Ernesto Vargas
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 140:587-94. 2012
  9. pmc Tuning the voltage-sensor motion with a single residue
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois
    Biophys J 103:L23-5. 2012
  10. pmc Thermal mechanisms of millimeter wave stimulation of excitable cells
    Mikhail G Shapiro
    Miller Research Institute, University of California, Berkeley, CA, USA
    Biophys J 104:2622-8. 2013

Detail Information

Publications31

  1. pmc Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction
    Rikard Blunck
    Département de Physique et Groupe d Etude des Protéines Membranaires GEPROM, Universite de Montreal, Montreal, Quebec, H3C 3J7, Canada
    J Gen Physiol 128:569-81. 2006
    ..The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning...
  2. pmc Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential
    Jérôme Lacroix
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA
    J Biol Chem 286:17945-53. 2011
    ....
  3. pmc Properties of deactivation gating currents in Shaker channels
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA
    Biophys J 100:L28-30. 2011
    ..We propose a new (to our knowledge) kinetic model that accounts for these observations...
  4. pmc Depolarization induces a conformational change in the binding site region of the M2 muscarinic receptor
    Noa Dekel
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    Proc Natl Acad Sci U S A 109:285-90. 2012
    ..Our results are also unique in suggesting that the allosteric site is also involved in controlling the voltage-dependent agonist binding...
  5. pmc Intermediate state trapping of a voltage sensor
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 140:635-52. 2012
    ..This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation...
  6. pmc Symmetry-constrained analysis of pulsed double electron-electron resonance (DEER) spectroscopy reveals the dynamic nature of the KcsA activation gate
    Olivier Dalmas
    Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA
    J Am Chem Soc 134:16360-9. 2012
    ..Our analysis reveals a significant increase in the dynamics of the inner bundle gate upon opening. Also, it explicitly demonstrates the degree to which the CTD restricts the motion of the lower gate at rest and during activation gating...
  7. pmc Molecular mechanism for depolarization-induced modulation of Kv channel closure
    Alain J Labro
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 140:481-93. 2012
    ..We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history...
  8. pmc An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations
    Ernesto Vargas
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 140:587-94. 2012
    ..This progress is highlighted in the broad context of preexisting work about voltage-gated channels...
  9. pmc Tuning the voltage-sensor motion with a single residue
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois
    Biophys J 103:L23-5. 2012
    ..Mutations of the conserved Phe in the S2 segment (F161) do not significantly perturb the voltage dependence of the VSD movement, suggesting a unique voltage sensing mechanism in Ci-VSP...
  10. pmc Thermal mechanisms of millimeter wave stimulation of excitable cells
    Mikhail G Shapiro
    Miller Research Institute, University of California, Berkeley, CA, USA
    Biophys J 104:2622-8. 2013
    ..Our results suggest that MMW stimulation produces significant thermally mediated effects on excitable cells via basic thermodynamic mechanisms that must be taken into account in the study and use of MMW radiation in biological systems. ..
  11. pmc The gating charge should not be estimated by fitting a two-state model to a Q-V curve
    Francisco Bezanilla
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637
    J Gen Physiol 142:575-8. 2013
    ..We demonstrate that the commonly used method to estimate the charge per molecule using a simple Boltzmann fit is not only inadequate, but in most cases, it underestimates the moving charge times the fraction of the field...
  12. pmc Sensing charges of the Ciona intestinalis voltage-sensing phosphatase
    Carlos A Villalba-Galea
    Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA 23298
    J Gen Physiol 142:543-55. 2013
    ..Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing. ..
  13. pmc Molecular bases for the asynchronous activation of sodium and potassium channels required for nerve impulse generation
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57 th Chicago, IL 60637, USA
    Neuron 79:651-7. 2013
    ..This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials. ..
  14. pmc Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels
    Deborah L Capes
    Department of Neuroscience, University of Wisconsin, Madison, Madison, WI 53706, USA
    J Gen Physiol 142:101-12. 2013
    ..We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation. ..
  15. pmc Control of a final gating charge transition by a hydrophobic residue in the S2 segment of a K+ channel voltage sensor
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, Gordon Center for Integrative Sciences W244, 929 East 57th Street, Chicago, IL 60637, USA
