Altered State Dependence of C-Type Inactivation in the Long and Short Forms of Human Kv1.5

Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5ΔN209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V1/2 of −19.5 ± 0.9 mV for activation and −35.7 ± 0.7 mV for inactivation, compared with a V1/2 of −11.2 ± 0.3 mV for activation and −0.9 ± 1.6 mV for inactivation in full-length Kv.15. Kv1.5ΔN209 channels exhibited several features rarely observed in voltage-gated K+ channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and “excessive cumulative inactivation,” in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5ΔN209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5ΔN209 showed 30–35% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:1779–1789) in which an absence of the NH2 terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K+ current diversity in human heart and many other tissues.

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Temperature dependence of early and late currents in human cardiac wild-type and long Q-T ΔKPQ Na+ channels

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.6.h2016 ◽

1998 ◽

Vol 275 (6) ◽

pp. H2016-H2024 ◽

Cited By ~ 33

Author(s):

Toshihisa Nagatomo ◽

Zheng Fan ◽

Bin Ye ◽

Gayle S. Tonkovich ◽

Craig T. January ◽

...

Keyword(s):

Steady State ◽

Voltage Dependence ◽

Inactivation Kinetics ◽

Wild Type ◽

Positive Direction ◽

Hek 293 ◽

Whole Cell Patch Clamp ◽

Recovery From Inactivation ◽

293 Cells ◽

Steady State Inactivation

Na+current ( I Na) through wild-type human heart Na+channels (hH1) is important for normal cardiac excitability and conduction, and it participates in the control of repolarization and refractoriness. I Na kinetics depend strongly on temperature, but I Na for hH1 has been studied previously only at room temperature. We characterized early I Na (the peak and initial decay) and late I Na of the wild-type hH1 channel and a mutant channel (ΔKPQ) associated with congenital long Q-T syndrome. Channels were stably transfected in HEK-293 cells and studied at 23 and 33°C using whole cell patch clamp. Activation and inactivation kinetics for early I Na were twofold faster at higher temperature for both channels and shifted activation and steady-state inactivation in the positive direction, especially for ΔKPQ. For early I Na (<24 ms), ΔKPQ decayed faster than the wild type for voltages negative to −20 mV but slower for more positive voltages, suggesting a reduced voltage dependence of fast inactivation. Late I Na at 240 ms was significantly greater for ΔKPQ than for the wild type at both temperatures. The majority of late I Na for ΔKPQ was not persistent; rather, it decayed slowly, and this late component exhibited slower recovery from inactivation compared with peak I Na. Additional kinetic changes for early and peak I Na for ΔKPQ compared with the wild type at both temperatures were 1) reduced voltage dependence of steady-state inactivation with no difference in midpoint, 2) positive shift for activation kinetics, and 3) more rapid recovery from inactivation. This study represents the first description of human Na+ channel kinetics near physiological temperature and also demonstrates complex gating changes in the ΔKPQ that are present at 33°C and that may underlie the electrophysiological and clinical phenotype of congenital long Q-T Na+ channel syndromes.

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The C-terminus of rat L-histidine decarboxylase specifically inhibits enzymic activity and disrupts pyridoxal phosphate-dependent interactions with L-histidine substrate analogues

Biochemical Journal ◽

10.1042/bj20031553 ◽

2004 ◽

Vol 381 (3) ◽

pp. 769-778 ◽

Cited By ~ 27

Author(s):

John V. FLEMING ◽

Ignacio FAJARDO ◽

Michael R. LANGLOIS ◽

Francisca SÁNCHEZ-JIMÉNEZ ◽

Timothy C. WANG

Keyword(s):

Pyridoxal Phosphate ◽

Histidine Decarboxylase ◽

Protein Sequences ◽

Enzymic Activity ◽

Full Length ◽

Substrate Analogue ◽

C Terminus ◽

Catalytically Active ◽

Truncated Isoforms

Full-length rat HDC (L-histidine decarboxylase) translated in reticulocyte cell lysate reactions is inactive, whereas C-terminally truncated isoforms are capable of histamine biosynthesis. C-terminal processing of the ∼74 kDa full-length protein occurs naturally in vivo, with the production of multiple truncated isoforms. The minimal C-terminal truncation required for the acquisition of catalytic competence has yet to be defined, however, and it remains unclear as to why truncation is needed. Here we show that ∼74 kDa HDC monomers can form dimers, which is the conformation in which the enzyme is thought to be catalytically active. Nevertheless, the resulting dimer is unable to establish pyridoxal phosphate-dependent interactions with an L-histidine substrate analogue. Protein sequences localized to between amino acids 617 and 633 specifically mediate this inhibition. Removing this region or replacing the entire C-terminus with non-HDC protein sequences permitted interactions with the substrate analogue to be re-established. This corresponded exactly with the acquisition of catalytic competence, and the ability to decarboxylate natural L-histidine substrate. These studies suggested that the ∼74 kDa full-length isoform is deficient in substrate binding, and demonstrated that C-terminally truncated isoforms with molecular masses between ∼70 kDa and ∼58 kDa have gradually increasing specific activities. The physiological relevance of our results is discussed in the context of differential expression of HDC isoforms in vivo.

