Inward rectifier potassium channel Kir2.2 is associated with synapse-associated protein SAP97.
The strong inwardly rectifying potassium channels Kir2.x are involved in maintenance and control of cell excitability. Recent studies reveal that the function and localization of ion channels are regulated by interactions with members of the membrane-associated guanylate kinase (MAGUK) protein family. To identify novel interacting MAGUK family members, we constructed GST-fusion proteins with the C termini of Kir2.1, Kir2.2 and Kir2.3. GST affinity-pulldown assays from solubilized rat cerebellum and heart membrane proteins revealed an interaction between all three Kir2.x C-terminal fusion proteins and the MAGUK protein synapse-associated protein 97 (SAP97). A truncated form of the C-terminal GST-Kir2.2 fusion protein indicated that the last three amino acids (S-E-I) are essential for association with SAP97. Affinity interactions using GST-fusion proteins containing the modular domains of SAP97 demonstrate that the second PSD-95/Dlg/ZO-1 (PDZ) domain is sufficient for interaction with Kir2.2. Coimmunoprecipitations demonstrated that endogenous Kir2.2 associates with SAP97 in rat cerebellum and heart. Additionally, phosphorylation of the Kir2.2 C terminus by protein kinase A inhibited the association with SAP97. In rat cardiac ventricular myocytes, Kir2.2 and SAP97 colocalized in striated bands corresponding to T-tubules. In rat cerebellum, Kir2.2 was present in a punctate pattern along SAP97-positive processes of Bergmann glia in the molecular layer, and colocalized with astrocytes and granule cells in the granule cell layer. These results identify a direct association of Kir2.1, Kir2.2 and Kir2.3 with the MAGUK family member SAP97 that may form part of a macromolecular signaling complex in many different tissues.
Leonoudakis
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D
Department of Molecular, Cellular and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA.
Mailliard
W
W
Wingerd
K
K
Clegg
D
D
Vandenberg
C
C
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism; D010766:Phosphorylation; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D020318:Rats, Long-Evans; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C501119:Dlg1 protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D017868:Cyclic AMP-Dependent Protein Kinases