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Kv2.1 potassium channel antibody

RRID:AB_10673392

Antibody ID

AB_10673392

Target Antigen

Kv2.1 potassium channel null

Proper Citation

(UC Davis/NIH NeuroMab Facility Cat# 75-014, RRID:AB_10673392)

Clonality

monoclonal antibody

Comments

Originating manufacturer of this product. Applications: IB, ICC, IHC, IP, KO, WB. Validation status: IF or IB (Pass), IB in brain (Pass), IHC in brain (Pass), KO (Pass).

Clone ID

K89/34

Host Organism

mouse

Heteromeric KV2/KV8.2 Channels Mediate Delayed Rectifier Potassium Currents in Primate Photoreceptors.

  • Gayet-Primo J
  • J. Neurosci.
  • 2018 Apr 4

Literature context:


Abstract:

Silent voltage-gated potassium channel subunits (KVS) interact selectively with members of the KV2 channel family to modify their functional properties. The localization and functional roles of these silent subunits remain poorly understood. Mutations in the KVS subunit, KV8.2 (KCNV2), lead to severe visual impairment in humans, but the basis of these deficits remains unclear. Here, we examined the localization, native interactions, and functional properties of KV8.2-containing channels in mouse, macaque, and human photoreceptors of either sex. In human retina, KV8.2 colocalized with KV2.1 and KV2.2 in cone inner segments and with KV2.1 in rod inner segments. KV2.1 and KV2.2 could be coimmunoprecipitated with KV8.2 in retinal lysates indicating that these subunits likely interact directly. Retinal KV2.1 was less phosphorylated than cortical KV2.1, a difference expected to alter the biophysical properties of these channels. Using voltage-clamp recordings and pharmacology, we provide functional evidence for Kv2-containing channels in primate rods and cones. We propose that the presence of KV8.2, and low levels of KV2.1 phosphorylation shift the activation range of KV2 channels to align with the operating range of rod and cone photoreceptors. Our data indicate a role for KV2/KV8.2 channels in human photoreceptor function and suggest that the visual deficits in patients with KCNV2 mutations arise from inadequate resting activation of KV channels in rod and cone inner segments.SIGNIFICANCE STATEMENT Mutations in a voltage-gated potassium channel subunit, KV8.2, underlie a blinding inherited photoreceptor dystrophy, indicating an important role for these channels in human vision. Here, we have defined the localization and subunit interactions of KV8.2 channels in primate photoreceptors. We show that the KV8.2 subunit interacts with different Kv2 channels in rods and cones, giving rise to potassium currents with distinct functional properties. Our results provide a molecular basis for retinal dysfunction in patients with mutations in the KCNV2 gene encoding KV8.2.

Funding information:
  • NEI NIH HHS - P30 EY003176(United States)
  • NEI NIH HHS - P30 EY010572(United States)
  • NEI NIH HHS - R01 EY024265(United States)
  • NEI NIH HHS - T32 EY023211(United States)
  • NIGMS NIH HHS - U01GM070708(United States)
  • NIH HHS - P51 OD011092(United States)

Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment.

  • Berger SL
  • Neuron
  • 2018 Feb 7

Literature context:


Abstract:

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.

Funding information:
  • NIMH NIH HHS - R37 MH063105(United States)

Defective trafficking of Kv2.1 channels in MPTP-induced nigrostriatal degeneration.

  • Chao RY
  • J. Neurochem.
  • 2017 Dec 22

Literature context:


Abstract:

Intracellular protein trafficking is tightly regulated, and improper trafficking might be the fundamental provocateur for human diseases including neurodegeneration. In neurons, protein trafficking to and from the plasma membrane affects synaptic plasticity. Voltage-gated potassium channel 2.1 (Kv2.1) is a predominant delayed rectifier potassium (K+ ) current, and electrical activity patterns of dopamine (DA) neurons within the substantia nigra are generated and modulated by the orchestrated function of different ion channels. The pathological hallmark of Parkinson's disease (PD) is the progressive loss of these DA neurons, resulting in the degeneration of striatal dopaminergic terminals. However, whether trafficking of Kv2.1 channels contributes to PD remains unclear. In this study, we demonstrated that MPTP/MPP+ increases the surface expression of the Kv2.1 channel and causes nigrostriatal degeneration by using a subchronic MPTP mouse model. The inhibition of the Kv2.1 channel by using a specific blocker, guangxitoxin-1E, protected nigrostriatal projections against MPTP/MPP+ insult and thus facilitated the recovery of motor coordination. These findings highlight the importance of trafficking of Kv2.1 channels in the pathogenesis of PD.

Funding information:
  • NHGRI NIH HHS - HG00983(United States)

Objective quantification of nanoscale protein distributions.

