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2.  Antibody-guided photoablation of voltage-gated potassium currents 
The Journal of General Physiology  2013;142(3):315-324.
A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb–porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody–porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.
PMCID: PMC3753605  PMID: 23940262
3.  Trafficking Mechanisms Underlying Neuronal Voltage-gated Ion Channel Localization at the Axon Initial Segment 
Epilepsia  2012;53(Suppl 9):21-31.
Voltage-gated ion channels are diverse and fundamental determinants of neuronal intrinsic excitability. Voltage-gated K+ (Kv) and Na+ (Nav) channels play complex yet fundamentally important roles in determining intrinsic excitability. The Kv and Nav channels located at the axon initial segment (AIS) play a unique and especially important role in generating neuronal output in the form of anterograde axonal and backpropagating action potentials, Aberrant intrinsic excitability in individual neurons within networks contributes to synchronous neuronal activity leading to seizures. Mutations in ion channel genes gives rise to a variety of seizure-related “Channelopathies”, and many of the ion channel subunits associated with epilepsy mutations are localized at the AIS, making this a hotspot for epileptogenesis. Here we review the cellular mechanisms that underlie the trafficking of Kv and Nav channels found at the AIS, and how Kv and Nav channel mutations associated with epilepsy can alter these processes.
PMCID: PMC3531813  PMID: 23216576
Potassium; channel-Sodium; channel- Neuron-Subcellular; localization-Seizures
4.  The Importance of Immunohistochemical Analyses in Evaluating the Phenotype of Kv Channel Knockout Mice 
Epilepsia  2012;53(Suppl 1):142-149.
To gain insights into the phenotype of Kv1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze 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. While 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 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.
PMCID: PMC3369274  PMID: 22612819
Potassium; channel-Hippocampus-Gene; Expression-Subcellular; localization-Antbodies-Seizures
5.  Interdomain Cytoplasmic Interactions Govern the Intracellular Trafficking, Gating, and Modulation of the Kv2.1 Channel 
Voltage-gated potassium (Kv) channels comprise four transmembrane α subunits, often associated with cytoplasmic β 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 α 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 β 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 α/β subunit interaction in other Kv channels.
PMCID: PMC3409667  PMID: 18463252
hippocampal neurons; potassium channel; assembly; immunocytochemistry; electrophysiology; phosphorylation
6.  Diverse Roles for Auxiliary Subunits in Phosphorylation-Dependent Regulation of Mammalian Brain Voltage-Gated Potassium Channels 
Pflugers Archiv  2011;462(5):631-643.
Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse, in part due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons, and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself, or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.
PMCID: PMC3371647  PMID: 21822597
7.  Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels 
Neuroscience Letters  2010;486(2):60-67.
Phosphorylation is the most common and abundant posttranslational modification to eukaryotic proteins, regulating a plethora of dynamic cellular processes. Here, we review and discuss recent advances in our knowledge of the breadth and importance of reversible phosphorylation in regulating the expression, localization and function of mammalian neuronal voltage-gated potassium (Kv) channels, key regulators of neuronal function. We highlight the role of modern mass spectrometric techniques and phosphospecific antibodies that reveal the extent and nature of phosphorylation at specific sites in Kv channels. We also emphasize the role of reversible phosphorylation in dynamically regulating diverse aspects of Kv channel biology. Finally, we discuss as important future directions the determination of the mechanistic basis for how altering phosphorylation state affects Kv channel expression, localization and function, the nature of macromolecular signaling complexes containing Kv channels and enzymes regulating their phosphorylation state, and the specific role of Kv channel phosphorylation in regulating neuronal function during physiological and pathophysiological events.
PMCID: PMC3365921  PMID: 20600597
ion channel; phosphorylation; expression; localization; function
8.  Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance 
PLoS ONE  2012;7(6):e38313.
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.
PMCID: PMC3365890  PMID: 22675541
9.  Mass spectrometry-based phosphoproteomics reveals multisite phosphorylation on mammalian brain voltage-gated sodium and potassium channels 
Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.
PMCID: PMC3043121  PMID: 20932926
Mass spectrometry; Phosphoproteomics; VGSC (Nav channel); VGKC (Kv channel); Brain ion channel
11.  Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting 
The Journal of Cell Biology  2011;192(5):813-824.
Phosphorylation of Kvβ2 releases Kv1 channels from microtubules to control their specific distribution at the axonal membrane.
Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)–mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end–tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.
PMCID: PMC3051814  PMID: 21357749
12.  Mining recent brain proteomic databases for ion channel phosphosite nuggets 
Voltage-gated ion channels underlie electrical activity of neurons and are dynamically regulated by diverse cell signaling pathways that alter their phosphorylation state. Recent global mass spectrometric–based analyses of the mouse brain phosphoproteome have yielded a treasure trove of new data as to the extent and nature of phosphorylation of numerous ion channel principal or α subunits in mammalian brain. Here we compile and review data on 347 phosphorylation sites (261 unique) on 42 different voltage-gated ion channel α subunits that were identified in these recent studies. Researchers in the ion channel field can now begin to explore the role of these novel in vivo phosphorylation sites in the dynamic regulation of the localization, activity, and expression of brain ion channels through multisite phosphorylation of their principal subunits.
PMCID: PMC3010060  PMID: 21149544
13.  Multi-site Phosphorylation of Voltage-Gated Sodium Channel α Subunits from Rat Brain 
Journal of proteome research  2010;9(4):1976-1984.
Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav α subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and important membrane proteins in mammalian brain.
PMCID: PMC2849892  PMID: 20131913
voltage-gated sodium channels; brain; phosphorylation; tandem mass spectrometry; immunopurification; monoclonal antibody; nanoflow liquid chromatography
14.  SynDIG1: an activity-regulated AMPA receptor-interacting transmembrane protein that regulates excitatory synapse development 
Neuron  2010;65(1):80-93.
During development of the central nervous system, precise synaptic connections between pre- and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extra-synaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C-terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.
PMCID: PMC2822728  PMID: 20152115
15.  A novel cyclic nucleotide-gated ion channel enriched in synaptic terminals of isotocin neurons in zebrafish brain and pituitary 
Neuroscience  2009;165(1):79-89.
Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the central nervous system, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca2+, which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocin-secreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects.
PMCID: PMC2789987  PMID: 19778592
oxytocin; cyclic nucleotides; calcium entry; neuroendocrine cells; synaptic plasticity; neurohypophysis
16.  Convergent Modulation of Kv4.2 Channel α Subunits by Structurally Distinct DPPX and KChIP Auxiliary Subunits† 
Biochemistry  2009;48(24):5721-5730.
Kv4.2 is the major voltage-gated K+ (Kv) channel α subunit responsible for the somatodendritic transient or A-type current ISA that activates at subthreshold membrane potentials. Stable association of Kv4.2 with diverse auxiliary subunits and reversible Kv4.2 phosphorylation regulate ISA function. Two classes of auxiliary subunits play distinct roles in modulating the biophysical properties of Kv4.2: dipeptidyl-peptidase-like Type II transmembrane proteins typified by DPPX-S, and cytoplasmic Ca2+ binding proteins known as K+ channel interacting proteins (KChIPs). Here, we characterize the convergent roles that DPPX-S and KChIPs play as component subunits of Kv4.2 channel complexes. We co-expressed DPPX-S with Kv4.2 in heterologous cells and found a dramatic redistribution of Kv4.2, releasing it from intracellular retention and allowing plasma membrane expression, as well as altered Kv4.2 phosphorylation, detergent solubility, and stability. These changes are remarkably similar to those obtained upon co-expression of Kv4.2 with the structurally distinct KChIPs1–3 auxiliary subunits. KChIP4a, which negatively affects the impact of other KChIPs on Kv4.2, also inhibits the effects of DPPXS, consistent with the formation of a ternary complex of Kv4.2, DPPX-S and KChIPs early in channel biosynthesis. Tandem MS analyses reveal that co-expression with DPPX-S or KChIP2 leads to a pattern of Kv4.2 phosphorylation in heterologous cells similar to that observed in brain, but lacking in cells expressing Kv4.2 alone. In conclusion, transmembrane DPPX-S and cytoplasmic KChIPs exert similar effects on Kv4.2 trafficking, despite having distinct structures and binding sites on Kv4.2, but distinct effects on Kv4.2 gating.
PMCID: PMC2733230  PMID: 19441798
17.  Altered Expression and Localization of Hippocampal A-Type Potassium Channel Subunits in the Pilocarpine-Induced Model of Temporal Lobe Epilepsy 
Neuroscience  2008;156(3):550-562.
Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of two-three weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.