    Proc Natl Acad Sci U S A 108:6444-9. 2011
    ..Our results suggest that F(290) controls the transfer of R(371), the fourth gating charge, during gating while not affecting the movement of the other three gating arginines...
  16. pmc In search of a consensus model of the resting state of a voltage-sensing domain
    Ernesto Vargas
    Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA
    Neuron 72:713-20. 2011
    ..The resulting structural model is in broad agreement with results that originate from various labs using different techniques, indicating the emergence of a consensus for the structural basis of voltage sensing...
  17. pmc The contribution of individual subunits to the coupling of the voltage sensor to pore opening in Shaker K channels: effect of ILT mutations in heterotetramers
    Dominique G Gagnon
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 136:555-68. 2010
    ....
  18. pmc Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer
    Jessica Richardson
    Departments of Anesthesiology and Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
    Proc Natl Acad Sci U S A 103:15865-70. 2006
    ..Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements...
  19. pmc In vivo measurement of intramolecular distances using genetically encoded reporters
    Walter Sandtner
    Department of Pediatrics, Institute for Molecular Pediatric Sciences, Gordon Center for Integrative Science, University of Chicago, Chicago, Illinois 60637, USA
    Biophys J 93:L45-7. 2007
    ..The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols...
  20. pmc Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel
    Fabiana V Campos
    Institute for Molecular Pediatric Sciences, Department of Pediatrics, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA
    Proc Natl Acad Sci U S A 104:7904-9. 2007
    ..5 A at the level of R362 in going from the open to the closed state of the channel, moving the gating charge across a focused electric field...
  21. pmc beta-Scorpion toxin modifies gating transitions in all four voltage sensors of the sodium channel
    Fabiana V Campos
    Institute of Molecular Pediatric Sciences and Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 130:257-68. 2007
    ..These findings reveal how the binding of beta-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels...
  22. ncbi A common pathway for charge transport through voltage-sensing domains
    Baron Chanda
    Department of Physiology, University of Wisconsin, Madison, WI 53706, USA
    Neuron 57:345-51. 2008
    ..Here we will discuss implications of these findings on the mechanisms underlying charge (and ion) transport by voltage-sensing domains...
  23. pmc Fluorescence detection of the movement of single KcsA subunits reveals cooperativity
    Rikard Blunck
    Département de Physique and GEPROM, Universite de Montreal, Montreal, QC, Canada H3C 3J7
    Proc Natl Acad Sci U S A 105:20263-8. 2008
    ..The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step...
  24. pmc S4-based voltage sensors have three major conformations
    Carlos A Villalba-Galea
    Department of Biochemistry and Molecular Biology, University of Chicago, Gordon Center for Integrative Sciences Room W244, 929 East 57th Street, Chicago, IL 60637, USA
    Proc Natl Acad Sci U S A 105:17600-7. 2008
    ....
  25. ncbi Ion channels: from conductance to structure
    Francisco Bezanilla
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
    Neuron 60:456-68. 2008
    ..These leaps have normally been associated with the introduction of a new technical advance or the development of a new biological preparation; therefore, it is quite certain that we have not seen them all...
  26. pmc Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels
    Fabiana V Campos
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 132:251-63. 2008
    ..These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates...
  27. pmc A single charged voltage sensor is capable of gating the Shaker K+ channel
    Dominique G Gagnon
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 133:467-83. 2009
    ..e., ready for pore opening), and provide experimental support to the proposed model of independent voltage sensors with a final, almost voltage-independent concerted step...
  28. pmc Coupling between the voltage-sensing and phosphatase domains of Ci-VSP
    Carlos A Villalba-Galea
    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA
    J Gen Physiol 134:5-14. 2009
    ....
  29. ncbi Gating currents from neuronal K(V)7.4 channels: general features and correlation with the ionic conductance
    Francesco Miceli
    Section of Pharmacology, Department of Neuroscience, University of Naples Federico II, Naples, Italy
    Channels (Austin) 3:274-83. 2009
    ..4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating...
  30. pmc Moving gating charges through the gating pore in a Kv channel voltage sensor
    Jérôme J Lacroix
    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637
    Proc Natl Acad Sci U S A 111:E1950-9. 2014
    ..This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors. ..