Direct Interaction between the Actin-binding Protein Filamin-A and the Inwardly Rectifying Potassium Channel, Kir2.1

• Published in: The Journal of biological chemistry Vol. 278; no. 43; pp. 41988 - 41997
• Main Authors: Sampson, Laura J., Leyland, Mark L., Dart, Caroline
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.10.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Actins - metabolism; Amino Acid Sequence; Animals; Cell Line, Tumor; Contractile Proteins - analysis; Contractile Proteins - metabolism; Coronary Vessels; DNA, Complementary - isolation & purification; Electrophysiology; filamin A; Filamins; Humans; Immunohistochemistry; Kir2.1 protein; Microfilament Proteins - analysis; Microfilament Proteins - metabolism; Muscle, Smooth, Vascular - chemistry; Mutation; Myocytes, Smooth Muscle - chemistry; Potassium Channels, Inwardly Rectifying - analysis; Potassium Channels, Inwardly Rectifying - genetics; Potassium Channels, Inwardly Rectifying - metabolism; Precipitin Tests; Protein Binding; Sus scrofa; Swine; Two-Hybrid System Techniques
• DOI: 10.1074/jbc.M307479200
• ISSN: 0021-9258

The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction. Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone. Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481–2647). Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone. Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in “hotspots” at the cell membrane. Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane. We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.