Publications by authors named "Chih-Yung Tang"

29 Publications

  • Page 1 of 1

Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation.

J Biol Chem 2021 Feb 26:100484. Epub 2021 Feb 26.

Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; Brain Research Center, National Yang-Ming University, Taipei, Taiwan. Electronic address:

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K channels, however, remains unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent non-glycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, non-glycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbc.2021.100484DOI Listing
February 2021

Neuronal Exosomes Secreted under Oxygen-Glucose Deprivation/Reperfusion Presenting Differentially Expressed miRNAs and Affecting Neuronal Survival and Neurite Outgrowth.

Neuromolecular Med 2021 Jan 3. Epub 2021 Jan 3.

Department of Neurology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei, 10055, Taiwan.

Ischemia/reperfusion is a key feature of acute ischemic stroke, which causes neuron dysfunction and death. Exosomes, small extracellular vesicles produced by most cell types, are implicated in the mediation of cellular interactions with their environment. Here, we investigated the contents and functions of exosomes from neurons under ischemic reperfusion injury. First, rat cortical primary neuronal cell cultures were placed in an oxygen- and glucose-deprived (OGD) medium, followed by reperfusion in a normoxic conditioned medium (OGD/R) to mimic ischemia/reperfusion in vitro. The neuron-derived exosomes were harvested from the conditioned medium under normoxia and OGD/R. Through next-generation sequencing, exosomal miRNA expression levels in normoxic and OGD/R condition were compared. Their functional activity in terms of neuron viability and quantitative analysis of neurite outgrowth were examined. The expression levels of 45 exosomal miRNAs were significantly different between normoxic and OGD/R conditions. Bioinformatics analysis of dysregulated exosomal miRNAs identified multiple pathways involved in cell survival and death processes and neuronal signaling. Moreover, treatment with exosomes from OGD/R to cultured cortical neurons significantly impaired neuronal cell viability and reduced neurite outgrowth in terms of the number of primary or total neurites as well as length of primary neurites, compared with exosomes from normoxic conditions. miRNA-packed exosomes released by neurons under OGD/R challenge may contribute to post ischemic neuronal injury and provide further understanding of the effect of stressed neurons on neighboring neuronal functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12017-020-08641-zDOI Listing
January 2021

CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy.

Cells 2020 05 26;9(6). Epub 2020 May 26.

Department of Physiology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.

Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9061332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348978PMC
May 2020

Defective Gating and Proteostasis of Human ClC-1 Chloride Channel: Molecular Pathophysiology of Myotonia Congenita.

Front Neurol 2020 11;11:76. Epub 2020 Feb 11.

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

The voltage-dependent ClC-1 chloride channel, whose open probability increases with membrane potential depolarization, belongs to the superfamily of CLC channels/transporters. ClC-1 is almost exclusively expressed in skeletal muscles and is essential for stabilizing the excitability of muscle membranes. Elucidation of the molecular structures of human ClC-1 and several CLC homologs provides important insight to the gating and ion permeation mechanisms of this chloride channel. Mutations in the human gene, which encodes the ClC-1 channel, are associated with a hereditary skeletal muscle disease, myotonia congenita. Most disease-causing mutations lead to loss-of-function phenotypes in the ClC-1 channel and thus increase membrane excitability in skeletal muscles, consequently manifesting as delayed relaxations following voluntary muscle contractions in myotonic subjects. The inheritance pattern of myotonia congenita can be autosomal dominant (Thomsen type) or recessive (Becker type). To date over 200 myotonia-associated ClC-1 mutations have been identified, which are scattered throughout the entire protein sequence. The dominant inheritance pattern of some myotonia mutations may be explained by a dominant-negative effect on ClC-1 channel gating. For many other myotonia mutations, however, no clear relationship can be established between the inheritance pattern and the location of the mutation in the ClC-1 protein. Emerging evidence indicates that the effects of some mutations may entail impaired ClC-1 protein homeostasis (proteostasis). Proteostasis of membrane proteins comprises of biogenesis at the endoplasmic reticulum (ER), trafficking to the surface membrane, and protein turn-over at the plasma membrane. Maintenance of proteostasis requires the coordination of a wide variety of different molecular chaperones and protein quality control factors. A number of regulatory molecules have recently been shown to contribute to post-translational modifications of ClC-1 and play critical roles in the ER quality control, membrane trafficking, and peripheral quality control of this chloride channel. Further illumination of the mechanisms of ClC-1 proteostasis network will enhance our understanding of the molecular pathophysiology of myotonia congenita, and may also bring to light novel therapeutic targets for skeletal muscle dysfunction caused by myotonia and other pathological conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fneur.2020.00076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026490PMC
February 2020