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Na+ Permeation and Block of hERG Potassium Channels

The Journal of General Physiology ◽

10.1085/jgp.200609500 ◽

2006 ◽

Vol 128 (1) ◽

pp. 55-71 ◽

Cited By ~ 22

Author(s):

Hongying Gang ◽

Shetuan Zhang

Keyword(s):

Stable State ◽

External Solution ◽

Fast Inactivation ◽

Na Current ◽

Hek 293 ◽

Whole Cell Patch Clamp ◽

293 Cells ◽

Inactivation Gating ◽

K Current ◽

Herg Channels

The inactivation gating of hERG channels is important for the channel function and drug–channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.

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Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

Aquatic Toxicology ◽

10.1016/j.aquatox.2013.09.014 ◽

2013 ◽

Vol 142-143 ◽

pp. 447-457 ◽

Cited By ~ 30

Author(s):

Afonso C.D. Bainy ◽

Akira Kubota ◽

Jared V. Goldstone ◽

Roger Lille-Langøy ◽

Sibel I. Karchner ◽

...

Keyword(s):

Danio Rerio ◽

Functional Characterization ◽

Pregnane X Receptor ◽

Full Length ◽

Receptor Expression

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EHF suppresses cancer progression by inhibiting ETS1-mediated ZEB expression

Oncogenesis ◽

10.1038/s41389-021-00313-2 ◽

2021 ◽

Vol 10 (3) ◽

Author(s):

Kaname Sakamoto ◽

Kaori Endo ◽

Kei Sakamoto ◽

Kou Kayamori ◽

Shogo Ehata ◽

...

Keyword(s):

Cancer Progression ◽

Epithelial Mesenchymal Transition ◽

Short Form ◽

Dominant Negative ◽

Mesenchymal Transition ◽

Exon 1 ◽

Ets Transcription Factor ◽

Ets Homologous Factor ◽

Form Variant

AbstractETS homologous factor (EHF) belongs to the epithelium-specific subfamily of the E26 transformation-specific (ETS) transcription factor family. Currently, little is known about EHF’s function in cancer. We previously reported that ETS1 induces expression of the ZEB family proteins ZEB1/δEF1 and ZEB2/SIP1, which are key regulators of the epithelial–mesenchymal transition (EMT), by activating the ZEB1 promoters. We have found that EHF gene produces two transcript variants, namely a long form variant that includes exon 1 (EHF-LF) and a short form variant that excludes exon 1 (EHF-SF). Only EHF-SF abrogates ETS1-mediated activation of the ZEB1 promoter by promoting degradation of ETS1 proteins, thereby inhibiting the EMT phenotypes of cancer cells. Most importantly, we identified a novel point mutation within the conserved ETS domain of EHF, and found that EHF mutations abolish its original function while causing the EHF protein to act as a potential dominant negative, thereby enhancing metastasis in vivo. Therefore, we suggest that EHF acts as an anti-EMT factor by inhibiting the expression of ZEBs, and that EHF mutations exacerbate cancer progression.

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A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues

Scientific Reports ◽

10.1038/s41598-021-89478-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Celinda M. Kofron ◽

Tae Yun Kim ◽

Fabiola Munarin ◽

Arvin H. Soepriatna ◽

Rajeev J. Kant ◽

...