PMCID: PMC2811367  PMID: 18727953
epilepsy; IA; dentate gyrus; ion channels; CA1; immunohistochemistry
Clustered Kv1 K+ channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments (AIS), and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 α subunits, then used mass-spectrometry to identify interacting proteins. We found that ADAM22 (A Disintegrin And Metalloproteinase 22) is a component of the Kv1 channel complex, and that ADAM22 co-immunoprecipitates Kv1.2 and the MAGUKs PSD-93 and PSD-95. When co-expressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, co-expression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K+ channel complex that recruits MAGUKs to juxtaparanodes.
PMCID: PMC2811262  PMID: 20089912
action potential; juxtaparanode; cell adhesion molecule; MAGUK; K+ channel
19.  Structural Consequences of Kcna1 Gene Deletion and Transfer in the Mouse Hippocampus 
Epilepsia  2007;48(11):2023-2046.
Mice lacking the Kv1.1 potassium channel α subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1−/− (“knock-out”) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons.
Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1−/− animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1−/− brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E.coli lacZ reporter gene. Vector-injected mice were were examined to determine the extent of neuronal infection.
Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1−/− mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed “ectopic” Kv1.1 α channel subunit expression.
Kcna1 deletion in mice results in a seizure disorder that resembles – electrographically and neuropathologically – the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection
PMCID: PMC2752664  PMID: 17651419
Epilepsy; Gene therapy; Hippocampal pathology; Knock-out; Potassium channel; Seizures
20.  Regulation of intrinsic excitability in hippocampal neurons by activity-dependent modulation of the Kv2.1 potassium channel 
Channels (Austin, Tex.)  2009;3(1):46-56.
Kv2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (Kv) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of Kv2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the Kv2.1-component of delayed rectifier K+ (IK) currents. In computer models of hippocampal neurons, these glutamate-stimulated shifts in the gating of the Kv2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate-stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of Kv1.1-containing channels. These studies together demonstrate a specific contribution of modulation of Kv2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.
PMCID: PMC2743964  PMID: 19276663
Voltage-gated potassium channel; hippocampal neuron; calcineurin; phosphorylation; neuronal excitability; hanatoxin; homeostatic plasticity
21.  Regulation of Kv1 channel trafficking by the mamba snake neurotoxin dendrotoxin K 
Modulation of voltage-gated potassium (Kv) channel surface expression can profoundly affect neuronal excitability. Some but not all mammalian Shaker or Kv1 α subunits contain a dominant endoplasmic reticulum (ER) retention signal in their pore region, preventing surface expression of Kv1.1 homotetrameric channels, and of heteromeric Kv1 channels containing more than one Kv1.1 subunit. The critical amino acid residues within this ER pore-region retention signal are also critical for high-affinity binding of snake dendrotoxins (DTX). This suggests that ER retention may be mediated by an ER protein with a domain structurally similar to that of DTX. One facet of such a model is that expression of soluble DTX in the ER lumen should compete for binding to the retention protein and allow for surface expression of retained Kv1.1. Here we show that luminal DTX expression dramatically increased both the level of cell surface Kv1.1 immunofluorescence staining and the proportion of Kv1.1 with processed N-linked oligosaccharides. Electrophysiological analyses showed that luminal DTX expression led to significant increases in Kv1.1 currents. Together these data showed that luminal DTX expression increases surface expression of functional Kv1.1 homotetrameric channels, and support a model whereby a DTX-like ER protein regulates abundance of cell surface Kv1 channels.
PMCID: PMC2737685  PMID: 17185748
potassium channel; neuron; endoplasmic reticulum
22.  Complete 3-D visualization of primate striosomes by KChIP1 immunostaining 
High resolution 3D reconstruction and morphometric analysis of striosomes was carried out in macaque monkeys using immunocytochemistry for the Kv4 potassium channel subunit, KChIP1, a novel marker. The striosomes form a connected reticulum made up of two distinct planar sheets spanning several millimeters in the putamen, and long finger-like branches in the caudate nucleus and putamen. Although their spatial organization is variable, morphometric analysis of the striosomes, utilizing skeletonizations, reveals several quantitative invariant measures of striosome organization, including: 1) individual bifurcation-free striosome branches are 355±108.5 microns in diameter and 1013±751 microns in length, and are both lognormally distributed, and 2) striosome branches exhibit three pronounced orientation preferences that are approximately orthogonal. The former finding suggests a fundamental anatomical and functional component of the striatum, whereas the latter indicates that striosomes are more lattice-like than their spatial variability suggests. The perceived variable spatial organization of the striosomes in primates belies many invariant features that may reflect striatal function, development, and pathophysiology.
