Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging.
Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.
Biskup
Christoph
C
Friedrich-Schiller-Universitat Jena, Institut fur Physiologie II, Teichgraben 8, 07740 Jena, Germany. christoph.biskup@mti.uni-jena.de
Kelbauskas
Laimonas
L
Zimmer
Thomas
T
Benndorf
Klaus
K
Bergmann
Axel
A
Becker
Wolfgang
W
Ruppersberg
J Peter
JP
Stockklausner
Clemens
C
Klöcker
Nikolaj
N
D000818:Animals; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000648:ultrastructure; D009893:Opossums; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism* / Q000648:ultrastructure; D025941:Protein Interaction Mapping / Q000379:methods*
C489461:Kir2.1 channel; D009419:Nerve Tissue Proteins; D024661:Potassium Channels, Inwardly Rectifying; C033652:postsynaptic density proteins