scholarly journals hERG potassium channel gating is mediated by N- and C-terminal region interactions

2011 ◽  
Vol 137 (3) ◽  
pp. 315-325 ◽  
Author(s):  
Ahleah S. Gustina ◽  
Matthew C. Trudeau
Keyword(s):  
Direct Interaction ◽  
Terminal Region ◽  
Pas Domain ◽  
Related Gene ◽  
Nucleotide Binding Domain ◽  
Glutathione S Transferase ◽  
Deactivation Kinetics ◽  
Voltage Dependent ◽  
Intersubunit Interactions ◽  
Herg Channels

Human ether-á-go-go–related gene (hERG) potassium channels have voltage-dependent closing (deactivation) kinetics that are unusually slow. A Per-Arnt-Sim (PAS) domain in the cytoplasmic N-terminal region of hERG regulates slow deactivation by making a direct interaction with another part of the hERG channel. The mechanism for slow deactivation is unclear, however, because the other regions of the channel that participate in regulation of deactivation are not known. To identify other functional determinants of slow deactivation, we generated hERG channels with deletions of the cytoplasmic C-terminal regions. We report that hERG channels with deletions of the cyclic nucleotide–binding domain (CNBD) had accelerated deactivation kinetics that were similar to those seen in hERG channels lacking the PAS domain. Channels with dual deletions of the PAS domain and the CNBD did not show further acceleration in deactivation, indicating that the PAS domain and the CNBD regulate deactivation by a convergent mechanism. A recombinant PAS domain that we previously showed could directly regulate PAS domain–deleted channels did not regulate channels with dual deletions of the PAS domain and CNBD, suggesting that the PAS domain did not interact with CNBD-deleted channels. Biochemical protein interaction assays showed that glutathione S-transferase (GST)–PAS (but not GST) bound to a CNBD-containing fusion protein. Coexpression of PAS domain–deleted subunits (with intact C-terminal regions) and CNBD-deleted subunits (with intact N-terminal regions) resulted in channels with partially restored slow deactivation kinetics, suggesting regulatory intersubunit interactions between PAS domains and CNBDs. Together, these data suggest that the mechanism for regulation of slow deactivation in hERG channels is an interaction between the N-terminal PAS domain and the C-terminal CNBD.

scholarly journals The hERG potassium channel intrinsic ligand regulates N- and C-terminal interactions and channel closure

2018 ◽  
Vol 151 (4) ◽  
pp. 478-488 ◽  
Author(s):  
Sara J. Codding ◽  
Matthew C. Trudeau
Keyword(s):  
Cyclic Nucleotide ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Pas Domain ◽  
Domain Interaction ◽  
Structural Interaction ◽  
Whole Cell Patch Clamp ◽  
In Trans ◽  
Patch Clamp Electrophysiology ◽  
Herg Channels

Human ether-à-go-go–related gene (hERG, KCNH2) voltage-activated potassium channels are critical for cardiac excitability. hERG channels have characteristic slow closing (deactivation), which is auto-regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). hERG channels are not activated by the binding of extrinsic cyclic nucleotide ligands, but rather bind an “intrinsic ligand” that is composed of residues 860–862 within the CNBHD and mimics a cyclic nucleotide. The intrinsic ligand is located at the PAS–CNBHD interface, but its mechanism of action in hERG is not well understood. Here we use whole-cell patch-clamp electrophysiology and FRET spectroscopy to examine how the intrinsic ligand regulates gating. To carry out this work, we coexpress PAS (a PAS domain fused to cyan fluorescent protein) in trans with hERG “core” channels (channels with a deletion of the PAS domain fused to citrine fluorescent protein). The PAS domain in trans with hERG core channels has slow (regulated) deactivation, like that of WT hERG channels, as well as robust FRET, which indicates there is a direct functional and structural interaction of the PAS domain with the channel core. In contrast, PAS in trans with hERG F860A core channels has intermediate deactivation and intermediate FRET, indicating perturbation of the PAS domain interaction with the CNBHD. Furthermore, PAS in trans with hERG L862A core channels, or PAS in trans with hERG F860G,L862G core channels, has fast (nonregulated) deactivation and no measurable FRET, indicating abolition of the PAS and CNBHD interaction. These results indicate that the intrinsic ligand is necessary for the functional and structural interaction between the PAS domain and the CNBHD, which regulates the characteristic slow deactivation gating in hERG channels.

