A genetically targetable fluorescent probe of channel gating with rapid kinetics.

We have developed a genetically targetable, optical channel-gating reporter that converts rapid membrane potential changes into changes in fluorescence intensity. We have named this construct SPARC (sodium channel protein-based activity reporting construct). Green fluorescent protein was inserted into an intracellular loop of a reversibly nonconducting form of the rat mu I skeletal muscle voltage-gated sodium channel. Rapid changes of the membrane potential modulate the fluorescence of the inserted green fluorescent protein. This change in fluorescence can faithfully report depolarizing pulses as short as 2 ms. The fluorescence signal does not inactivate during extended depolarizations. Several features of the probe's response properties indicate that it likely reports gating charge movement of a single domain of rat mu I skeletal muscle. This probe provides a new approach for studying rapid channel movements and may possibly act as a fluorescent activity reporter in excitable cells.

[1]  F Bezanilla,et al.  Gating currents. , 1998, Methods in enzymology.

[2]  W. Stühmer,et al.  Electrophysiological recording from Xenopus oocytes. , 1992, Methods in enzymology.

[3]  R. Mitra,et al.  Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. , 1996, Gene.

[4]  Yuh Nung Jan,et al.  Evidence for the formation of heteromultimeric potassium channels in Xenopus oocytes , 1990, Nature.

[5]  B. Hille.  Ionic channels of excitable membranes , 2001 .

[6]  A. L. Goldin,et al.  Sodium Channel Activation Gating Is Affected by Substitutions of Voltage Sensor Positive Charges in All Four Domains , 1997, The Journal of general physiology.

[7]  G. Tomaselli,et al.  Molecular motions within the pore of voltage-dependent sodium channels. , 1997, Biophysical journal.

[8]  S J Remington,et al.  Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Hughes,et al.  The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function , 1995, Neuron.

[10]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[11]  C. Johnson,et al.  A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[13]  L B Cohen,et al.  Optical measurement of membrane potential. , 1978, Reviews of physiology, biochemistry and pharmacology.

[14]  F Conti,et al.  Recording of gating currents from Xenopus oocytes and gating noise analysis. , 1992, Methods in enzymology.

[15]  A. Grinvald,et al.  Optical methods for monitoring neuron activity. , 1978, Annual review of neuroscience.

[16]  F. Bezanilla,et al.  Sodium channel activation in the squid giant axon. Steady state properties , 1985, The Journal of general physiology.

[17]  Ehud Y Isacoff,et al.  A Genetically Encoded Optical Probe of Membrane Voltage , 1997, Neuron.

[18]  Bertil Hille,et al.  Voltage-Gated Ion Channels and Electrical Excitability , 1998, Neuron.

[19]  W. Catterall,et al.  From Ionic Currents to Molecular Mechanisms The Structure and Function of Voltage-Gated Sodium Channels , 2000, Neuron.

[20]  Weijing Sun,et al.  Probing Sodium Channel Cytoplasmic Domain Structure , 1995, The Journal of Biological Chemistry.

[21]  Christopher Miller,et al.  Ionic channels of excitable membranes. Second edition By Bertil Hille. Sunderland, Massachusetts: Sinauer. (1991). 607 pp. $46.95 , 1992, Cell.

[22]  F Bezanilla,et al.  Inactivation of the sodium channel. II. Gating current experiments , 1977, The Journal of general physiology.

[23]  D. Apps,et al.  Ionic channels of excitable membranes (second edition) : By Bertil Hille; Sinauer Associates (distributed by W.H. Freeman); Sunderland, MA, 1992; xiv + 607 pages. £37.95. ISBN 0878933239 , 1992 .

[24]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.

[25]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[26]  Francisco Bezanilla,et al.  Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy , 1999, Nature.

[27]  E. Isacoff,et al.  Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel , 1999, Nature.

[28]  F. Conti,et al.  Quantal charge redistributions accompanying the structural transitions of sodium channels , 1989, European Biophysics Journal.

[29]  R. Tsien,et al.  Circular permutation and receptor insertion within green fluorescent proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  W. Webb,et al.  Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  F J Sigworth,et al.  Voltage gating of ion channels , 1994, Quarterly Reviews of Biophysics.

[32]  Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum. , 1998, Nucleic acids research.

[33]  Francisco Bezanilla,et al.  Voltage Sensors in Domains III and IV, but Not I and II, Are Immobilized by Na+ Channel Fast Inactivation , 1999, Neuron.

[34]  A. Hodgkin,et al.  A quantitative description of membrane current and its application to conduction and excitation in nerve , 1952, The Journal of physiology.

[35]  G. Tomaselli,et al.  Structure and function of voltage‐gated sodium channels , 1998, The Journal of physiology.

[36]  W. Almers,et al.  Gating currents and charge movements in excitable membranes. , 1978, Reviews of physiology, biochemistry and pharmacology.

[37]  R. Horn,et al.  Molecular Basis of Charge Movement in Voltage-Gated Sodium Channels , 1996, Neuron.

[38]  C Kaether,et al.  Green fluorescent protein: applications in cell biology , 1996, FEBS letters.

[39]  R. Horn,et al.  Evidence for voltage-dependent S4 movement in sodium channels , 1995, Neuron.

[40]  F Bezanilla,et al.  The voltage sensor in voltage-dependent ion channels. , 2000, Physiological reviews.

[41]  F Bezanilla,et al.  Microsecond response of a voltage-sensitive merocyanine dye: fast voltage-clamp measurements on squid giant axon. , 1993, The Japanese journal of physiology.

[42]  Francisco Bezanilla,et al.  Inactivation of the Sodium Channel , 2003 .

[43]  J. Balser,et al.  Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification. , 1997, Biophysical journal.

[44]  D. Hanck,et al.  Gating of skeletal and cardiac muscle sodium channels in mammalian cells , 1999, The Journal of physiology.

[45]  J. Patlak,et al.  Transfer of twelve charges is needed to open skeletal muscle Na+ channels , 1995, The Journal of general physiology.

[46]  N. Doi,et al.  Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution , 1999, FEBS letters.

[47]  Francisco Bezanilla,et al.  [19] Gating currents , 1998 .