Novel SCA19/22-associated KCND3 mutations disrupt human K 4.3 protein biosynthesis and channel gating.

Hum Mutat 2019 11 17;40(11):2088-2107. Epub 2019 Aug 17.

Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

Mutations in the human voltage-gated K channel subunit K 4.3-encoding KCND3 gene have been associated with the autosomal dominant neurodegenerative disorder spinocerebellar ataxia types 19 and 22 (SCA19/22). The precise pathophysiology underlying the dominant inheritance pattern of SCA19/22 remains elusive. Using cerebellar ataxia-specific targeted next-generation sequencing technology, we identified two novel KCND3 mutations, c.950 G>A (p.C317Y) and c.1123 C>T (p.P375S) from a cohort with inherited cerebellar ataxias in Taiwan. The patients manifested notable phenotypic heterogeneity that includes cognitive impairment. We employed in vitro heterologous expression systems to inspect the biophysical and biochemical properties of human K 4.3 harboring the two novel mutations, as well as two previously reported but uncharacterized disease-related mutations, c.1013 T>A (p.V338E) and c.1130 C>T (p.T377M). Electrophysiological analyses revealed that all of these SCA19/22-associated K 4.3 mutant channels manifested loss-of-function phenotypes. Protein chemistry and immunofluorescence analyses further demonstrated that these mutants displayed enhanced protein degradation and defective membrane trafficking. By coexpressing K 4.3 wild-type with the disease-related mutants, we provided direct evidence showing that the mutants instigated anomalous protein biosynthesis and channel gating of K 4.3. We propose that the dominant inheritance pattern of SCA19/22 may be explained by the dominant-negative effects of the mutants on protein biosynthesis and voltage-dependent gating of K 4.3 wild-type channel.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.23865DOI Listing
November 2019

FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane.

Int J Mol Sci 2018 Nov 28;19(12). Epub 2018 Nov 28.

Department of Physiology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.

Mutations in the skeletal muscle-specific CLC-1 chloride channel are associated with the human hereditary disease myotonia congenita. The molecular pathophysiology underlying some of the disease-causing mutations can be ascribed to defective human CLC-1 protein biosynthesis. CLC-1 protein folding is assisted by several molecular chaperones and co-chaperones, including FK506-binding protein 8 (FKBP8). FKBP8 is generally considered an endoplasmic reticulum- and mitochondrion-resident membrane protein, but is not thought to contribute to protein quality control at the cell surface. Herein, we aim to test the hypothesis that FKBP8 may regulate CLC-1 protein at the plasma membrane. Surface biotinylation and subcellular fractionation analyses reveal that a portion of FKBP8 is present at the plasma membrane, and that co-expression with CLC-1 enhances surface localization of FKBP8. Immunoblotting analyses of plasma membrane proteins purified from skeletal muscle further confirm surface localization of FKBP8. Importantly, FKBP8 promotes CLC-1 protein stability at the plasma membrane. Together, our data underscore the importance of FKBP8 in the peripheral quality control of CLC-1 channel.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19123783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320802PMC
November 2018

Calmodulin modulates the Ca-dependent inactivation and expression level of bovine Ca2.2 expressed in HEK293T cells.

IBRO Rep 2017 Jun 18;2:63-71. Epub 2017 Mar 18.

