Abstract
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.
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