Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins.
Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.
Leonoudakis
Dmitri
D
Department of Molecular, Cellular, University of California, Santa Barbara, California 93106, USA.
Conti
Lisa R
LR
Anderson
Scott
S
Radeke
Carolyn M
CM
McGuire
Leah M M
LM
Adams
Marvin E
ME
Froehner
Stanley C
SC
Yates
John R
JR
Vandenberg
Carol A
CA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000196:embryology / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D049029:Dystrophin-Associated Proteins; D005982:Glutathione Transferase / Q000378:metabolism; D051528:Guanylate Kinases; D015151:Immunoblotting; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020543:Proteome; D040901:Proteomics / Q000379:methods; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016622:Silver Staining; D021241:Spectrometry, Mass, Electrospray Ionization
D018076:DNA, Complementary; D003902:Detergents; D049029:Dystrophin-Associated Proteins; C496472:MPP6 protein, human; D008565:Membrane Proteins; D009124:Muscle Proteins; D010455:Peptides; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms; D020543:Proteome; D011993:Recombinant Fusion Proteins; C091824:syntrophin; D005982:Glutathione Transferase; D051528:Guanylate Kinases