N-terminal PDZ-binding domain in Kv1 potassium channels.
We have investigated the interactions of prototypical PDZ domains with both the C- and N-termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two-hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C- and N-termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co-immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C-terminal PDZ-binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co-localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ-binding motif. Deletion analysis localized the N-terminal PDZ-binding site in Kv1.5 to the T1 region of the channel. Co-expression of PSD95 with Kv1.5 N- and C-terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95.
Eldstrom
Jodene
J
Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada.
Doerksen
Kyle W
KW
Steele
David F
DF
Fedida
David
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D000076122:Disks Large Homolog 4 Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051666:Kv1.5 Potassium Channel; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; C496981:KCNA5 protein, human; D051666:Kv1.5 Potassium Channel; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; C033652:postsynaptic density proteins