Ion channels are key determinants of membrane excitability. The actin cytoskeleton has a central role in morphology, migration, intracellular transport, and signaling. In this article, we show that the actin-binding protein cortactin regulates the potassium channel Kv1.2 and thereby provides a direct link between actin dynamics and membrane excitability. In previous reports, we showed that the tyrosine phosphorylation-mediated suppression of Kv1.2 ionic current occurs by endocytosis of the channel protein. Pull-down assays using recombinant-purified cortactin and Kv1.2 demonstrated that their interaction is direct and reduced by tyrosine phosphorylation of Kv1.2. This finding suggests a link between cortactin and Kv1.2 endocytosis. Here, we confirm that relationship and identify the molecular mechanisms involved. We use FRET to demonstrate that Kv1.2 and cortactin interact in vivo. By manipulating the cortactin-binding site within Kv1.2, we confirm that cortactin proximity influences channel function. We used flow cytometry in conjunction with cortactin gene replacement to identify C-terminal tyrosines, the fourth repeat actin-binding domain, and the N-terminal Arp2/3-binding region, as critical to Kv1.2 regulation. Surprisingly, cortactin's dynamin-binding Src homology 3 domain is not required for Kv1.2 endocytosis, despite that process being dynamin-dependent. These findings predict that cortactin-mediated actin remodeling in excitable cells is not only important for cell structure, but may directly impact membrane excitability.actin ͉ cytoskeleton ͉ endocytosis ͉ excitability ͉ trafficking S uppression of Kv1.2 mediated by the M1 muscarinic receptor was the first example of tyrosine phosphorylation-dependent modulation of voltage-dependent ion channels. 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. Subsequently, we found that the mechanism for channel suppression involves the dynamin-dependent endocytosis of Kv1.2 (2). That finding is particularly intriguing because cortactin's role in endocytosis is becoming increasingly apparent (3, 4).Since cortactin's discovery, its functions have been correlated with specific structural domains. Cortactin was first described as an actin cross-linking protein enriched within the cell cortex (5), a property requiring the fourth cortactin repeat region. Later studies identified cortactin as an activator of the Arp2/3 complex, a property dependent on a tryptophan residue (W22) within cortactin's N terminus (5). Tyrosine phosphorylation of cortactin's C-terminal region by Src family kinases affects its ability to regulate actin (6), identifying cortactin as a link between tyrosine kinase sign...

Doczi MA, Morielli AD, Damon DH. Kv1.3 channels in postganglionic sympathetic neurons: expression, function, and modulation.

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms.

Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis.

The hypothalamus is a brain region critical for the homeostatic regulation of appetite and energy expenditure. Hypothalamic neuronal activity that is altered during development can produce permanent physiological changes later in life. For example, circulating hormones such as insulin have been shown to influence hypothalamic neuronal projections, leading to altered metabolism in adult rodents. While insulin signaling in the post-hatch chicken has been shown to mirror that of mammals, the developmental role of insulin in the avian embryonic hypothalamus remains largely unexplored. Here we present the earliest known evidence for insulin receptor (InsR) expression in embryonic avian hypothalamic nuclei governing energy homeostasis. RT-PCR analysis reveals InsR mRNA in E8, E10, and E12 neurons while western blot data demonstrate protein expression in E12 avian whole brain and hypothalamic lysates. Immunohistochemical analysis of avian hypothalamic brain slices demonstrates a shift in InsR localization from paraventricular expression in E8 to a more defined concentration of InsR in developmental regions resembling the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) in E12 time points. In addition, InsR expression appears in a heterogeneous pattern, suggesting receptor localization to subpopulations of avian hypothalamic neurons as early as E8. With increasing evidence suggesting energy homeostasis pathways may be altered via the gestational environment, it is important to understand how insulin signaling may affect embryogenesis. Our research provides evidence for the earliest known embryonic expression of InsR protein in the avian hypothalamus and may suggest a developmental role for insulin signaling in the early patterning of metabolic pathways in the central nervous system.