  • Szoboszlay M
  • Sci Rep
  • 2017 Nov 10

Literature context:


Abstract:

Nanoscale distribution of molecules within small subcellular compartments of neurons critically influences their functional roles. Although, numerous ways of analyzing the spatial arrangement of proteins have been described, a thorough comparison of their effectiveness is missing. Here we present an open source software, GoldExt, with a plethora of measures for quantification of the nanoscale distribution of proteins in subcellular compartments (e.g. synapses) of nerve cells. First, we compared the ability of five different measures to distinguish artificial uniform and clustered patterns from random point patterns. Then, the performance of a set of clustering algorithms was evaluated on simulated datasets with predefined number of clusters. Finally, we applied the best performing methods to experimental data, and analyzed the nanoscale distribution of different pre- and postsynaptic proteins, revealing random, uniform and clustered sub-synaptic distribution patterns. Our results reveal that application of a single measure is sufficient to distinguish between different distributions.

Erratum to: Rectocutaneous fistula with transmigration of the suture: a rare delayed complication of vault fixation with the sacrospinous ligament.

  • Kadam PD
  • Int Urogynecol J
  • 2016 Mar 25

Literature context:


Abstract:

There was an oversight in the Authorship of a recent Images in Urogynecology article titled: Rectocutaneous fistula with transmigration of the suture: a rare delayed complication of vault fixation with the sacrospinous ligament (DOI 10.1007/ s00192-015-2823-5). We would like to include Adj A/P Han How Chuan’s name in the list of authors. Adj A/P Han is a Senior Consultant and Department Head of Urogynaecology at the KK Hospital for Women and Children, Singapore.

Funding information:
  • NHGRI NIH HHS - R01HG005855(United States)

Distinct axo-somato-dendritic distributions of three potassium channels in CA1 hippocampal pyramidal cells.

  • Kirizs T
  • Eur. J. Neurosci.
  • 2015 Jan 16

Literature context:


Abstract:

Potassium channels comprise the most diverse family of ion channels and play critical roles in a large variety of physiological and pathological processes. In addition to their molecular diversity, variations in their distributions and densities on the axo-somato-dendritic surface of neurons are key parameters in determining their functional impact. Despite extensive electrophysiological and anatomical investigations, the exact location and densities of most K(+) channels in small subcellular compartments are still unknown. Here we aimed at providing a quantitative surface map of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K(+) channel subunits on hippocampal CA1 pyramidal cells (PCs). Freeze-fracture replica immunogold labelling was employed to determine the relative densities of these K(+) channel subunits in 18 axo-somato-dendritic compartments. Significant densities of the Kv1.1 subunit were detected on axon initial segments (AISs) and axon terminals, with an approximately eight-fold lower density in the latter compartment. The Kv2.1 subunit was found in somatic, proximal dendritic and AIS plasma membranes at approximately the same densities. This subunit has a non-uniform plasma membrane distribution; Kv2.1 clusters are frequently adjacent to, but never overlap with, GABAergic synapses. A quasi-linear increase in the Kir3.2 subunit density along the dendrites of PCs was detected, showing no significant difference between apical dendritic shafts, oblique dendrites or dendritic spines at the same distance from the soma. Our results demonstrate that each subunit has a unique cell-surface distribution pattern, and predict their differential involvement in synaptic integration and output generation at distinct subcellular compartments.

Funding information:
  • NICHD NIH HHS - NIH P30 HD003352(United States)

A unique ion channel clustering domain on the axon initial segment of mammalian neurons.

  • King AN
  • J. Comp. Neurol.
  • 2014 Aug 1

Literature context:


Abstract:

The axon initial segment (AIS) plays a key role in initiation of action potentials and neuronal output. The plasma membrane of the AIS contains high densities of voltage-gated ion channels required for these electrical events, and much recent work has focused on defining the mechanisms for generating and maintaining this unique neuronal plasma membrane domain. The Kv2.1 voltage-gated potassium channel is abundantly present in large clusters on the soma and proximal dendrites of mammalian brain neurons. Kv2.1 is also a component of the ion channel repertoire at the AIS. Here we show that Kv2.1 clusters on the AIS of brain neurons across diverse mammalian species including humans define a noncanonical ion channel clustering domain deficient in Ankyrin-G. The sites of Kv2.1 clustering on the AIS are sites where cisternal organelles, specialized intracellular calcium release membranes, come into close apposition with the plasma membrane, and are also sites of clustering of γ-aminobutyric acid (GABA)ergic synapses. Using an antibody specific for a single Kv2.1 phosphorylation site, we find that the phosphorylation state differs between Kv2.1 clusters on the proximal and distal portions of the AIS. Together, these studies show that the sites of Kv2.1 clustering on the AIS represent specialized domains containing components of diverse neuronal signaling pathways that may contribute to local regulation of Kv2.1 function and AIS membrane excitability.

