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Peter Sonderegger - One of the best experts on this subject based on the ideXlab platform.
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Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the Axonal Cell adhesion molecule axonin-1.
Journal of Neuroscience Methods, 1998Co-Authors: Roman J. Giger, Urs Ziegler, Wim T.j.m.c. Hermens, Beat Kunz, Stephan Kunz, Peter SondereggerAbstract:By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the Axonal Cell adhesion molecule axonin-1 in neurons and glial Cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial Cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the Cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.
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Expression of the Axonal Cell adhesion molecules axonin-1 and Ng-CAM during the development of the chick retinotectal system.
The Journal of Comparative Neurology, 1996Co-Authors: Günter Rager, Patrizia Morino, Jutta Schnitzer, Peter SondereggerAbstract:Cell surface glycoproteins expressed on growth cones and axons during brain development have been postulated to be involved in the Cell-Cell interactions that guide axons into their target area. Nevertheless, an unequivocal description of the mechanism by which such molecules exert control over the pathway of a growing axon has not been done. As a crucial requirement in support of a relevant involvement of an Axonal surface molecule in growth cone guidance, this molecule should be expressed in the growth cone. The developing retinotectal system provides an exCellent opportunity to test whether a particular neuronal surface molecule fulfills the requirement of the spatiotemporal coincidence between its appearance and the emergence of growth cones because its setup follows the rule of chronotopy, i.e., the position of axons in a certain site is determined by the time of their arrival. We have analyzed axonin-1 and the neuron-glia Cell adhesion molecule (Ng-CAM), two Axonal surface molecules that promote neurite growth in vitro, for their expression in the retina and in the retinotectal system of the chick throughout its development. At stage 18, both axonin-like (A-LI) and Ng-CAM-like immunoreactivity (Ng-CAM-LI) are clearly present in the area where first retinal ganglion Cells (RGCs) are generated. The immunoreactivity spreads synchronously with the formation of RGCs over the developing retina. From stage 32 on, the inner plexiform layer is also stained according to its temporospatial gradient of maturation. In later stages, the outer plexiform layer and the inner segments of photoreceptors also show immunoreactivity. The development of A-LI and Ng-CAM-LI along the optic nerve, chiasm, optic tract, and in the superficial layers of the optic tectum follows the chronotopic pattern of axons, as was found by earlier morphological investigations. Older axons loose their A-LI. This allows to localize the position of newly formed axons. The fact that A-LI and Ng-CAM-LI parallel the formation and maturation of axons suggests that axonin-1 and Ng-CAM may play an important role in the organization of the retinotectal system.
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The Gene of Chicken Axonin-1
FEBS Journal, 1995Co-Authors: Roman J. Giger, Lorenz Vogt, Richard A. Zuellig, Christoph Rader, Anne Henehan-beatty, David P. Wolfer, Peter SondereggerAbstract:We have isolated and characterised the gene encoding the chicken Axonal Cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exonintron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa Cells, rat PC-12 phaeochromocytoma Cells, and chicken embryonic fibroblasts with serially truncated segments of the 5′-flanking region linked to a luciferase reporter gene, we have found that the sequence from −91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.
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The gene of chicken axonin-1. Complete structure and analysis of the promoter.
FEBS Journal, 1995Co-Authors: Roman J. Giger, Lorenz Vogt, Richard A. Zuellig, Christoph Rader, Anne Henehan-beatty, David P. Wolfer, Peter SondereggerAbstract:: We have isolated and characterised the gene encoding the chicken Axonal Cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exon/intron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa Cells, rat PC-12 phaeochromocytoma Cells, and chicken embryonic fibroblasts with serially truncated segments of the 5'-flanking region linked to a luciferase reporter gene, we have found that the sequence from -91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.
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Distribution of TAG-1/axonin-1 in fibre tracts and migratory streams of the developing mouse nervous system.
The Journal of Comparative Neurology, 1994Co-Authors: David P. Wolfer, Peter Sonderegger, Anne Henehan-beatty, Esther T. Stoeckli, Hans-peter LippAbstract:The Axonal Cell adhesion molecule, TAG-1/axonin-1, stimulates Axonal growth and supports neurite fasciculation in vitro. Using a polyclonal antiserum raised against chick axonin-1, which shares 75% of its sequence with TAG-1 of the rat, we have mapped the distribution of TAG-1/axonin-1 throughout the developing nervous system of the mouse. Although absent from proliferating neuroepithelia and from non-neuronal Cells, immunoreactivity for TAG-1/axonin-1 is expressed by stage-specific subpopulations of differentiating neurons from embryonic day 10 to postnatal day 15. It stains their axons and the surface of their parent somata during the early phases of axogenesis. In agreement with a putative role of TAG-1/axonin-1 as an axon-bound growth substrate, immunoreactivity is found in developing spinal and cranial nerves, in corticothalamic projections, as well as in subsets of fasciculating long projecting tracts of the central nervous system, such as the dorsal funiculi of the spinal cord, the lateral olfactory and optic tracts, the fasciculus retroflexus, and the predorsal bundle. High levels of immunoreactivity characterise the development of the cerebellar molecular layer, the corpus callosum, anterior and hippocampal commissure, and of crossed projections in the spinal cord and at several levels of the brainstem. Intense immunoreactivity in fine collaterals of cutaneous afferents, including their growth cones that are in contact with the embryonic skin, suggests a role of TAG-1/axonin-1 in target recognition. While staining is weak on the somata of radially migrating neurons such as cortical neurons and cerebellar granule Cells, strong immunoreactivity is associated with neural somata and processes of the three tangential migrations that form the precerebellar nuclei, indicating a possible involvement of TAG-1/axonin-1 in contacts between these neurons and the processes they migrate upon. © 1994 Wiley-Liss, Inc.