How to Fluorescently Label the Potassium Channel: A Case in hERG

2020 ◽  
Vol 27 (18) ◽  
pp. 3046-3054
Author(s):  
Xiaomeng Zhang ◽  
Beilei Wang ◽  
Zhenzhen Liu ◽  
Yubin Zhou ◽  
Lupei Du
Keyword(s):  
Potassium Channel ◽  
Herg Channel ◽  
Pore Channel ◽  
Design Rationale ◽  
Related Gene ◽  
Open Conformation ◽  
Cardiac Action ◽  
Qt Syndrome ◽  
Herg Channels ◽  
Action Potential Repolarization

hERG (Human ether-a-go-go-related gene) potassium channel, which plays an essential role in cardiac action potential repolarization, is responsible for inherited and druginduced long QT syndrome. Recently, the Cryo-EM structure capturing the open conformation of hERG channel was determined, thus pushing the study on hERG channel at 3.8 Å resolution. This report focuses primarily on summarizing the design rationale and application of several fluorescent probes that target hERG channels, which enables dynamic and real-time monitoring of potassium pore channel affinity to further advance the understanding of the channels.

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2413-2419 ◽  
Author(s):  
José L. Alonso-Lebrero ◽  
Juan M. Serrador ◽  
Carmen Domı́nguez-Jiménez ◽  
Olga Barreiro ◽  
Alfonso Luque ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Adhesion Molecule ◽  
Direct Interaction ◽  
Intercellular Adhesion ◽  
Actin Binding ◽  
Intercellular Adhesion Molecule ◽  
Human Neutrophils ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Amino Terminal

Abstract In response to the chemoattractants interleukin 8, C5a,N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

scholarly journals The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins

2001 ◽  
Vol 355 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Claudia TROST ◽  
Christiane BERGS ◽  
Nina HIMMERKUS ◽  
Veit FLOCKERZI
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Transient Receptor

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca2+-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca2+-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM–Sepharose in the presence of Ca2+, and (2) TRP4–glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688–759 and 786–848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca2+ concentrations above 10µM, with half maximal binding occurring at 16.6µM (domain 1) and 27.9µM Ca2+ (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694–728 and 829–853, interacted directly with dansyl–CaM with apparent Kd values of 94–189nM. These results indicate that TRP4/Ca2+-CaM are parts of a signalling complex involved in agonist-induced Ca2+ entry.

scholarly journals Gonadotropin-Releasing Hormone Inhibits Ether-à-Go-Go-Related Gene K+ Currents in Mouse Gonadotropes

Endocrinology ◽  
2010 ◽  
Vol 151 (3) ◽  
pp. 1079-1088 ◽  
Author(s):  
Wiebke Hirdes ◽  
Crenguta Dinu ◽  
Christiane K. Bauer ◽  
Ulrich Boehm ◽  
Jürgen R. Schwarz
Keyword(s):  
Resting Potential ◽  
Related Gene ◽  
Activation Curve ◽  
Signal Cascade ◽  
Primary Mouse ◽  
Voltage Dependent ◽  
K Current ◽  
Unknown Signal ◽  
Ca2 Channels ◽  
K Currents

