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 t argeting and clustering of membrane proteins. Biochemical and immunohistoch emical studies indicate that PSD-95 recognizes COOH-terminal S/TXV sequence s present in Kvl K+ channels. However, the effect of binding a PDZ domain o n 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 eff ect of PSD-95 on channel movement. Fluorescence recovery after photobleachi ng 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 po sition. As expected from previous reports, two green fluorescent protein-ta gged COOH-terminal variants of Kv1.4, Delta 15 and V655A, are not clustered by PSD-95. However, coexpression of PSD-95 with V655A, but not Delta 15, l eads to the appearance of PSD-95 immunoreactivity in the plasma membrane. F urthermore, fluorescence recovery after photobleaching studies show that V6 55A channels are immobilized by PSD-95. Thus, V655A channels can interact w ith PSD-95 in a manner that leads to channel immobilization, but not cluste ring. These experiments document for the first time that PSD-SS immobilizes target proteins. Additionally, the data presented here demonstrate that th e structural requirements for protein clustering and immobilization by PSD- 95 are distinct.