Department of Life Science, National Taiwan University, Taipei, Taiwan.

Ca influx through voltage-gated Ca channels (Cas) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca concentration ([Ca]), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca-dependent inactivation (CDI) of several types of Cas; however, little is known about how CaM modulates Ca2.2. Here, we expressed Ca2.2 with CaM or CaM mutants with a Ca-binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that Ca2.2 displayed a fast inactivation with Ca but not Ba as the charge carrier; when Ca2.2 was co-expressed with CaM mutants with a Ca-binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of Ca2.2 in the presence or absence of Ca. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. Ca2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM (Ca-binding deficiency at the N-lobe) overexpression; in addition, CaM enhanced the total expression level of Ca2.2, but the ratio of Ca2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca-binding abilities, modulates not only the inactivation of Ca2.2 but also its expression to regulate Ca-related physiological activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibror.2017.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084911PMC
June 2017

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca2.1 (P/Q-Type) Calcium Channels.

J Neurosci 2017 03 6;37(9):2485-2503. Epub 2017 Feb 6.

Department of Physiology and

Voltage-gated Ca2.1 channels comprise a pore-forming α subunit with auxiliary αδ and β subunits. Ca2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca2.1-binding partner. In neurons, RNF138 and Ca2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca2.1 protein level and enhances Ca2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca2.1 subunits. Loss-of-function mutations in the human Ca2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the Ca2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the Ca2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for Ca2.1, RNF138. Ca2.1 protein stability is dynamically regulated by RNF138 and auxiliary αδ and β subunits. We provide a proof of concept that protecting the human Ca2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.3070-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596842PMC
March 2017

Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels.

Sci Rep 2017 01 18;7:40825. Epub 2017 Jan 18.

Institute of Anatomy and Cell Biology, School of Medicine, National  Yang-Ming University, Taipei, Taiwan.

Mammalian Eag1 (Kv10.1) potassium (K) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep40825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241692PMC
January 2017

Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Sci Rep 2016 09 1;6:32444. Epub 2016 Sep 1.

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep32444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007535PMC
September 2016

The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels.

Sci Rep 2016 Jan 18;6:19378. Epub 2016 Jan 18.

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvβ1.1 or Kvβ2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvβ1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep19378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726062PMC
January 2016

The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

Sci Rep 2015 May 29;5:10667. Epub 2015 May 29.

1] Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan [2] Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei, Taiwan.

Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep10667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448132PMC
May 2015

Densin-180 is not a transmembrane protein.

Cell Biochem Biophys 2013 Nov;67(2):773-83

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

In the central nervous system, densin-180 (densin) is one of the major components of the post-synaptic density (PSD) of excitatory synapses. Through its intricate interaction with various post-synaptic proteins, this scaffold protein may play a key role in synaptic regulation. Initial structural analyses suggest that densin is a transmembrane protein and may participate in cell-adhesion function between pre- and post-synaptic membranes. Whereas recent biochemical and mass spectrometry studies indicate that densin may instead be a membrane-associated protein with no extracellular domain. To further investigate the structural topology of densin, we began with examining the extracellular accessibility of multiple epitopes in densin. We have provided immunofluorescence evidence showing that none of the tested epitope sites in densin was accessible to extracellularly applied antibodies. In addition, both protease digestion and surface biotinylation data failed to affirm the presence of extracellular domain for densin. However, protein extraction experiments indicated that densin exhibited a significant hydrophobic interaction with the cell membrane that was not expected of cytosolic proteins. Our data therefore do not support the transmembrane model, but rather are consistent with the idea that the topology of densin involves the membrane association configuration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12013-013-9570-3DOI Listing
November 2013

Myotonia congenita mutation enhances the degradation of human CLC-1 chloride channels.

PLoS One 2013 12;8(2):e55930. Epub 2013 Feb 12.

