The Na$^+$/Ca$^2+$ exchanger (NCX) regulates cardiac contractility
by adjusting the amount of Ca$^2+$ inside myocytes. NCX accomplishes
this by using the electrochemical gradient of Na$^+$: during
each cycle three Na$^+$ ions enter the cell and one Ca$^2+$
ion is extruded against its gradient. In addition to being transported,
cytoplasmic Na$^+$ and Ca$^2+$ ions also regulate exchanger
activity. The physiological relevance and molecular processes underlying
ionic regulation remain unclear. Also unresolved are the events that
regulate NCX trafficking to the membrane and its oligomeric state.
This is essential information to interpret structure-function data.
The full-length exchanger was fused to both CFP and YFP, creating
active fluorescent exchangers used in FRET experiments to assess
both conformational changes associated with ionic regulation and
the oligomeric state of NCX. Electrophysiological characterization
demonstrates that these constructs behave similarly to the wild-type
(WT) exchanger. We have been able for the first time to monitor conformational
changes of the exchanger Ca$^2+$-binding site in vivo. These
studies provide a better understanding of the molecular properties
of the NCX.