Funding information:
  • NIGMS NIH HHS - R01 GM115545(United States)

Somatodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development.

  • Dufour MA
  • J. Neurosci. Res.
  • 2014 Aug 16

Literature context:


Abstract:

Dopaminergic neurons of the substantia nigra pars compacta (SNc) are involved in the control of movement, sleep, reward, learning, and nervous system disorders and disease. To date, a thorough characterization of the ion channel phenotype of this important neuronal population is lacking. Using immunohistochemistry, we analyzed the somatodendritic expression of voltage-gated ion channel subunits that are involved in pacemaking activity in SNc dopaminergic neurons in 6-, 21-, and 40-day-old rats. Our results demonstrate that the same complement of somatodendritic ion channels is present in SNc dopaminergic neurons from P6 to P40. The major developmental changes were an increase in the dendritic range of the immunolabeling for the HCN, T-type calcium, Kv4.3, delayed rectifier, and SK channels. Our study sheds light on the ion channel subunits that contribute to the somatodendritic delayed rectifier (Kv1.3, Kv2.1, Kv3.2, Kv3.3), A-type (Kv4.3) and calcium-activated SK (SK1, SK2, SK3) potassium currents, IH (mainly HCN2, HCN4), and the L- (Cav1.2, Cav1.3) and T-type (mainly Cav3.1, Cav3.3) calcium currents in SNc dopaminergic neurons. Finally, no robust differences in voltage-gated ion channel immunolabeling were observed across the population of SNc dopaminergic neurons for each age examined, suggesting that differing levels of individual ion channels are unlikely to distinguish between specific subpopulations of SNc dopaminergic neurons. This is significant in light of previous studies suggesting that age- or region-associated variations in the expression profile of voltage-gated ion channels in SNc dopaminergic neurons may underlie their vulnerability to dysfunction and disease.

Funding information:
  • NIGMS NIH HHS - GM37432(United States)
  • NIMH NIH HHS - R01MH61469(United States)

β(IV)-Spectrin regulates TREK-1 membrane targeting in the heart.

  • Hund TJ
  • Cardiovasc. Res.
  • 2014 Apr 1

Literature context:


Abstract:

AIMS: Cardiac function depends on the highly regulated and co-ordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K(+) channels have been well characterized in the heart, much less is known about regulation and/or targeting of two-pore K(+) channel (K(2P)) family members, despite their potential importance in modulation of heart function. METHODS AND RESULTS: Here, we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K(2P) channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that β(IV)-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in the heart, and is required for TREK-1 membrane targeting. Mice expressing β(IV)-spectrin lacking TREK-1 binding (qv(4J)) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal β(IV)-spectrin levels in human heart failure. CONCLUSIONS: These data provide new insight into membrane targeting of TREK-1 in the heart and establish a broader role for β(IV)-spectrin in organizing functional membrane domains critical for normal heart function.

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability.

  • Speca DJ
  • Genes Brain Behav.
  • 2014 Apr 9

Literature context:


Abstract:

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

Funding information:
  • NCI NIH HHS - P30 CA177558(United States)

Specific sorting and post-Golgi trafficking of dendritic potassium channels in living neurons.

  • Jensen CS
  • J. Biol. Chem.
  • 2014 Apr 11

Literature context:


Abstract:

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Funding information:
  • NIMH NIH HHS - R01 MH059950(United States)

Cyclin e1 regulates Kv2.1 channel phosphorylation and localization in neuronal ischemia.

  • Shah NH
  • J. Neurosci.
  • 2014 Mar 19

Literature context:


Abstract:

Kv2.1 is a major delayed rectifying K(+) channel normally localized to highly phosphorylated somatodendritic clusters in neurons. Excitatory stimuli induce calcineurin-dependent dephosphorylation and dispersal of Kv2.1 clusters, with a concomitant hyperpolarizing shift in the channel's activation kinetics. We showed previously that sublethal ischemia, which renders neurons transiently resistant to excitotoxic cell death, can also induce Zn(2+)-dependent changes in Kv2.1 localization and activation kinetics, suggesting that activity-dependent modifications of Kv2.1 may contribute to cellular adaptive responses to injury. Recently, cyclin-dependent kinase 5 (Cdk5) was shown to phosphorylate Kv2.1, with pharmacological Cdk5 inhibition being sufficient to decluster channels. In another study, cyclin E1 was found to restrict neuronal Cdk5 kinase activity. We show here that cyclin E1 regulates Kv2.1 cellular localization via inhibition of Cdk5 activity. Expression of cyclin E1 in human embryonic kidney cells prevents Cdk5-mediated phosphorylation of Kv2.1, and cyclin E1 overexpression in rat cortical neurons triggers dispersal of Kv2.1 channel clusters. Sublethal ischemia in neurons induces calcineurin-dependent upregulation of cyclin E1 protein expression and cyclin E1-dependent Kv2.1 channel declustering. Importantly, overexpression of cyclin E1 in neurons is sufficient to reduce excitotoxic cell death. These results support a novel role for neuronal cyclin E1 in regulating the phosphorylation status and localization of Kv2.1 channels, a likely component of signaling cascades leading to ischemic preconditioning.