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl(-)) channel CLC-1. Myotonia congenita can be inherited in an autosomal recessive (Becker type) or dominant (Thomsen type) fashion. One hypothesis for myotonia congenita is that the inheritance pattern of the disease is determined by the functional consequence of the mutation on the gating of CLC-1 channels. Several disease-related mutations, however, have been shown to yield functional CLC-1 channels with no detectable gating defects. In this study, we have functionally and biochemically characterized a myotonia mutant: A531V. Despite a gating property similar to that of wild-type (WT) channels, the mutant CLC-1 channel displayed a diminished whole-cell current density and a reduction in the total protein expression level. Our biochemical analyses further demonstrated that the reduced expression of A531V can be largely attributed to an enhanced proteasomal degradation as well as a defect in protein trafficking to surface membranes. Moreover, the A531V mutant protein also appeared to be associated with excessive endosomal-lysosomal degradation. Neither the reduced protein expression nor the diminished current density was rescued by incubating A531V-expressing cells at 27°C. These results demonstrate that the molecular pathophysiology of A531V does not involve anomalous channel gating, but rather a disruption of the balance between the synthesis and degradation of the CLC-1 channel protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055930PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570542PMC
August 2013

Dominantly inherited myotonia congenita resulting from a mutation that increases open probability of the muscle chloride channel CLC-1.

Neuromolecular Med 2012 Dec 12;14(4):328-37. Epub 2012 Jul 12.

Department of Neurology and Center for Neuroscience, University of California, Davis, CA 95616, USA.

Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (P (o)) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common-gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased P (o) of the channel's fast-gate, from ~0.4 in the wild type to >0.9 in the mutant at -90 mV. In contrast, the mutant exhibits a minimal effect on the P (o) of the common-gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast-gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a fivefold increase in chloride conductance in the wild type to a twofold increase in the mutant-this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12017-012-8190-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3508202PMC
December 2012

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity.

J Biol Chem 2012 Mar 23;287(13):10316-10324. Epub 2012 Jan 23.

Department of Biomedical Sciences, Chang Gung University, Kwei-Shan, Tao-Yuan 333, Taiwan, R.O.C.; Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan, R.O.C.. Electronic address:

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.244962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323054PMC
March 2012

Physiology and pathophysiology of CLC-1: mechanisms of a chloride channel disease, myotonia.

J Biomed Biotechnol 2011 1;2011:685328. Epub 2011 Dec 1.

Department of Physiology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.

The CLC-1 chloride channel, a member of the CLC-channel/transporter family, plays important roles for the physiological functions of skeletal muscles. The opening of this chloride channel is voltage dependent and is also regulated by protons and chloride ions. Mutations of the gene encoding CLC-1 result in a genetic disease, myotonia congenita, which can be inherited as an autosmal dominant (Thomsen type) or an autosomal recessive (Becker type) pattern. These mutations are scattered throughout the entire protein sequence, and no clear relationship exists between the inheritance pattern of the mutation and the location of the mutation in the channel protein. The inheritance pattern of some but not all myotonia mutants can be explained by a working hypothesis that these mutations may exert a "dominant negative" effect on the gating function of the channel. However, other mutations may be due to different pathophysiological mechanisms, such as the defect of protein trafficking to membranes. Thus, the underlying mechanisms of myotonia are likely to be quite diverse, and elucidating the pathophysiology of myotonia mutations will require the understanding of multiple molecular/cellular mechanisms of CLC-1 channels in skeletal muscles, including molecular operation, protein synthesis, and membrane trafficking mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2011/685328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237021PMC
June 2012

CD4+ T cell-derived IL-2 signals during early priming advances primary CD8+ T cell responses.

PLoS One 2009 Nov 10;4(11):e7766. Epub 2009 Nov 10.

Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.

Stimulating naïve CD8+ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8+ T cells require IL-2 provided by activated CD4+ T cells at the initial priming stage within 0-2.5 hours after stimulation. This critical IL-2 signal from CD4+ cells is mediated through the IL-2Rbetagamma of CD8+ cells, which is independent of IL-2Ralpha. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8+ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNgamma and granzyme B produced by CD8+ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8+ T-cell response is elicited by a critical temporal function of IL-2 released from CD4+ T cells, providing mechanistic insights into the regulation of CD8+ T cell activation and differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007766PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770320PMC
November 2009

Toluene disrupts synaptogenesis in cultured hippocampal neurons.