Keyword(s):

Risk Assessment ◽

Action Potentials ◽

Induced Pluripotent Stem Cell ◽

Human Action ◽

Environmental Toxicants ◽

Pharmaceutical Drugs ◽

Industrial Chemicals ◽

Endocrine Disrupting Chemical

AbstractCardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively). Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

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Microtubule Actin Cross-Linking Factor (Macf)

The Journal of Cell Biology ◽

10.1083/jcb.147.6.1275 ◽

1999 ◽

Vol 147 (6) ◽

pp. 1275-1286 ◽

Cited By ~ 150

Author(s):

Conrad L. Leung ◽

Dongming Sun ◽

Min Zheng ◽

David R. Knowles ◽

Ronald K.H. Liem

Keyword(s):

Calcium Binding ◽

Coiled Coil ◽

Specific Protein ◽

Binding Domain ◽

Full Length ◽

Actin Binding ◽

Cross Linking ◽

Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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Activation of Slo2.1 channels by niflumic acid

The Journal of General Physiology ◽

10.1085/jgp.200910316 ◽

2010 ◽

Vol 135 (3) ◽

pp. 275-295 ◽

Cited By ~ 18

Author(s):

Li Dai ◽

Vivek Garg ◽

Michael C. Sanguinetti

Keyword(s):

Voltage Dependence ◽

Reversal Potential ◽

Niflumic Acid ◽

Flufenamic Acid ◽

Channel Activation ◽

Outward Currents ◽

Transmembrane Voltage ◽

High K ◽

Charged Residues ◽

K Current

Slo2.1 channels conduct an outwardly rectifying K+ current when activated by high [Na+]i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na+. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC50 = 2.1 mM) or flufenamic acid (EC50 = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K+]e, the conductance–voltage (G-V) relationship had a V1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V1/2 to more negative potentials (EC50 = 2.1 mM) and increased the minimum value of G/Gmax (EC50 = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V1/2 of the G-V relationship was shifted to more positive potentials when [K+]e was elevated from 1 to 300 mM (EC50 = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K+]e dependency (EC50 = 23.5 mM). Conductance was also [Na+]e dependent. Outward currents were reduced when Na+ was replaced with choline or mannitol, but unaffected by substitution with Rb+ or Li+. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K+]e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K+]e and [Na+]e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na+.

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Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3484-3493.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3484-3493

Author(s):

T J Wu ◽

G Monokian ◽

D F Mark ◽

C R Wobbe

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Transcriptional Activation ◽

Deletion Mutant ◽

Full Length ◽

Early Gene ◽

Immediate Early ◽

Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

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Abstract 202: Aging Alters the Electrical and Mechanical properties of LV Myocytes

Circulation Research ◽

10.1161/res.113.suppl_1.a202 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Andrea Sorrentino ◽

Sergio Signore ◽

Mark Sundman ◽

Ramaswamy Kannappan ◽

Chiara Mangiaracina ◽

...

Keyword(s):

Mechanical Performance ◽

Na Channel ◽

Relaxation Phase ◽

Qt Intervals ◽

K Current ◽

Electrocardiographic Parameters ◽

Perfused Hearts ◽

Old Mice ◽

Ionic Basis

Aging results in delays in the electrical recovery of the heart, enhancing the risk of malignant ventricular arrhythmias and sudden death. But whether altered electrical properties affect myocardial mechanical performance remains unclear. The aim of this study was to establish the ionic basis for the protracted repolarization of the senescent heart, and to determine whether these electrical changes impact on myocardial contractility. For this purpose, electrophysiological analyses were conducted in vivo and in isolated LV myocyte preparations from mice ranging from 3 to 30 months of age. Electrocardiographic parameters were preserved from 3 to 12 months of age, whereas animals 2 years or older presented prolonged PR, QRS and QT intervals. These in vivo results were confirmed in the isolated organ, using Langendorff perfused hearts. Myocytes from 30 months old mice showed longer early and late repolarization phases of the action potential (AP), in comparison with cells from animals at 3 months. By voltage-clamp, the density of the transient outward K+ current (Ito) was significantly reduced in old, whereas the late Na+ current (INaL) was increased, changes consistent with the prolonged AP of old cells. Additionally, by Western blotting, the SCN1B subunit, involved in Na+ channel gating, was reduced with age. Using field stimulation, old myocytes presented slower Ca2+ transient decay and relaxation, in comparison to young. Blockade of INaL with mexiletine in old myocytes shortened the AP duration, and fastened the decay of Ca2+ transients and relaxation. To assess the functional impact of INaL on the myocardium, papillary muscles were studied in an isometric system. Using tension-length protocols, old muscles presented elevated diastolic tension with respect to young. Importantly, inhibition of INaL in old muscles reduced diastolic tension, and attenuated the active developed force. Conversely, increasing INaL in the myocardium of young mice with anemonetoxin had opposite effects. Overall, these data indicate that aging results in a prolongation of the AP mediated, at least in part, by enhanced INaL. The prolonged repolarization of the AP contributes to the slower clearance of diastolic Ca2+ from the cytoplasm, resulting in protracted relaxation phase.

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