Funding information:
  • European Research Council - 323183(International)

Voltage-gated membrane currents in neurons involved in odor information processing in snail procerebrum.

  • Pirger Z
  • Brain Struct Funct
  • 2014 Mar 25

Literature context:


Abstract:

The procerebrum (PC) of the snail brain is a critical region for odor discrimination and odor learning. The morphological organization and physiological function of the PC has been intensively investigated in several gastropod species; however, the presence and distribution of ion channels in bursting and non-bursting cells has not yet been described. Therefore, the aim of our study was to identify the different ion channels present in PC neurons. Based on whole cell patch-clamp and immunohistochemical experiments, we show that Na(+)-, Ca(2+)-, and K(+)-dependent voltage-gated channels are differentially localized and expressed in the cells of the PC. Different Na-channel subtypes are present in large (10-15 μm) and small (5-8 μm) diameter neurons, which are thought to correspond to the bursting and non-bursting cells, respectively. Here, we show that the bursting neurons possess fast sodium current (I NaT) and NaV1.9-like channels and the non-bursting neurons possess slow sodium current (I NaT) and NaV1.8-like channels in addition to the L-type Ca(-), KV4.3 (A-type K-channel) and KV2.1 channels. We suggest that the bursting and/or non-bursting character of the PC neurons is at least partly determined by the battery of ion-channels present and their cellular and subcellular compartmentalization.

Funding information:
  • NIGMS NIH HHS - T32 GMO 7413(United States)
  • NINR NIH HHS - NR009270(United States)

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex.

  • Guan D
  • J. Physiol. (Lond.)
  • 2013 Oct 1

Literature context:


Abstract:

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/F005806/1(United Kingdom)

Chemokine co-receptor CCR5/CXCR4-dependent modulation of Kv2.1 channel confers acute neuroprotection to HIV-1 glycoprotein gp120 exposure.

  • Shepherd AJ
  • PLoS ONE
  • 2013 Oct 2

Literature context:


Abstract:

Infection with human immunodeficiency virus-1 (HIV-1) within the brain has long been known to be associated with neurodegeneration and neurocognitive disorder (referred as HAND), a condition characterized in its early stages by declining cognitive function and behavioral disturbances. Mechanistically, the HIV-1 coat glycoprotein 120 (gp120) has been suggested to be a critical factor inducing apoptotic cell death in neurons via the activation of p38 mitogen-activated protein kinase (MAPK), upon chronic exposure to the virus. Here we show that acute exposure of neurons to HIV-1 gp120 elicits a homeostatic response, which provides protection against non-apoptotic cell death, involving the major somatodendritic voltage-gated K⁺ (Kv) channel Kv2.1 as the key mediator. The Kv2.1 channel has recently been shown to provide homeostatic control of neuronal excitability under conditions of seizures, ischemia and neuromodulation/neuroinflammation. Following acute exposure to gp120, cultured rat hippocampal neurons show rapid dephosphorylation of the Kv2.1 protein, which ultimately leads to changes in specific sub-cellular localization and voltage-dependent channel activation properties of Kv2.1. Such modifications in Kv2.1 are dependent on the activation of the chemokine co-receptors CCR5 and CXCR4, and subsequent activation of the protein phosphatase calcineurin. This leads to the overall suppression of neuronal excitability and provides neurons with a homeostatic protective mechanism. Specific blockade of calcineurin and Kv2.1 channel activity led to significant enhancement of non-apoptotic neuronal death upon acute gp120 treatment. These observations shed new light on the intrinsic homeostatic mechanisms of neuronal resilience during the acute stages of neuro-HIV infections.

Funding information:
  • NINDS NIH HHS - NS 37172(United States)

Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents.

  • McCord MC
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2013 Aug 20

Literature context:


Abstract:

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.

Lack of sigma-1 receptor exacerbates ALS progression in mice.

  • Mavlyutov TA
  • Neuroscience
  • 2013 Jun 14

Literature context:


Abstract:

The function of the sigma-1 receptor (S1R) has been implicated in modulating the activity of various ion channels. In the CNS S1R is enriched in cholinergic postsynaptic densities in spinal cord motoneurons (MNs). Mutations in S1R have been found in familial cases of amyotrophic lateral sclerosis (ALS). In this study we show that a knockout of S1R in the SOD1*G93A mouse model of ALS significantly reduces longevity (end stage). Electrophysiological experiments demonstrate that MN of mice lacking S1R exhibit increased excitability. Taken together the data suggest the S1R acts as a brake on excitability, an effect that might enhance longevity in an ALS mouse model.

Funding information:
  • NCRR NIH HHS - U54 RR023506-01(United States)

Regulation of Kv2.1 K(+) conductance by cell surface channel density.

  • Fox PD
  • J. Neurosci.
  • 2013 Jan 16

Literature context:


Abstract:

The Kv2.1 voltage-gated K(+) channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites, and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are nonconducting. Using total internal reflection fluorescence microscopy, the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared with K(+) channel conductance measured by whole-cell voltage clamp of the same cell. This approach indicated that, as channel density increases, nonclustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the nonconducting state with 17% conducting K(+) at higher surface densities. The nonconducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immunofluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared with the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 d, respectively. Together, these data indicate that the nonconducting state depends primarily on surface density as opposed to cluster location and that this nonconducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K(+) conductance further supports a nonconducting role for Kv2.1 in excitable tissues.

Funding information:
  • NIA NIH HHS - AG13154(United States)

Dynamic interaction between sigma-1 receptor and Kv1.2 shapes neuronal and behavioral responses to cocaine.

  • Kourrich S
  • Cell
  • 2013 Jan 17

Literature context:


Abstract:

The sigma-1 receptor (Sig-1R), an endoplasmic reticulum (ER) chaperone protein, is an interorganelle signaling modulator that potentially plays a role in drug-seeking behaviors. However, the brain site of action and underlying cellular mechanisms remain unidentified. We found that cocaine exposure triggers a Sig-1R-dependent upregulation of D-type K(+) current in the nucleus accumbens (NAc) that results in neuronal hypoactivity and thereby enhances behavioral cocaine response. Combining ex vivo and in vitro studies, we demonstrated that this neuroadaptation is caused by a persistent protein-protein association between Sig-1Rs and Kv1.2 channels, a phenomenon that is associated to a redistribution of both proteins from intracellular compartments to the plasma membrane. In conclusion, the dynamic Sig-1R-Kv1.2 complex represents a mechanism that shapes neuronal and behavioral response to cocaine. Functional consequences of Sig-1R binding to K(+) channels may have implications for other chronic diseases where maladaptive intrinsic plasticity and Sig-1Rs are engaged.

Funding information:
  • NIAAA NIH HHS - U01 AA016669(United States)

Distinct modifications in Kv2.1 channel via chemokine receptor CXCR4 regulate neuronal survival-death dynamics.

  • Shepherd AJ
  • J. Neurosci.
  • 2012 Dec 5

Literature context:


Abstract:

The chemokine stromal cell-derived factor-1α (SDF-1α) has multiple effects on neuronal activity, survival, and death under conditions that generate a proinflammatory microenvironment within the brain, via signaling through C-X-C-type chemokine receptor 4 (CXCR4), although the underlying cellular/molecular mechanisms are unclear. Using rat hippocampal neurons, we investigated distinct modifications in the voltage-gated K⁺ (Kv) channel Kv2.1 in response to short- and long-term SDF-1α/CXCR4-mediated signaling as an underlying mechanism for CXCR4-dependent regulation of neuronal survival and death. Acute exposure of neurons to SDF-1α led to dynamic dephosphorylation and altered localization of Kv2.1 channel, resulting in enhanced voltage-dependent activation of Kv2.1-based delayed-rectifier Kv currents (I(DR)). These changes were dependent on CXCR4- and/or NMDA receptor-mediated activation of calcineurin and provide neuroprotection. However, prolonged SDF-1α treatment leads to CXCR4-mediated activation of p38 mitogen-activated protein kinase, resulting in phosphorylation of Kv2.1 at S800 and enhanced surface trafficking of the channel protein, resulting in increased I(DR)/Kv2.1 current density. This, in combination with sustained dephosphorylation-induced enhancement of the voltage-dependent activation of I(DR)/Kv2.1, predisposed neurons to excessive K⁺ efflux, a vital step for the neuronal apoptotic program. Such apoptotic death was dependent on CXCR4 and Kv2.1 function and was absent in cells expressing the Kv2.1-S800A mutant channel. Furthermore, similar modifications in Kv2.1 and CXCR4/Kv2.1-dependent apoptosis were observed following treatment of neurons with the human immunodeficiency virus-1 (HIV-1) glycoprotein gp120. Therefore, distinct modifications in Kv2.1 in response to short- and long-term CXCR4-mediated signaling could provide a basis for neuroprotection or apoptosis in neuropathologies, such as neuroinflammation, stroke, brain tumors, and HIV-associated neurodegeneration.