Toxicol Lett 2009 Jan 7;184(2):90-6. Epub 2008 Nov 7.

Department of Physiology, National Taiwan University, Taipei, Taiwan.

Prenatal toluene exposure may lead to significant developmental neurotoxicity known as fetal solvent syndrome. Emerging evidence suggests that toluene embryopathy may arise from an elusive deviation of the neurogenesis process. One key event during neural development is synaptogenesis, which is essential for the progression of neuronal differentiation and the establishment of neuronal network. We therefore aim to test the hypothesis that toluene may interfere with synaptogenesis by applying toluene to cultured hippocampal neurons dissected from embryonic rat brains. In the presence of toluene, hippocampal neurons displayed a significant loss of the immunostaining of synapsin and densin-180 punctas. Notably, a dramatic reduction was also discerned for the colocalization of the two synaptic markers. Moreover, Western blotting analyses revealed that toluene exposure resulted in considerable down-regulation of the expression of synapse-specific proteins. None of the preceding observations can be attributed to toluene-induced cell death effects, since toluene treatments failed to affect the viability of hippocampal neurons. Overall, our data are consistent with the idea that toluene may alter the expression and localization of essential synaptic proteins, thereby leading to a disruption of synapse formation and maintenance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxlet.2008.10.027DOI Listing
January 2009

Membrane targeting and coupling of NHE1-integrinalphaIIbbeta3-NCX1 by lipid rafts following integrin-ligand interactions trigger Ca2+ oscillations.

J Biol Chem 2009 Feb 7;284(6):3855-64. Epub 2008 Nov 7.

Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan.

The cyclic calcium release and uptake during calcium oscillation are thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in nonexcitable cells, how the initial calcium mobilization that triggers CICR occurs. We report here a novel mechanism, other than conventional calcium channels or the phopholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrin alphaIIbbeta3 binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 are actively transported to the plasma membrane, and they become physically coupled to integrin alphaIIbbeta3. Lipid raft-dependent mechanisms modulate the membrane targeting and formation of the NHE1-integrin alphaIIbbeta3-NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR as a prelude to calcium oscillation downstream of integrin alphaIIbbeta3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1-integrin alphaIIbbeta3-NCX1, which could not be properly detected using conventional biochemical assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M804334200DOI Listing
February 2009

Effects of sodium azide, barium ion, d-amphetamine and procaine on inward rectifying potassium channel 6.2 expressed in Xenopus oocytes.

J Formos Med Assoc 2008 Aug;107(8):600-8

School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.

Background/purpose: Inward rectifying potassium channel 6.2 (Kir6.2DelataC26 channel) is closely related to ATP-sensitive potassium channels. Whether sodium azide, barium ion, d-amphetamine or procaine acts directly on the Kir6.2DeltaC26 channel remains unclear. We studied the effects of these compounds on Kir6.2DeltaC26 channel expressed in Xenopus oocytes.

Methods: The coding sequence of a truncated form of mouse Kir6.2 (GenBank accession number NP_034732.1), Kir6.2(1-364) (i.e. Kir6.2DeltaC26), was subcloned into the pET20b(+) vector. Plasmid containing the correct T7 promoter-Kir6.2(1-364) cDNA fragment [Kir6.2/pET20b(+)] was then subject to NotI digestion to generate the templates for in vitro run-off transcriptions. The channel was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to measure the effects of sodium azide, barium ion, d-amphetamine and procaine on Kir6.2DeltaC26 channel current.

Results: Sodium azide activated and barium ion and d-amphetamine inhibited the Kir6.2DeltaC26 channel. Procaine did not have any significant effect on the Kir6.2DeltaC26 channel.