PMCID: PMC2737266  PMID: 19350670
potassium channel subunit; putamen; caudate nucleus; basal ganglia; matrix
PSD-93/Chapsyn-110 is a PDZ domain-containing membrane-associated guanylate kinase (MAGUK) that functions as a scaffold to assemble channels, receptors, and other signaling proteins at cell membranes. PSD-93 is highly enriched at synapses, but mice lacking this protein have no synaptic structural abnormalities, probably due to overlapping expression and redundancy with other MAGUKs. Consequently, the function of PSD-93 is not well understood. Here we show that PSD-93, but not other MAGUKs, is enriched at the axon initial segment (AIS) where it colocalizes with Kv1.1, Kv1.2, Kv1.4, and Kvβ2 subunit-containing K+ channels, Caspr2, and TAG-1. When co-expressed with Kv1 channels in heterologous cells, PSD-93 induces formation of large cell-surface clusters. Knockdown of PSD-93 in cultured hippocampal neurons by RNA interference disrupted Kv1 channel localization at the AIS. Similarly, PSD-93 −/− mice failed to cluster Kv1 channels at the AIS of cortical and hippocampal neurons. In contrast, Caspr2, which mediates Kv1 channel clustering at the juxtaparanode is not required for localization of Kv1 channels at the AIS. These results show PSD-93 mediates AIS accumulation of Kv1 channels independently of Caspr2.
PMCID: PMC2729199  PMID: 18509034
action potential; juxtaparanode; scaffold; ankyrin; cell adhesion molecule; MAGUK
24.  Immunolocalization of the Ca2+-Activated K+ Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons 
Ca2+-activated voltage-dependent K+ channels (Slo1, KCa1.1, Maxi-K, or BK channel) play a crucial role in controlling neuronal signaling by coupling channel activity to both membrane depolarization and intracellular Ca2+ signaling. In mammalian brain, immunolabeling experiments have shown staining for Slo1 channels predominantly localized to axons and presynaptic terminals of neurons. We have developed anti-Slo1 mouse monoclonal antibodies that have been extensively characterized for specificity of staining against recombinant Slo1 in heterologous cells, and native Slo1 in mammalian brain, and definitively by the lack of detectable immunoreactivity against brain samples from Slo1 knockout mice. Here we provide precise immunolocalization of Slo1 in rat brain with one of these monoclonal antibodies and show that Slo1 is accumulated in axons and synaptic terminal zones associated with glutamatergic synapses in hippocampus and GABAergic synapses in cerebellum. By using cultured hippocampal pyramidal neurons as a model system, we show that heterologously expressed Slo1 is initially targeted to the axonal surface membrane, and with further development in culture, become localized in presynaptic terminals. These studies provide new insights into the polarized localization of Slo1 channels in mammalian central neurons and provide further evidence for a key role in regulating neurotransmitter release in glutamatergic and GABAergic terminals.
PMCID: PMC2605666  PMID: 16566008
BK channel; ion channel; localization; protein trafficking; mAb
25.  Localization and Targeting of Voltage-Gated Ion Channels in Mammalian Central Neurons 
Physiological reviews  2008;88(4):1407-1447.
The intrinsic electrical properties and the synaptic input-output relationships of neurons are governed by the action of voltage-dependent ion channels. The localization of specific population of ion channels with distinct functional properties at discrete sites in neurons dramatically impacts excitability and synaptic transmission. Molecular cloning studies have revealed a large family of genes encoding voltage-dependent ion channel principal and auxiliary subunits, most of which are expressed in mammalian central neurons. Much recent effort has focused on determining which of these subunits co-assemble into native neuronal channel complexes, and the cellular and subcellular distributions of these complexes, as a crucial step in understanding the contribution of these channels to specific aspects of neuronal function. Here we review progress made on recent studies aimed at determining the cellular and subcellular distribution of specific ion channel subunits in mammalian brain neurons using in situ hybridization, and immunohistochemistry. We also discuss the repertoire of ion channel subunits in specific neuronal compartments and implications for neuronal physiology. Finally, we discuss the emerging mechanisms for determining the discrete subcellular distributions observed for many neuronal ion channels.
PMCID: PMC2587220  PMID: 18923186

Results 1-25 (31)