Distinct Structural Requirements for Clustering and Immobilization of K(+) Channels by PSD-95

PDZ-domain–containing proteins such as PSD-95 have been implicated in the targeting and clustering of membrane proteins. Biochemical and immunohistochemical studies indicate that PSD-95 recognizes COOH-terminal S/TXV sequences present in Kv1 K(+) channels. However, the effect of binding a PDZ domain on a target protein has not been studied in live cells. In the present study, a green fluorescent protein–Kv1.4 fusion protein is used to study the effect of PSD-95 on channel movement. Fluorescence recovery after photobleaching showed that PSD-95 can immobilize K(+) channels in the plasma membrane in an all-or-none manner. Furthermore, time lapse imaging showed that channel clusters formed in the presence of PSD-95 are stable in size, shape, and position. As expected from previous reports, two green fluorescent protein–tagged COOH-terminal variants of Kv1.4, Δ15 and V655A, are not clustered by PSD-95. However, coexpression of PSD-95 with V655A, but not Δ15, leads to the appearance of PSD-95 immunoreactivity in the plasma membrane. Furthermore, fluorescence recovery after photobleaching studies show that V655A channels are immobilized by PSD-95. Thus, V655A channels can interact with PSD-95 in a manner that leads to channel immobilization, but not clustering. These experiments document for the first time that PSD-95 immobilizes target proteins. Additionally, the data presented here demonstrate that the structural requirements for protein clustering and immobilization by PSD-95 are distinct.