Conclusion: Kir6.2DeltaC26 channel expressed in Xenopus oocytes can be used as a pharmacological tool for the study of inward rectifying potassium channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0929-6646(08)60177-1DOI Listing
August 2008

Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels.

J Cell Physiol 2008 Feb;214(2):422-33

Institute of Anatomy and Cell biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder associated with mutations in the gene encoding pore-forming alpha(1A) subunits of human P/Q-type calcium (Ca(V)2.1) channels. The exact mechanism of how mutant channels cause such clinical EA2 features as cerebellar dysfunctions, however, remains unclear. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert prominent dominant-negative effects on wild-type Ca(V)2.1 channels. To further pursue the mechanism underlying this dominant-negative effect, we examined the effects of EA2 mutants on the subcellular localization pattern of GFP-tagged wild-type Ca(V)2.1 channels in HEK293T cells. In the presence of EA2 mutants, wild-type channels displayed a significant deficiency in membrane targeting and a concurrent increase in cytoplasm retention. Moreover, the cytoplasmic fraction of wild-type channels co-localized with an endoplasmic reticulum (ER) marker, suggesting that a significant amount of wild-type Ca(V)2.1 channels was trapped in the ER. This EA2 mutant-induced ER retention pattern was reversed by lowering the cell incubation temperature from 37 to 27 degrees C. We also inspected the effects of untagged EA2 mutants on the functional expression of GFP-tagged wild-type Ca(V)2.1 channels in HEK293T cells. Whole-cell current density of wild-type channels was diminished in the presence of EA2 mutants, which was also reversed by 27 degrees C incubation. Finally, biochemical analyses indicated that EA2 mutants did not significantly affect the protein expression level of wild-type channels. Taken together, our data suggest that EA2 mutants induce significant ER retention of their wild-type counterparts, thereby suppressing the functional expression of Ca(V)2.1 channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.21216DOI Listing
February 2008

Effects of selective unilateral dorsal root(s) rhizotomy on micturition reflex in anesthetized rats.

Neurourol Urodyn 2006 ;25(7):820-7

Department of Pharmacology, National Taiwan University, Taipei, Taiwan.

Purpose: To clarify the contributions of sensory inputs and glutamate transmissions to the spinal micturition reflex.

Materials And Methods: Cystometrogram and external urethral sphincter electromyogram activities evaluated the L6 and/or S1 levels.

Results: Changes in intravesicular pressure (IVP) in response to saline infusion (0.1 ml/min) were found after unilateral dorsal root rhizotomy at the L6 level, which showed significant increases in threshold pressure (rhizotomized vs. control: 14.25 +/- 0.82 vs. 8.40 +/- 0.69 cmH(2)O, P < 0.01, n = 28), post-voiding pressure (7.66 +/- 0.56 vs. 5.42 +/- 0.52 cmH(2)O, P < 0.01, n = 28), holding duration (135.06 +/- 23.6 vs. 77.73 +/- 13.56 sec, P < 0.05, n = 28), and inter-contraction interval (140.62 +/- 23.29 vs. 82.40 +/- 13.57 sec, P < 0.05, n = 28). Several (mean = 2.32 +/- 1.31 vs. 0.12 +/- 0.21, P < 0.01, n = 28, P < 0.01, n = 28) non-voiding contractions with gradual increase in IVP were found ahead of voiding contraction after rhizotomy. An additional dorsal root rhizotomy at the ipsilateral S1 level caused further increases in urodynamic parameters (threshold pressure, 18.18 +/- 1.67 cmH(2)O, P < 0.01; post-voiding pressure 8.07 +/- 0.96 cmH(2)O, P < 0.01; holding duration, 211.44 +/- 42.54 sec, P < 0.01; inter-contraction interval, 264.2 +/- 59.99 sec, P < 0.05; non-voiding contractions, 4.41 +/- 2.12, P < 0.01, n = 7). Intrathecal glutamate (100 microM, 10 microl) ameliorated all the pathological conditions induced by unilateral dorsal root rhizotomy at the L6 level in a dose dependent manner (ED(50) = 1.25 +/- 10(-5)). Intrathecal CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 100 microM, 10 microl) and APV (D-2-amino-5-phosphonovaleric acid; 100 microM, 10 microl) injections after rhizotomy at the L6 level induced disturbances similar to that caused by an additional rhizotomy at ipsilateral S1 level. Wherease, glutamate (100 microM, 10 microl) reversed the disturbances caused by CNQX but showed no effect on that by APV.

