Neuronal inwardly rectifying K(+) channels differentially couple to PDZ proteins of the PSD-95/SAP90 family.
Several signaling proteins clustered at the postsynaptic density specialization in neurons harbor a conserved C-terminal PDZ domain recognition sequence (X-S/T-X-V/I) that mediates binding to members of the PSD-95/SAP90 protein family. This motif is also present in the C termini of some inwardly rectifying K(+) (Kir) channels. Constitutively active Kir2 channels as well as G protein-gated Kir3 channels, which are fundamental for neuronal excitability, were analyzed as candidates for binding to PSD-95/SAP90 family members. Therefore C termini of Kir2.1(+), Kir2.3(+), Kir2.4(-), Kir3.1(-), Kir3.2(+), Kir3.3(+) and Kir3.4(-) subunits (+, motif present; -, motif absent) were used as baits in the yeast two-hybrid assay to screen for in vivo interaction with PDZ domains 1-3 of PSD-95/SAP90. In contrast to Kir2.1 and Kir2.3, all Kir3 fragments failed to bind PSD-95 in this assay, which was supported by the lack of coimmunoprecipitation and colocalization of the entire proteins in mammalian cells. A detailed analysis of interaction domains demonstrated that the C-terminal motif in Kir3 channels is insufficient for binding PDZ domains. Kir2.1 and Kir2.3 subunits on the other hand coprecipitate with PSD-95. When coexpressed in a bicistronic internal ribosome entry site expression vector in HEK-293 cells macroscopic and elementary current analysis revealed that PSD-95 suppressed the activity of Kir2.3 channels by >50%. This inhibitory action of PSD-95, which predominantly affects the single-channel conductance, is likely attributable to a molecular association with additional internal interaction sites in the Kir2.3 protein.
Nehring
R B
RB
Molecular Neurobiology of Signal Transduction, Max-Planck-Institut for Biophysical Chemistry, 37070 Göttingen, Germany.
Wischmeyer
E
E
Döring
F
F
Veh
R W
RW
Sheng
M
M
Karschin
A
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D000076122:Disks Large Homolog 4 Protein; D004594:Electrophysiology; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D051528:Guanylate Kinases; D047908:Intracellular Signaling Peptides and Proteins; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry*; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000074407:SAP90-PSD95 Associated Proteins; D013569:Synapses / Q000737:chemistry* / Q000201:enzymology; D020798:Two-Hybrid System Techniques
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D047908:Intracellular Signaling Peptides and Proteins; C490968:Kcnj4 protein, rat; C497073:Kcnj9 protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D000074407:SAP90-PSD95 Associated Proteins; C033652:postsynaptic density proteins; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases