Visualization of the dynamics of PSD-95 and Kir2.1 interaction by fluorescence lifetime-based resonance energy transfer imaging

Many cellular processes are orchestrated by protein–protein interactions that allow the formation of protein networks involved in subcellular compartmentalization, communication, and signalling. Postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase protein (MAGuK) family, is a central scaffold protein of the postsynaptic density (PSD) of excitatory synapses in the mammalian central nervous system. PSD-95 serves as a matrix for targeting and accumulation of yet other PSD proteins including ion channels and receptors via its three N-terminal PDZ (PSD-95, discs large, zonula occludens-1) domains. However, the stoichiometry of PSD-95 and its binding partners in such complexes and the dynamic regulation of their interactions remain elusive. Here, we have investigated the protein–protein interaction between PSD-95 and the inward rectifier potassium channel Kir2.1, which we consider as a model for other PDZ domain based interactions at synapses. By using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), we show that PSD-95 and Kir2.1 directly interact within clusters which are formed at the plasma membrane of culture cells. Our in vivo FRET data indicate that Kir2.1 binds to more than one PDZ domain of PSD-95, suggesting a structural model of synergistic target binding by the first two PDZ domains of the scaffold protein. We show that the cluster formation is induced by the channel whereas PSD-95 alone does not form clusters. The interaction of PSD-95 and Kir2.1 is dynamically regulated by protein kinase A (PKA) mediated phosphorylation, which is directly visualized and quantified in living cells and in real time.