Robert C. Foehring - One of the best experts on this subject based on the ideXlab platform.
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Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones
The Journal of Physiology, 2006Co-Authors: Dongxu Guan, Tatiana Tkatch, D.j. Surmeier, William E. Armstrong, Robert C. FoehringAbstract:Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-Cell RT-PCR, immunocytochemistry, and whole Cell recordings with specific peptide toxins revealed that individual pyramidal Cells express multiple Kv1 α-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as Axonal Cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these Cells. The α-dendrotoxin (α-DTX)-sensitive current activated more rapidly and at more negative potentials than the α-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The α-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from −70 mV. From −50 mV, this percentage increased to ∼20%. All Cells expressed an α-DTX-sensitive current with slow inactivation kinetics. In some Cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the α-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.
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Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones.
The Journal of physiology, 2005Co-Authors: Dongxu Guan, Tatiana Tkatch, D.j. Surmeier, William E. Armstrong, Robert C. FoehringAbstract:Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-Cell RT-PCR, immunocytochemistry, and whole Cell recordings with specific peptide toxins revealed that individual pyramidal Cells express multiple Kv1 alpha-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as Axonal Cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these Cells. The alpha-dendrotoxin (alpha-DTX)-sensitive current activated more rapidly and at more negative potentials than the alpha-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The alpha-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from -70 mV. From -50 mV, this percentage increased to approximately 20%. All Cells expressed an alpha-DTX-sensitive current with slow inactivation kinetics. In some Cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the alpha-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.
Roman J. Giger - One of the best experts on this subject based on the ideXlab platform.
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Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the Axonal Cell adhesion molecule axonin-1.
Journal of Neuroscience Methods, 1998Co-Authors: Roman J. Giger, Urs Ziegler, Wim T.j.m.c. Hermens, Beat Kunz, Stephan Kunz, Peter SondereggerAbstract:By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the Axonal Cell adhesion molecule axonin-1 in neurons and glial Cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial Cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the Cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.
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The Gene of Chicken Axonin-1
FEBS Journal, 1995Co-Authors: Roman J. Giger, Lorenz Vogt, Richard A. Zuellig, Christoph Rader, Anne Henehan-beatty, David P. Wolfer, Peter SondereggerAbstract:We have isolated and characterised the gene encoding the chicken Axonal Cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exonintron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa Cells, rat PC-12 phaeochromocytoma Cells, and chicken embryonic fibroblasts with serially truncated segments of the 5′-flanking region linked to a luciferase reporter gene, we have found that the sequence from −91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.
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The gene of chicken axonin-1. Complete structure and analysis of the promoter.
FEBS Journal, 1995Co-Authors: Roman J. Giger, Lorenz Vogt, Richard A. Zuellig, Christoph Rader, Anne Henehan-beatty, David P. Wolfer, Peter SondereggerAbstract:: We have isolated and characterised the gene encoding the chicken Axonal Cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exon/intron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa Cells, rat PC-12 phaeochromocytoma Cells, and chicken embryonic fibroblasts with serially truncated segments of the 5'-flanking region linked to a luciferase reporter gene, we have found that the sequence from -91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.
Bettina Winckler - One of the best experts on this subject based on the ideXlab platform.
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Maturational conversion of dendritic early endosomes and their roles in L1-mediated axon growth.
The Journal of Neuroscience, 2014Co-Authors: Zofia M. Lasiecka, Joshua Katz, Bettina WincklerAbstract:The function of endosomes is intricately linked to Cellular function in all Cell types, including neurons. Intriguingly, neurons express Cell type-specific proteins that localize to endosomes, but little is known about how these neuronal proteins interface with canonical endosomes and ubiquitously expressed endosomal components, such as EEA1 (Early Endosomal Antigen 1). NEEP21 (Neuronal Early Endosomal Protein 21 kDa) localizes to somatodendritic endosomes, and downregulation of NEEP21 perturbs the correct trafficking of multiple receptors, including glutamate receptors (GluA2) during LTP and amyloidogenic processing of βAPP. Our own work implicated NEEP21 in correct trafficking of the Axonal Cell adhesion molecule L1/neuron–glia Cell adhesion molecule (NgCAM). NEEP21 dynamically localizes with EEA1-positive early endosomes but is also found in EEA1-negative endosomes. Live imaging reveals that NEEP21-positive, EEA1-negative endosomes arise as a consequence of maturational conversion of EEA1/NEEP21 double-positive endosomes. Interfering with EEA1 function causes missorting of L1/NgCAM, axon outgrowth defects on the L1 substrate, and disturbance of NEEP21 localization. Last, we uncover evidence that functional interference with NEEP21 reduces axon and dendrite growth of primary rat hippocampal neurons on L1 substrate but not on N-cadherin substrate, thus implicating endosomal trafficking through somatodendritic early endosomes in L1-mediated axon growth.