Funding information:
  • NCRR NIH HHS - P41 RR-08605(United States)

Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury.

  • Tsantoulas C
  • J. Neurosci.
  • 2012 Nov 28

Literature context:


Abstract:

Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition.

  • Al-Owais MM
  • J. Biol. Chem.
  • 2012 Jul 13

Literature context:


Abstract:

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

Funding information:
  • Canadian Institutes of Health Research - GMX-191597(Canada)

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

  • Menegola M
  • Epilepsia
  • 2012 Jun 22

Literature context:


Abstract:

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains.

Funding information:
  • NIGMS NIH HHS - P41 GM109824(United States)

Regulation of neuronal proapoptotic potassium currents by the hepatitis C virus nonstructural protein 5A.

  • Norris CA
  • J. Neurosci.
  • 2012 Jun 27

Literature context:


Abstract:

Apoptosis-enabling neuronal potassium efflux is mediated by an enhancement of K+ currents. In cortical neurons, increased currents are triggered by dual phosphorylation of Kv2.1 by Src and p38 at channel residues Y124 and S800. It was recently shown that a K+ current surge is also present in hepatocytes undergoing apoptosis, and that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) could inhibit Kv2.1-mediated currents and block cell death. Here, we show that NS5A1b (from HCV genotype 1b) expression in rat neurons depresses delayed rectifier potassium currents, limits the magnitude of the K+ current surge following exposure to activated microglia, and is neuroprotective. In a non-neuronal recombinant expression system, cells expressing Kv2.1 mutated at residue Y124, but not S800 mutants, are insensitive to NS5A1b-mediated current inhibition. Accordingly, NS5A1b coexpression prevents phosphorylation of wild-type Kv2.1 by Src at Y124, but is unable to inhibit p38 phosphorylation of the channel at S800. The actions of the viral protein are genotype-selective, as NS5A1a does not depress neuronal potassium currents nor inhibit Src phosphorylation of Kv2.1. Our results indicate that NS5A1b limits K+ currents following injury, leading to increased neuronal viability. NS5A1b may thus serve as a model for a new generation of neuroprotective agents.

Funding information:
  • Intramural FDA HHS - FD999999(United States)

Benefits and pitfalls of secondary antibodies: why choosing the right secondary is of primary importance.

  • Manning CF
  • PLoS ONE
  • 2012 Jun 7

Literature context:


Abstract:

Simultaneous labeling of multiple targets in a single sample, or multiplexing, is a powerful approach to directly compare the amount, localization and/or molecular properties of different targets in the same sample. Here we highlight the robust reliability of the simultaneous use of multiple mouse monoclonal antibodies (mAbs) of different immunoglobulin G (IgG) subclasses in a wide variety of multiplexing applications employing anti-mouse IgG subclass-specific secondary antibodies (2°Abs). We also describe the unexpected finding that IgG subclass-specific 2°Abs are superior to general anti-mouse IgG 2 °Abs in every tested application in which mouse mAbs were used. This was due to a detection bias of general anti-mouse IgG-specific 2°Abs against mAbs of the most common mouse IgG subclass, IgG1, and to a lesser extent IgG2b mAbs. Thus, when using any of numerous mouse mAbs available through commercial and non-profit sources, for cleaner and more robust results each mAb should be detected with its respective IgG subclass-specific 2°Ab and not a general anti-mouse IgG-specific 2°Ab.

Funding information:
  • NCRR NIH HHS - P40-RR17072(United States)

Cholesterol enhances neuron susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1.

  • Zhou MH
  • J. Neurochem.
  • 2012 Feb 16

Literature context:


Abstract:

Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death.

Funding information:
  • NIAAA NIH HHS - AA10760(United States)

AMIGO is an auxiliary subunit of the Kv2.1 potassium channel.

  • Peltola MA
  • EMBO Rep.
  • 2011 Dec 1

Literature context:


Abstract:

Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (I(K)) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal I(K) at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.

Funding information:
  • NIH HHS - P40 OD010440(United States)

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability.

  • Ikematsu N
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2011 Nov 1

Literature context:


Abstract:

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

Funding information:
  • NIDDK NIH HHS - U24 DK093000(United States)

Postsynaptic density-95 scaffolding of Shaker-type K⁺ channels in smooth muscle cells regulates the diameter of cerebral arteries.