Conclusions: Acute partial sensory deprivation caused acute impaired micturition reflex in rat models. In addition, glutamatergic NMDA and AMPA receptors are important for mediating these impairments in micturition reflex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/nau.20211DOI Listing
January 2007

Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2.

Am J Physiol Cell Physiol 2006 Apr 23;290(4):C1209-20. Epub 2005 Nov 23.

School of Medicine, Fu Jen Catholic University, Hsin-Chuang, Taipei County, Taiwan.

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming alpha(1A)-subunits of human P/Q-type Ca(2+) channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human alpha(1A)-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of alpha(1A)-WT with each alpha(1A)-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba(2+) currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of alpha(1A)-mutants increased, suggesting the presence of an alpha(1A)-mutant-specific suppression effect. When we coexpressed alpha(1A)-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of alpha(1A)-WT and mutant using a splice variant of alpha(1A)-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-alpha(1A)-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00247.2005DOI Listing
April 2006

Selective enhancement of tonic inhibition by increasing ambient GABA is insufficient to suppress excitotoxicity in hippocampal neurons.

Biochem Biophys Res Commun 2005 Dec 26;338(3):1417-25. Epub 2005 Oct 26.

Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.

Gamma-aminobutyric acid (GABA) activates synaptic GABA(A) receptors to generate inhibitory postsynaptic potentials. GABA also acts on extrasynaptic GABA(A) receptors, resulting in tonic inhibition. The physiological role of tonic inhibition, however, remains elusive. We explored the neurophysiological significance of tonic inhibition by testing whether selective activation of extrasynaptic GABA(A) receptors is sufficient to curb excitotoxicity. Tonic inhibition was selectively enhanced by increasing ambient GABA. In both acute hippocampal slices and cultured hippocampal neurons, boosting tonic inhibition alone is insufficient to withstand the hyper-excitability of hippocampal neurons induced by low-magnesium (Mg2+) baths. Furthermore, selective activation of extrasynaptic GABA(A) receptors resulted in no significant neuroprotective effects against glutamate or low-Mg2+-induced neuronal cell deaths. These data imply that under physiological conditions extrasynaptic GABA(A) receptors are optimally activated by ambient GABA and that a further increase in extracellular GABA concentration will not significantly enhance the effect of tonic inhibition on neuronal excitability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2005.10.103DOI Listing
December 2005

Beauvericin activates Ca2+-activated Cl- currents and induces cell deaths in Xenopus oocytes via influx of extracellular Ca2+.

Chem Res Toxicol 2005 May;18(5):825-33

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Beauvericin is a mycotoxin that infects a wide variety of cereal grains. The toxicological importance of beauvericin is implicated by its cytotoxicity in animal and human cells, which has been suggested to result from an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). Despite the fact that beauvericin may activate extracellular Ca(2+) influx, beauvericin-induced cell deaths has been suggested to be exclusively due to Ca(2+) release from internal Ca(2+) stores. We endeavored to elucidate the mechanism of beauvericin-induced [Ca(2+)](i) increase by studying the effects of beauvericin in Xenopus oocytes. By applying a -140-mV prepulse prior to a series of test pulses, we found that beauvericin induced small inward currents at -140 mV, followed by outwardly rectifying currents that displayed an apparent reversal potential close to the expected equilibrium potential of Cl(-). Both the inward and outward currents induced by beauvericin were blocked by niflumic acid, a specific blocker for Ca(2+)-activated Cl(-) currents (I(Cl,Ca)). Removal of extracellular Ca(2+), as well as perfusion of lanthanide, abrogated beauvericin-induced currents. Beauvericin also displayed prominent cytotoxic effects in Xenopus oocytes in a dose-dependent manner. In the absence of extracellular Ca(2+), cytotoxicity-induced by 10 and 30 microM, but not 50 microM, of beauvericin was significantly diminished. Our results are consistent with the idea that beauvericin induces extracellular Ca(2+) influx, which in turn activates I(Cl,Ca) and contributes to beauvericin-induced cell deaths in Xenopus oocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/tx049733dDOI Listing
May 2005

Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons.

Neuroreport 2005 Feb;16(3):229-33

School of Medicine, Fu Jen Catholic University, Taipei County, Taiwan.

Two isoforms of rat ether-a-go-go (Eag) K channels, rEag1 and rEag2, are widely expressed in many regions of the brain. The neurophysiological roles of these channels, however, are unclear. We addressed this issue by studying their subcellular localizations in hippocampal neurons. Immunofluorescence studies using markers for different compartments of neurons demonstrated a differential expression pattern of rEag1 and rEag2 K channels in the somatodendritic region. Furthermore, rEag1 K channels were in close proximity to synaptophysin and densin-180, but not GAD65. Our data suggest that both rEag1 and rEag2 K channels may play a pivotal role in the regulation of the excitability of dendrites and somas, and that rEag1 K channels may modulate the postsynaptic signaling of glutamatergic synapses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/00001756-200502280-00005DOI Listing
February 2005

Depressor effect on blood pressure and flow elicited by electroacupuncture in normal subjects.

Auton Neurosci 2003 Aug;107(1):60-4

School of Physical Therapy, Chung-Shan Medical University, Taichung, Taiwan.

To clarify the effect of electroacupuncture (Ea) on the activity of the cardiovascular system in normal individuals, hemodynamic parameters including arterial blood pressure (BP), finger blood flow (FBF) and heart rate (HR) as well as paravertebral temperature (PVT) were non-invasively recorded under Ea stimulation. Surface stimulation electrode was placed on the Hoku point (Li-4). Square wave pulses (0.05 ms) were applied from a stimulator with a stimulation frequency of 2 Hz (3 min). The stimulation intensity was five times of sensory threshold. BP and FBF were decreased (68.5+/-6.0%, P<0.01 and 96.8+/-1.1%, P<0.01 of control, respectively, n=7) while HR and PVT were increased significantly (115.0+/-5.1 of control, P<0.05 and 0.054+/-0.004 degree C, P<0.01, respectively, n=7) during Ea treatment. The results suggested an inhibition in sympathetic outflow, which induced vasodilatation of systemic arteriole and decrease in BP and FBF were elicited by Ea stimulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S1566-0702(03)00103-6DOI Listing
August 2003

Structural organization of the voltage sensor in voltage-dependent potassium channels.

Novartis Found Symp 2002 ;245:178-90; discussion 190-2, 261-4

Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1751, USA.

The structural organization of the voltage sensor in K+ channels has been investigated by second site suppressor analysis in Shaker and by identification of a metal ion binding site in ether-à-go-go (eag). In Shaker, two groups of interacting charged residues have been identified. K374 in the S4 segment interacts with E293 in S2 and D316 in S3, whereas E283 in S2 interacts with R368 and R371, two voltage-sensing residues in S4. Interactions of E283 with R368 and R371 are voltage dependent. The results suggest that E283 is located in a water-filled pocket near the extracellular surface of the protein. During voltage-dependent activation of Shaker channels, R368 and R371 move into this pocket and come into proximity with E283. In eag channels, extracellular Mg2+ directly modulates the activation process by binding to two acidic residues that are located in an analogous pocket. These acidic residues are found only in eag family members, accounting for the specificity of Mg2+ modulation to that family. These compatible results from Shaker and eag suggest a model for the packing and conformational changes of transmembrane segments in the voltage sensor of K+ channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
November 2002