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Pathway selection to the axon depends on multiple targeting signals in NgCAM
Journal of Cell Science, 2008Co-Authors: Rita L. Nokes, Dolora Wisco, Eric Anderson, Heike Fölsch, Bettina WincklerAbstract:Similar to most differentiated Cells, both neurons and epithelial Cells elaborate distinct plasma membrane domains that contain different membrane proteins. We have previously shown that the Axonal Cell-adhesion molecule L1/NgCAM accumulates on the Axonal surface by an indirect transcytotic pathway via somatodendritic endosomes. MDCK epithelial Cells similarly traffic NgCAM to the apical surface by transcytosis. In this study, we map the signals in NgCAM required for routing via the multi-step transcytotic pathway. We identify both a previously mapped tyrosine-based signal as a sufficient somatodendritic targeting signal, as well as a novel Axonal targeting signal in the cytoplasmic tail of NgCAM. The Axonal signal is glycine and serine rich, but only the glycine residues are required for activity. The somatodendritic signal is cis-dominant and needs to be inactivated in order for the Axonal signal to be executed. Additionally, we show that the Axonal cytoplasmic signal promotes apical targeting in MDCK Cells. Transcytosis of NgCAM to the axon thus requires the sequential regulated execution of multiple targeting signals.
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Inhibition of sphingolipid synthesis affects kinetics but not fidelity of L1/NgCAM transport along direct but not transcytotic Axonal pathways.
Molecular and Cellular Neuroscience, 2005Co-Authors: Michael C. Chang, Dolora Wisco, Helge Ewers, Caren Norden, Bettina WincklerAbstract:Abstract Glycosphingolipids are constituents of lipid rafts which might function in sorting apical and Axonal cargoes in the trans-Golgi network. In fact, two GPI-linked proteins, Thy1 and PrP C , require lipid raft lipids for sorting to the axon. It was previously shown that inhibition of glycosphingolipid synthesis by FumonisinB1 (FB1) impairs axon outgrowth but not axon specification, leading to the hypothesis that formation of Axonally-targeted vesicles is coupled to sphingolipid synthesis. Since the Axonal Cell adhesion molecule L1/NgCAM can partition into membrane rafts biochemically, we asked whether correct targeting to the axon is FB1-sensitive, similarly to GPI-linked proteins. We previously showed that cultured hippocampal neurons use more than one trafficking pathway to the axon: a transcytotic pathway and a direct pathway. We show here that reducing raft lipid levels does not disrupt Axonal targeting of L1/NgCAM along either pathway. Unexpectedly, FB1 selectively slowed the kinetics of surface expression of a truncated NgCAM using the direct pathway, but not of NgCAM using the transcytotic pathway. Therefore, the formation and/or transport of a subset of Axonally-targeted vesicles are coupled to sphingolipid synthesis. Our results yield a mechanism for the axon outgrowth defect observed in FB1.
Dongxu Guan - One of the best experts on this subject based on the ideXlab platform.
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Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones
The Journal of Physiology, 2006Co-Authors: Dongxu Guan, Tatiana Tkatch, D.j. Surmeier, William E. Armstrong, Robert C. FoehringAbstract:Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-Cell RT-PCR, immunocytochemistry, and whole Cell recordings with specific peptide toxins revealed that individual pyramidal Cells express multiple Kv1 α-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as Axonal Cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these Cells. The α-dendrotoxin (α-DTX)-sensitive current activated more rapidly and at more negative potentials than the α-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The α-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from −70 mV. From −50 mV, this percentage increased to ∼20%. All Cells expressed an α-DTX-sensitive current with slow inactivation kinetics. In some Cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the α-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.
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Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones.
The Journal of physiology, 2005Co-Authors: Dongxu Guan, Tatiana Tkatch, D.j. Surmeier, William E. Armstrong, Robert C. FoehringAbstract:Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-Cell RT-PCR, immunocytochemistry, and whole Cell recordings with specific peptide toxins revealed that individual pyramidal Cells express multiple Kv1 alpha-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as Axonal Cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these Cells. The alpha-dendrotoxin (alpha-DTX)-sensitive current activated more rapidly and at more negative potentials than the alpha-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The alpha-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from -70 mV. From -50 mV, this percentage increased to approximately 20%. All Cells expressed an alpha-DTX-sensitive current with slow inactivation kinetics. In some Cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the alpha-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.
