Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume.
To examine the structure and function of the Na-K-Cl cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl(-) provided a ratiometric indicator of intracellular [Cl(-)]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed contemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA.
Meike
M
Pedersen
Monica
M
Carmosino
Biff
B
Forbush
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012754:Sharks / Q000235:genetics / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D014162:Transfection
D001426:Bacterial Proteins; D002712:Chlorides; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C578714:SLC12A1 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins