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.
Mesh Terms:
Amino Acid Sequence, Animals, COS Cells, Electrophysiology, G Protein-Coupled Inwardly-Rectifying Potassium Channels, Guanylate Kinase, Intracellular Signaling Peptides and Proteins, Membrane Potentials, Membrane Proteins, Molecular Sequence Data, Nerve Tissue Proteins, Neurons, Nucleoside-Phosphate Kinase, Potassium Channels, Potassium Channels, Inwardly Rectifying, Precipitin Tests, Rats, Recombinant Fusion Proteins, Synapses, Two-Hybrid System Techniques
Amino Acid Sequence, Animals, COS Cells, Electrophysiology, G Protein-Coupled Inwardly-Rectifying Potassium Channels, Guanylate Kinase, Intracellular Signaling Peptides and Proteins, Membrane Potentials, Membrane Proteins, Molecular Sequence Data, Nerve Tissue Proteins, Neurons, Nucleoside-Phosphate Kinase, Potassium Channels, Potassium Channels, Inwardly Rectifying, Precipitin Tests, Rats, Recombinant Fusion Proteins, Synapses, Two-Hybrid System Techniques
J. Neurosci.
Date: Jan. 01, 2000
PubMed ID: 10627592
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