  • Joseph BK
  • J. Physiol. (Lond.)
  • 2011 Nov 1

Literature context:


Abstract:

Postsynaptic density-95 (PSD95) is a 95 kDa scaffolding molecule in the brain that clusters postsynaptic proteins including ion channels, receptors, enzymes and other signalling partners required for normal cognition. The voltage-gated, Shaker-type K(+) (K(V)1) channel is one key binding partner of PSD95 scaffolds in neurons. However, K(V)1 channels composed of α1.2 and α1.5 pore-forming subunits also are expressed in the vascular smooth muscle cells (cVSMCs) of the cerebral circulation, although the identity of their molecular scaffolds is unknown. Since α1.2 contains a binding motif for PSD95, we explored the possibility that cVSMCs express PSD95 as a scaffold to promote K(V)1 channel expression and cerebral vasodilatation. Cerebral arteries from Sprague-Dawley rats were isolated for analysis of PSD95 and K(V)1 channel proteins. PSD95 was detected in cVSMCs and it co-immunoprecipitated and co-localized with the pore-forming α1.2 subunit of the K(V)1 channel. Antisense-mediated knockdown of PSD95 profoundly reduced K(V)1 channel expression and suppressed K(V)1 current in patch-clamped cVSMCs. Loss of PSD95 also depolarized cVSMCs in pressurized cerebral arteries and induced a strong constriction associated with a loss of functional K(V)1 channels. Our findings provide initial evidence that PSD95 is expressed in cVSMCs, and the K(V)1 channel is one of its important binding partners. PSD95 appears to function as a critical 'dilator' scaffold in cerebral arteries by increasing the number of functional K(V)1 channels at the plasma membrane.

Funding information:
  • NEI NIH HHS - R01 EY015128(United States)

Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons.

  • Guan D
  • J. Neurophysiol.
  • 2011 Jun 17

Literature context:


Abstract:

Potassium channels regulate numerous aspects of neuronal excitability, and several voltage-gated K(+) channel subunits have been identified in pyramidal neurons of rat neocortex. Previous studies have either considered the development of outward current as a whole or divided currents into transient, A-type and persistent, delayed rectifier components but did not differentiate between current components defined by α-subunit type. To facilitate comparisons of studies reporting K(+) currents from animals of different ages and to understand the functional roles of specific current components, we characterized the postnatal development of identified Kv channel-mediated currents in pyramidal neurons from layers II/III from rat somatosensory cortex. Both the persistent/slowly inactivating and transient components of the total K(+) current increased in density with postnatal age. We used specific pharmacological agents to test the relative contributions of putative Kv1- and Kv2-mediated currents (100 nM α-dendrotoxin and 600 nM stromatoxin, respectively). A combination of voltage protocol, pharmacology, and curve fitting was used to isolate the rapidly inactivating A-type current. We found that the density of all identified current components increased with postnatal age, approaching a plateau at 3-5 wk. We found no significant changes in the relative proportions or kinetics of any component between postnatal weeks 1 and 5, except that the activation time constant for A-type current was longer at 1 wk. The putative Kv2-mediated component was the largest at all ages. Immunocytochemistry indicated that protein expression for Kv4.2, Kv4.3, Kv1.4, and Kv2.1 increased between 1 wk and 4-5 wk of age.

Funding information:
  • Cancer Research UK - C309/A8274(United Kingdom)

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1.

  • Dallas ML
  • FASEB J.
  • 2011 May 2

Literature context:


Abstract:

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.

Funding information:
  • NIGMS NIH HHS - GM68958(United States)

Contribution of the delayed-rectifier potassium channel Kv2.1 to acute spinal cord injury in rats.

  • Song MY
  • BMB Rep
  • 2010 Nov 29

Literature context:


Abstract:

Recent studies have reported that delayed-rectifier Kv channels regulate apoptosis in the nervous system. Herein, we investigated changes in the expression of the delayed-rectifier Kv channels Kv1.2, Kv2.1, and Kv3.1 after acute spinal cord injury (SCI) in rats. We performed RT-PCR analysis and found an increase in the level of Kv2.1 mRNA after SCI but no significant changes in the levels of Kv1.2 and Kv3.1 mRNA. Western blot analysis revealed that Kv2.1 protein levels rapidly decreased and then dramatically increased from 1 day, whereas Kv3.1b protein levels gradually and sharply decreased at 5 days. Kv1.2 protein levels did not change significantly. In addition, Kv2.1 clusters were disrupted in the plasma membranes of motor neurons after SCI. Interestingly, the expressional changes and translocation of Kv2.1 were consistent with the apoptotic changes on day 1. Therefore, these results suggest that Kv2.1 channels probably contribute to neuronal cell responses to SCI.

Funding information:
  • Medical Research Council - G9900837(United Kingdom)
  • NCRR NIH HHS - R01 RR07861(United States)

The sigma-1 receptor is enriched in postsynaptic sites of C-terminals in mouse motoneurons. An anatomical and behavioral study.

