Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

A simultaneous increase in cytosolic Zn 2+ and Ca 2+ accompanies the initiation of neuronal cell death signaling cascades. However, the molecular convergence points of cellular processes activated by these cations are poorly understood. Here, we show that Ca 2+ -dependent activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is required for a cell death-enabling process previously shown to also depend on Zn 2+ . We have reported that oxidant-induced intraneuronal Zn 2+ liberation triggers a syntaxin-dependent incorporation of Kv2.1 voltage-gated potassium channels into the plasma membrane. This channel insertion can be detected as a marked enhancement of delayed rectifier K + currents in voltage clamp measurements observed at least 3 h following a short exposure to an apoptogenic stimulus. This current increase is the process responsible for the cytoplasmic loss of K + that enables protease and nuclease activation during apoptosis. In the present study, we demonstrate that an oxidative stimulus also promotes intracellular Ca 2+ release and activation of CaMKII, which, in turn, modulates the ability of syntaxin to interact with Kv2.1. Pharmacological or molecular inhibition of CaMKII prevents the K + current enhancement observed following oxidative injury and, importantly, significantly increases neuronal viability. These findings reveal a previously unrecognized cooperative convergence of Ca 2+ -and Zn 2+ -mediated injurious signaling pathways, providing a potentially unique target for therapeutic intervention in neurodegenerative conditions associated with oxidative stress.

Section: Discussionmentioning

confidence: 99%

Convergent Ca ²⁺ and Zn ²⁺ signaling regulates apoptotic Kv2.1 K ⁺ currents

McCord

Aizenman

2013

“…We optically detected activity of the Kv2.1 channel subtype using VST-conjugate probes. Kv2.1 is ubiquitously expressed in the soma of neurons throughout the brain (52) and is the dominant delayed rectifier of central neurons (53,54). The channel's activity shapes the repetitive firing properties of neurons (13), and Kv2.1 is homeostatically regulated in response to electrical activity (55).…”

Section: Discussionmentioning

confidence: 99%

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Tilley

Eum

Fletcher-Taylor

et al. 2014

111

Significance Electrically excitable cells, such as neurons, exhibit tremendous variation in their patterns of electrical signals. These variations arise from the collection of ion channels present in any specific cell, but understanding which ion channels are at the root of particular electrical signals remains a significant challenge. Here, we describe novel probes, derived from a tarantula venom peptide, that are able to report the activity of voltage-gated ion channels in living cells. This technology uses state-selective binding to optically monitor the activation of ion channels during cellular electrical signaling. Activity-reporting probes based on these prototypes could potentially identify when endogenous ion channels contribute to electrical signaling, thus facilitating the identification of ion channel targets for therapeutic drug intervention.

“…RBM or rat hippocampal membranes (RHM), mouse brain membranes (MBM), and human hippocampal or cerebellar membranes (HHM and HCM, respectively) were prepared as described in ref. 29. Human brain tissue from anonymous donors was obtained from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore.…”

Section: Materials and Methods Preparation Of Brain Membrane Fractionmentioning

confidence: 99%

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

Yang¹,

Vacher²,

Park³

et al. 2007

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...