%0 Journal Article %1 Otto_2007_3695 %A Ottolia, Michela %A John, Scott %A Ren, Xiaoyan %A Philipson, Kenneth D %D 2007 %J J. Biol. Chem. %K Animals; Cell Dogs; Exchanger, Fluid, Fluorescent Fusion Green Humans; Insertional; Intracellular Line; Luminescent Mutagenesis, Oocytes, Patch-Clamp Protein Proteins, Recombinant Sodium-Calcium Structure, Techniques; Tertiary, Transport, Xenopus chemistry/genetics/physiology; chemistry/genetics; chemistry/physiology; genetics; laevis %N 6 %P 3695--3701 %R 10.1074/jbc.M610425200 %T Fluorescent Na$^+$-Ca+ exchangers: electrophysiological and optical characterization. %U http://dx.doi.org/10.1074/jbc.M610425200 %V 282 %X The cardiac sarcolemmal Na$^+$-Ca$^2+$ exchanger (NCX1) influences cardiac contractility by extruding Ca$^2+$ from myocytes. As a Ca$^2+$ efflux mechanism, the exchanger plays a prominent role in Ca$^2+$ homeostasis. To track NCX1 and study changes in conformation, NCX1 was tagged with derivatives of green fluorescent protein. Cyan (CFP) and yellow (YFP) fluorescent proteins were used for both visualization of the protein in HEK cells and fluorescent resonance energy transfer (FRET). CFP or YFP was inserted at position 266, 371, 467, or 548 of the large intracellular loop of NCX1 located between transmembrane segments 5 and 6. These constructs were tested for functional activity and visualized for cell surface expression. All constructs were targeted to the plasma membrane. Transport properties were assessed by both 45Ca$^2+$ uptake and electrophysiological measurements. The fluorescent-tagged exchangers had similar biophysical properties to the wild type NCX1. Unexpectedly, all constructs retain their sensitivity to regulation by cytoplasmic Na$^+$ and Ca$^2+$ ions. FRET analysis indicates the proximity of NCX1 to plasma membrane phosphatidylinositol 4,5-bisphosphate. These results indicate that insertion of CFP or YFP into the large intracellular loop of NCX1 protein does not impair exchanger properties. These constructs will be useful to further characterize the biological properties of the exchanger in intact cells.

@article{Otto_2007_3695, abstract = {The cardiac sarcolemmal {N}a$^{+}$-{C}a$^{2+}$ exchanger (NCX1) influences cardiac contractility by extruding {C}a$^{2+}$ from myocytes. As a {C}a$^{2+}$ efflux mechanism, the exchanger plays a prominent role in {C}a$^{2+}$ homeostasis. To track NCX1 and study changes in conformation, NCX1 was tagged with derivatives of green fluorescent protein. Cyan (CFP) and yellow (YFP) fluorescent proteins were used for both visualization of the protein in HEK cells and fluorescent resonance energy transfer (FRET). CFP or YFP was inserted at position 266, 371, 467, or 548 of the large intracellular loop of NCX1 located between transmembrane segments 5 and 6. These constructs were tested for functional activity and visualized for cell surface expression. All constructs were targeted to the plasma membrane. Transport properties were assessed by both 45{C}a$^{2+}$ uptake and electrophysiological measurements. The fluorescent-tagged exchangers had similar biophysical properties to the wild type NCX1. Unexpectedly, all constructs retain their sensitivity to regulation by cytoplasmic {N}a$^{+}$ and {C}a$^{2+}$ ions. FRET analysis indicates the proximity of NCX1 to plasma membrane phosphatidylinositol 4,5-bisphosphate. These results indicate that insertion of CFP or YFP into the large intracellular loop of NCX1 protein does not impair exchanger properties. These constructs will be useful to further characterize the biological properties of the exchanger in intact cells.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Ottolia, Michela and John, Scott and Ren, Xiaoyan and Philipson, Kenneth D}, biburl = {https://www.bibsonomy.org/bibtex/2c07e82177f3bf725e335684ff3008879/hake}, description = {The whole bibliography file I use.}, doi = {10.1074/jbc.M610425200}, file = {Otto_2007_3695.pdf:Otto_2007_3695.pdf:PDF}, institution = {Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1760, USA.}, interhash = {219c8386e97024d7a5ee8315649752c8}, intrahash = {c07e82177f3bf725e335684ff3008879}, journal = {J. Biol. Chem.}, keywords = {Animals; Cell Dogs; Exchanger, Fluid, Fluorescent Fusion Green Humans; Insertional; Intracellular Line; Luminescent Mutagenesis, Oocytes, Patch-Clamp Protein Proteins, Recombinant Sodium-Calcium Structure, Techniques; Tertiary, Transport, Xenopus chemistry/genetics/physiology; chemistry/genetics; chemistry/physiology; genetics; laevis}, month = Feb, number = 6, pages = {3695--3701}, pdf = {Otto_2007_3695.pdf}, pii = {M610425200}, pmid = {17158867}, timestamp = {2009-06-03T11:21:24.000+0200}, title = {Fluorescent {N}a$^{+}$-Ca+ exchangers: electrophysiological and optical characterization.}, url = {http://dx.doi.org/10.1074/jbc.M610425200}, volume = 282, year = 2007 }