  • Mavlyutov TA
  • Neuroscience
  • 2010 May 5

Literature context:


Abstract:

The sigma-1 receptor regulates various ion channel activity and possesses protein chaperone function. Using an antibody against the full sequence of the sigma-1 receptor we detected immunostaining in wild type but not in knockout mice. The receptor was found primarily in motoneurons localized to the brainstem and spinal cord. At the subcellular level the receptor is restricted to large cholinergic postsynaptic densities on the soma of motoneurons and is colocalized with the Kv2.1 potassium channel and the muscarinic type 2 cholinergic receptor. Ultrastructural analysis of the neurons indicates that the immunostained receptor is located close but separate from the plasma membrane, possibly in subsurface cisternae formed from the endoplasmic reticulum (ER), which are a prominent feature of cholinergic postsynaptic densities. Behavioral testing on a rotorod revealed that Sigma-1 receptor knockout mice remained on the rotorod for significantly less time (a shorter latency period) compared to the wild type mice. Together these data indicate that the sigma-1 receptor may play a role in the regulation of motor behavior.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence.

  • Mankouri J
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2009 Sep 15

Literature context:


Abstract:

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

Ethanol disrupts NMDA receptor and astroglial EAAT2 modulation of Kv2.1 potassium channels in hippocampus.

  • Mulholland PJ
  • Alcohol
  • 2009 Feb 2

Literature context:


Abstract:

Delayed-rectifier Kv2.1 channels are the principal component of voltage-sensitive K+ currents (I(K)) in hippocampal neurons and are critical regulators of somatodendritic excitability. In a recent study, we demonstrated that surface trafficking and phosphorylation of Kv2.1 channels is modulated by NMDA-type glutamate receptors and that astroglial excitatory amino acid transporters 2 (EAAT2) regulate the coupling of NMDA receptors and Kv2.1 channels. Because ethanol is known to acutely inhibit NMDA receptors, we sought to determine if NMDA receptor and astroglial EAAT2 modulation of Kv2.1 channels is impaired by ethanol in the rodent hippocampus. As expected, bath application of NMDA to hippocampal cultures reduced the size of Kv2.1 clusters and produced a hyperpolarizing shift in the voltage-dependent activation of I(K) that was associated with dephosphorylated Kv2.1 channels. Ethanol, applied acutely, prevented the hyperpolarizing shift in activation of I(K) induced by NMDA and restored Kv2.1 clustering and phosphorylation to near control levels. Ethanol also attenuated the dephosphorylation of Kv2.1 channels produced by the EAAT2 selective inhibitor dihydrokainic acid. These data demonstrate that acute ethanol disrupts changes in Kv2.1 channels that follow NMDA receptor activation and impairs astroglial regulation of the functional coupling between NMDA receptors and Kv2.1 channels.

Interdomain cytoplasmic interactions govern the intracellular trafficking, gating, and modulation of the Kv2.1 channel.

  • Mohapatra DP
  • J. Neurosci.
  • 2008 May 7

Literature context:


Abstract:

Voltage-gated potassium (Kv) channels comprise four transmembrane alpha subunits, often associated with cytoplasmic beta subunits that impact channel expression and function. Here, we show that cell surface expression, voltage-dependent activation gating, and phosphorylation-dependent modulation of Kv2.1 are regulated by cytoplasmic N/C interaction within the alpha subunit. Kv2.1 surface expression is greatly reduced by C-terminal truncation. Tailless Kv2.1 channels exhibit altered voltage-dependent gating properties and lack the bulk of the phosphorylation-dependent modulation of channel gating. Remarkably, the soluble C terminus of Kv2.1 associates with tailless channels and rescues their expression, function, and phosphorylation-dependent modulation. Soluble N and C termini of Kv2.1 can also interact directly. We also show that the N/C-terminal interaction in Kv2.1 is governed by a 34 aa motif in the juxtamembrane cytoplasmic C terminus, and a 17 aa motif located in the N terminus at a position equivalent to the beta subunit binding site in other Kv channels. Deletion of either motif disrupts N/C-terminal interaction and surface expression, function, and phosphorylation-dependent modulation of Kv2.1 channels. These findings provide novel insights into intrinsic mechanisms for the regulation of Kv2.1 trafficking, gating, and phosphorylation-dependent modulation through cytoplasmic N/C-terminal interaction, which resembles alpha/beta subunit interaction in other Kv channels.

Bidirectional activity-dependent regulation of neuronal ion channel phosphorylation.

  • Misonou H
  • J. Neurosci.
  • 2006 Dec 27

Literature context:


Abstract:

Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.

Funding information:
  • NCI NIH HHS - 1U19 CA148065(United States)