Tyrosine Phosphorylation of Kv1.2 Modulates Its Interaction with the Actin-binding Protein Cortactin

Abstract

Tyrosine phosphorylation evokes functional changes in a variety of ion channels. Modulation of the actin cytoskeleton also affects the function of some channels. Little is known about how these avenues of ion channel regulation may interact. We report that the potassium channel Kv1.2 associates with the actin-binding protein cortactin and that the binding is modulated by tyrosine phosphorylation. Immunocytochemical and biochemical analyses show that Kv1.2 and cortactin co-localize to the cortical actin cytoskeleton at the leading edges of the cell. Binding assays using purified recombinant proteins reveal a 19-amino acid span within the carboxyl terminus of Kv1.2 that is necessary for direct cortactin binding. Phosphorylation of specific tyrosines within the C terminus of Kv1.2 attenuates that binding. In HEK293 cells, activation of the M1 muscarinic acetylcholine receptor evokes tyrosine phosphorylationdependent suppression of Kv1.2 ionic current. We show that M1 receptor activation also reduces the interaction of cortactin with Kv1.2 and that mutant Kv1.2 channels deficient for cortactin binding exhibit strongly attenuated ionic current. These results demonstrate a dynamic, phosphorylation-dependent interaction between Kv1.2 and the actin cytoskeleton-binding protein cortactin and suggest a role for that interaction in the regulation of Kv1.2 ionic current.Ion channels regulate a wide range of cellular processes, including development (1, 2), and neuronal plasticity (3, 4). Accordingly, the activity of nearly all ion channels is under some form of post-translational control, most commonly direct phosphorylation of the ion channel protein (5-7). The earliest and most widely reported examples involve serine/threonine phosphorylation; however, it is also now recognized that tyrosine phosphorylation is an important means of ion channel regulation (8). The nicotinic acetylcholine receptor was the first ligand-gated ion channel found to be regulated by tyrosine phosphorylation (9). The shaker family potassium channel Kv1.2, which is expressed abundantly in cardiac muscle (10) and neurons (11-13), was the first voltage-gated channel found to be so regulated.Kv1.2 ␣-subunit protein becomes tyrosine-phosphorylated upon the activation of M1 muscarinic acetylcholine receptors (mAChRs), 1 leading to a profound suppression of Kv1.2 ionic current (14). Part of the mechanism for such suppression involves phosphorylation of the N-terminal tyrosine Tyr 132 . However, phosphorylation of additional tyrosines within Kv1.2 is also likely to be required, since Y132F mutant channels are only partially resistant to tyrosine phosphorylation and ionic current suppression. Little is known about the signaling proteins involved in tyrosine phosphorylation-dependent suppression of Kv1.2 ionic current; however, binding of the activated form of the guanine nucleotide-binding protein RhoA to Kv1.2 is required (15).RhoA is an important regulator of actin dynamics (16 -18), and the physical association between RhoA and Kv1.2 suggests a link bet...

An essential role for cortactin in the modulation of the potassium channel Kv1.2

“…Therefore, the cortactin-Kv1.2 interaction appears to be maintained, albeit weaker, after channel endocytosis. This finding is consistent with biochemical studies showing that tyrosine phosphorylation of Kv1.2 reduces, but does not eliminate, cortactin binding (1).…”

“…3). Data from previous studies (1) and Fig. 1 leading edge structures or vesicles revealed the most intense FRET signal at the leading edge, with significantly (P Ͻ 0.001, n ϭ 20) weaker FRET within vesicles (Fig.…”

“…In a previous report, we used fluorescence microscopy to show that Kv1.2 colocalizes with cortactin and immunoprecipitation and pull-down assays to show that the proteins interact directly (1). Here, we use FRET to determine whether Kv1.2 and cortactin interact within intact cells and, if so, where those interactions occur.…”

“…Later studies revealed that channel suppression involves the dynamic interaction of Kv1.2 with the actin-binding protein cortactin. Cortactin binds to the N and C termini of Kv1.2, and binding is diminished upon tyrosine phosphorylation of the channel (1). That evidence, along with mutagenesis studies, led to the hypothesis that cortactin was necessary for Kv1.2 function, and that its dissociation contributed to channel suppression.…”

Interference Microscopy under Double-Wavelet Analysis: A New Approach to Studying Cell Dynamics

“…We suppose that frequencies in the low and high ranges can be partly related to the cytoskeleton reorganizations that occur under the function of the ion channels and organelles movements [18]. Recently it was shown that cytoskeleton in its turn regulates ion channel activity and location of mitochondria and endoplasmic reticulum and Ca 2 signaling between them [19,20]. Further studies of neuron function by means of interference microscopy and doublewavelet analysis may help to reveal the relation of the cytoskeleton organization to the activity of the plasma membrane and cytoplasmic processes.…”