Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1.
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.
Sampson
Laura J
LJ
Department of Cell Physiology and Pharmacology, University of Leicester, P. O. Box 138, Leicester LE1 9HN, United Kingdom. ljs17@le.ac.uk
Leyland
Mark L
ML
Dart
Caroline
C
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D003285:Contractile Proteins / Q000032:analysis / Q000378:metabolism*; D003331:Coronary Vessels; D018076:DNA, Complementary / Q000302:isolation & purification; D004594:Electrophysiology; D064448:Filamins; D006801:Humans; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000737:chemistry*; D024661:Potassium Channels, Inwardly Rectifying / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D013552:Swine; D020798:Two-Hybrid System Techniques
D000199:Actins; D003285:Contractile Proteins; D018076:DNA, Complementary; D064448:Filamins; D008840:Microfilament Proteins; D024661:Potassium Channels, Inwardly Rectifying