Fluorescent Na+-Ca+ exchangers: electrophysiological and optical characterization.
The cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1) influences cardiac contractility by extruding Ca2+ from myocytes. As a Ca2+ efflux mechanism, the exchanger plays a prominent role in Ca2+ 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 45Ca2+ 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 Ca2+ 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.
Ottolia
Michela
M
Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1760, USA.
John
Scott
S
Ren
Xiaoyan
X
Philipson
Kenneth D
KD
D000818:Animals; D002460:Cell Line; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000502:physiology; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016254:Mutagenesis, Insertional; D009865:Oocytes / Q000737:chemistry / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D049452:Green Fluorescent Proteins