Endocytosis as a Mechanism for Tyrosine Kinase-dependent Suppression of a Voltage-gated Potassium Channel

Nesti, Edmund; Everill, Brian; Morielli, Anthony D.

doi:10.1091/mbc.e03-11-0788

Cited by 82 publications

(110 citation statements)

References 71 publications

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Section: Discussionmentioning

confidence: 99%

“…Kv1.2 currents are decreased upon Tyr kinase activation (22) through increased Kv1.2 endocytosis dependent on an intact Y132 residue in the Kv1.2 N terminus (23). As such, the Y132F mutant exhibits increased steady-state levels of Kv1.2 expression (23). Phosphorylation at S440/S441 presumably acts through a distinct mechanism, because the S440A/S441A double mutation reduces cell-surface Kv1.2 expression of both WT Kv1.2 and the Y132F mutant to a similar extent (H.V.…”

Section: Discussionmentioning

confidence: 99%

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Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression

Yang¹,

Vacher²,

Park³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Kv1.2 ␣-subunits are components of low-threshold, rapidly activating voltage-gated potassium (Kv) channels in mammalian neurons. Expression and localization of Kv channels is regulated by trafficking signals encoded in their primary structure. Kv1.2 is unique in lacking strong trafficking signals and in exhibiting dramatic cell-specific differences in trafficking, which is suggestive of conditional trafficking signals. Here we show that a cluster of cytoplasmic C-terminal phosphorylation sites regulates Kv1.2 trafficking. Using tandem MS to analyze Kv1.2 purified from rat, human, and mouse brain, we identified in each sample in vivo phosphoserine (pS) phosphorylation sites at pS434, pS440, and pS441, as well as doubly phosphorylated pS440/pS441. We also found these sites, as well as pS449, on recombinant Kv1.2 expressed in heterologous cells. We found that phosphorylation at pS440/pS441 is present only on the post-endoplasmic reticulum (ER)/cell surface pool of Kv1.2 and is not detectable on newly synthesized and ER-localized Kv1.2, on which we did observe pS449 phosphorylation. Elimination of PS440/PS441 phosphorylation by mutation reduces cell-surface expression efficiency and functional expression of homomeric Kv1.2 channels. Interestingly, mutation of S449 reduces phosphorylation at pS440/pS441 and also decreases Kv1.2 cell-surface expression efficiency and functional expression. These mutations also suppress trafficking of Kv1.2/ Kv1.4 heteromeric channels, suggesting that incorporation of Kv1.2 into heteromeric complexes confers conditional phosphorylation-dependent trafficking to diverse Kv channel complexes. These data support Kv1.2 phosphorylation at these clustered C-terminal sites as playing an important role in regulating trafficking of Kv1.2-containing Kv channels.ion channel ͉ mass spectrometry ͉ proteomics ͉ brain ͉ phosphospecific V oltage-gated potassium, or Kv, channels that form as tetramers of Kv1 ␣-subunits play important and diverse roles in regulating excitability of axons, nerve terminals, and dendrites of mammalian neurons (1). The major Kv1 ␣-subunits in mammalian brain, Kv1.1, Kv1.2, and Kv1.4, are found predominantly in heterotetrameric channel complexes (2-4). Homotetrameric Kv1.2 channels form low-threshold, sustained-or delayed-rectifier Kv channels (5) and, when coassembled with other Kv1 ␣-subunits, can generate a diverse array of channel subtypes (6). In mammalian brain, Kv1.2 is found prominently along unmyelinated axons, in nerve terminals and/or in preterminal axon segments, at axon initial segments, and at juxtaparanodes of myelinated axons, but it is also present in the dendrites of some neurons (7). The physiological importance of Kv1.2 is underscored by the recent finding that Kv1.2 knockout mice have enhanced seizure susceptibility and die in the third postnatal week (8).Biosynthetic trafficking of Kv1 channels from their site of translation in the rough endoplasmic reticulum (ER) to the cell surface is governed by a set of intrinsic targeting motifs encoded in primary s...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression

Yang¹,

Vacher²,

Park³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…HEK293 cells stably expressing Kv1.2, Kv␤2, and the M1 muscarinic acetylcholine receptor under Zeocin and G418 selection, respectively, were cultured as reported (2).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 89%

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

Williams¹,

Markey²,

Doczi

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Activation of muscarinic acetylcholine receptors, for example, elicits tyrosine phosphorylation of Kv1.2 and the resultant suppression of its ionic current (11). Tyrosine phosphorylation of Kv1.2 also elicits endocytosis of the channel, and this endocytosis is a mechanism for current suppression (10). This process is dependent upon the proteins dynamin and cortactin, both of which are well known for their roles in endocytosis (12)(13)(14).…”

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confidence: 99%

Homeostatic Regulation of Kv1.2 Potassium Channel Trafficking by Cyclic AMP

Connors

Ballif

Morielli

2008

Journal of Biological Chemistry

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The Shaker family potassium channel, Kv1.2, is a key determinant of membrane excitability in neurons and cardiovascular tissue. Kv1.2 is subject to multiple forms of regulation and therefore integrates cellular signals involved in the homeostasis of excitability. The cyclic AMP/protein kinase A (PKA) pathway enhances Kv1.2 ionic current; however, the mechanisms for this are not fully known. Here we show that cAMP maintains Kv1.2 homeostasis through opposing effects on channel trafficking. We found that Kv1.2 is regulated by two distinct cAMP pathways, one PKA-dependent and the other PKA-independent. PKA inhibitors elevate Kv1.2 surface levels, suggesting that basal levels of cAMP control steady-state turnover of the channel. Elevation of cAMP above basal levels also increases the amount of Kv1.2 at the cell surface. This effect is not blocked by PKA inhibitors, but is blocked by inhibition of Kv1.2 endocytosis. We conclude that Kv1.2 levels at the cell surface are kept in dynamic balance by opposing effects of cAMP.The voltage-gated potassium channel, Kv1.2, plays an important role in modulating membrane potential, and its effects are associated with a wide range of human pathologies. In the brain, Kv1.2 is associated with seizure activity (1, 2). In dorsal root ganglion neurons, Kv1.2 plays a central role in nociception and is involved in the generation of neuropathic pain (3-5). In cardiac and vascular smooth muscle, Kv1.2 activity is associated with increased vascular tone and hypertension (6 -8). Kv1.2 activity within the cell is not static, but is instead subject to both positive and negative regulation (9, 10). Disruption of that regulation may underlie some of the pathologies associated with Kv1.2 function. Therefore, elucidating the mechanisms of Kv1.2 regulation is essential for understanding a broad range of pathological conditions.

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Endocytosis as a Mechanism for Tyrosine Kinase-dependent Suppression of a Voltage-gated Potassium Channel

Cited by 82 publications

References 71 publications

Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression

Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression

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

Homeostatic Regulation of Kv1.2 Potassium Channel Trafficking by Cyclic AMP

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