Single-molecule imaging of l-type Ca(2+) channels in live cells.

L-type Ca(2+) channels are an important means by which a cell regulates the Ca(2+) influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca(2+) channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca(2+) channels tend to form larger aggregates which are mobile in the plasma membrane.

[1]  M. Saxton,et al.  Lateral diffusion in an archipelago. Distance dependence of the diffusion coefficient. , 1989, Biophysical journal.

[2]  Jerker Widengren,et al.  Protonation kinetics of GFP and FITC investigated by FCS — aspects of the use of fluorescent indicators for measuring pH , 1999 .

[3]  H. Kahr,et al.  A sequence in the carboxy‐terminus of the α1C subunit important for targeting, conductance and open probability of L‐type Ca2+ channels , 2000, FEBS letters.

[4]  A. Caswell,et al.  Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro , 1993, The Journal of cell biology.

[5]  S. Weiss Fluorescence spectroscopy of single biomolecules. , 1999, Science.

[6]  A. Verkman,et al.  Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. , 1997, Biophysical journal.

[7]  G. Schütz,et al.  Free Brownian motion of individual lipid molecules in biomembranes. , 1999, Biophysical journal.

[8]  Thomas Schmidt,et al.  Local stoichiometries determined by counting individual molecules , 1996 .

[9]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[10]  J. Frank,et al.  Ultrastructure of the calcium release channel of sarcoplasmic reticulum , 1988, The Journal of cell biology.

[11]  G. Schütz,et al.  Single-molecule anisotropy imaging. , 1999, Biophysical journal.

[12]  W. Catterall,et al.  Activation of purified calcium channels by stoichiometric protein phosphorylation. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Rigler,et al.  Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces. , 1998, Cellular and molecular biology.

[14]  G. A. Blab,et al.  Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy. , 2001, Biophysical journal.

[15]  H. Qian,et al.  Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. , 1991, Biophysical journal.

[16]  W. Almers,et al.  Dihydropyridine receptors in muscle are voltage-dependent but most are not functional calcium channels , 1985, Nature.

[17]  W. Webb,et al.  Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. , 1999, Biophysical journal.

[18]  H Schindler,et al.  Imaging of single molecule diffusion. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Edidin Rotational and Lateral Diffusion of Membrane Proteins and Lipids: Phenomena and Function , 1987 .

[20]  X. Xie,et al.  Single-molecule enzymatic dynamics. , 1998, Science.

[21]  B. Flucher,et al.  Current modulation and membrane targeting of the calcium channel α1C subunit are independent functions of the β subunit , 1999 .

[22]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[23]  I. Sase,et al.  Axial rotation of sliding actin filaments revealed by single-fluorophore imaging. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M.,et al.  L-type VoltageSensitive Calcium Channels Mediate Synaptic Activation of Immediate Early , 2003 .

[25]  M. Saxton,et al.  Lateral diffusion in an archipelago. Effects of impermeable patches on diffusion in a cell membrane. , 1982, Biophysical journal.

[26]  A. Chien,et al.  Complexes of the α1C and β Subunits Generate the Necessary Signal for Membrane Targeting of Class C L-type Calcium Channels* , 1999, The Journal of Biological Chemistry.

[27]  Gerald Kada,et al.  Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy , 2000, The EMBO journal.

[28]  W. Catterall Structure and function of voltage-gated ion channels. , 1995, Annual review of biochemistry.

[29]  R. Rigler,et al.  Conformational transitions monitored for single molecules in solution. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Beam,et al.  Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca2+ channels expressed in dysgenic myotubes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J Greve,et al.  Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Saxton,et al.  Lateral diffusion in an archipelago. Single-particle diffusion. , 1993, Biophysical journal.

[33]  T. Osborne,et al.  Cooperation by Sterol Regulatory Element-binding Protein and Sp1 in Sterol Regulation of Low Density Lipoprotein Receptor Gene (*) , 1995, The Journal of Biological Chemistry.

[34]  A. Fabiato,et al.  Calcium and cardiac excitation-contraction coupling. , 1979, Annual review of physiology.

[35]  W E Moerner,et al.  Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Werner Baumgartner,et al.  Characterization of Photophysics and Mobility of Single Molecules in a Fluid Lipid Membrane , 1995 .

[37]  C. Franzini-armstrong,et al.  Formation of junctions involved in excitation-contraction coupling in skeletal and cardiac muscle. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[38]  H. Gruber,et al.  Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor. , 1997, Biochemistry.

[39]  Toshio Yanagida,et al.  Single-molecule imaging of EGFR signalling on the surface of living cells , 2000, Nature Cell Biology.

[40]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[41]  E. Ríos,et al.  Involvement of dihydropyridine receptors in excitation–contraction coupling in skeletal muscle , 1987, Nature.

[42]  Y. Jia,et al.  Folding dynamics of single GCN-4 peptides by fluorescence resonant energy transfer confocal microscopy , 1999 .

[43]  E. Neher Correction for liquid junction potentials in patch clamp experiments. , 1992, Methods in enzymology.

[44]  A. Oijen,et al.  Unraveling the electronic structure of individual photosynthetic pigment-protein complexes , 1999, Science.

[45]  M. Biel,et al.  Molecular basis for Ca2+ channel diversity. , 1994, Annual review of neuroscience.