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, Delta15 and V655A, are not clustered by PSD-95. However, coexpression of PSD-95 with V655A, but not Delta15, 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.
Burke
N A
NA
Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Takimoto
K
K
Li
D
D
Han
W
W
Watkins
S C
SC
Levitan
E S
ES
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D000076122:Disks Large Homolog 4 Protein; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D051665:Kv1.4 Potassium Channel; D008164:Luminescent Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D051381:Rats
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; C496977:KCNA4 protein, human; C496979:Kcna4 protein, rat; D051665:Kv1.4 Potassium Channel; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C033652:postsynaptic density proteins; D049452:Green Fluorescent Proteins