Secretion of LH from gonadotropes is initiated by a GnRH-induced increase in intracellular Ca2+ concentration ([Ca2+]i). This increase in [Ca2+]i is the result of Ca2+ release from intracellular stores and Ca2+ influx through voltage-dependent Ca2+ channels. Here we describe an ether-à-go-go-related gene (erg) K+ current in primary mouse gonadotropes and its possible function in the control of Ca2+ influx. To detect gonadotropes, we used a knock-in mouse strain, in which GnRH receptor-expressing cells are fluorescently labeled. Erg K+ currents were recorded in 80–90% of gonadotropes. Blockage of erg currents by E-4031 depolarized the resting potential by 5–8 mV and led to an increase in [Ca2+]i, which was abolished by nifedipine. GnRH inhibited erg currents by a reduction of the maximal erg current and in some cells additionally by a shift of the activation curve to more positive potentials. In conclusion, the erg current contributes to the maintenance of the resting potential in gonadotropes, thereby securing a low [Ca2+]i by restricting Ca2+ influx. In addition, the erg channels are modulated by GnRH by an as-yet unknown signal cascade.

scholarly journals Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261960
Author(s):  
Ana Laura López-Serrano ◽  
Rodrigo Zamora-Cárdenas ◽  
Iván A. Aréchiga-Figueroa ◽  
Pedro D. Salazar-Fajardo ◽  
Tania Ferrer ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Gradual Increase ◽  
Relaxation Property ◽  
Voltage Sensitivity ◽  
Response Curves ◽  
Deactivation Kinetics ◽  
Relative Affinity ◽  
Voltage Dependent ◽  
K Current ◽  
Inhibitory Regulation

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

Delay of desensitization onset by potassium ion in voltage-clamped frog muscle fibers

1985 ◽  
Vol 249 (5) ◽  
pp. C435-C446 ◽  
Author(s):  
A. A. Manthey
Keyword(s):  
Voltage Clamp ◽  
Acetylcholine Receptors ◽  
Transmembrane Potential ◽  
Direct Action ◽  
Direct Interaction ◽  
Frog Muscle ◽  
Average Potential ◽  
Voltage Dependent ◽  
Source Voltage ◽  
K Concentration

Increase in extracellular K+ concentration causes delay in desensitization onset during prolonged application of carbamylcholine to the postjunctional membrane in muscle. This could be due to a direct action of K+ on acetylcholine receptors or to some change in the receptors related to K+-induced effects on transmembrane potential. The question of direct vs. voltage-dependent action of K+ was investigated in frog muscle (Rana pipiens) using a point-source voltage clamp. In conductance measurements first without voltage control, desensitization rate in bath media containing 33 mM K+ was -0.198 s-1 among fibers showing an average potential of -30 mV and -0.104 s-1 in 165 mM K+ where the average potential was -2 mV, a decrease of 47%. By comparison, in voltage-clamp tests at a nominal holding potential of +20 mV, increasing extracellular K+ from 33 to 165 mM caused a decrease of 61% in desensitization rate from -0.151 to -0.059 s-1. Another series in 165 mM K+ at a holding level of +10 mV showed a decrease of 67% to a rate of 0.047 s-1. It is concluded that increases in extracellular K+ can delay desensitization onset independently of effects on transmembrane potential. It is suggested that this could result from a direct interaction of K+ with sites on the outer receptor moiety or within channels, but probably not at the inner membrane face, if the latter are considered in equilibrium with bulk intracellular K+.

Prostaglandin E2 production in rat IMCD cells. I. Stimulation by dopamine

1991 ◽  
Vol 261 (4) ◽  
pp. F647-F654 ◽  
Author(s):  
T. L. Huo ◽  
D. P. Healy
Keyword(s):  
Prostaglandin E2 ◽  
Intracellular Calcium ◽  
Calcium Release ◽  
Collecting Duct ◽  
Direct Interaction ◽  
Pge2 Production ◽  
Channel Blockers ◽  
Maximal Stimulation ◽  
Voltage Dependent ◽  
Important Regulator

Previous studies from our laboratory have determined that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to prostaglandin E2 (PGE2) production. In the present study, we have further characterized the dopamine-stimulated PGE2 response. Dopamine stimulated PGE2 production in cultured IMCD cells dose dependently (concentration for half-maximal stimulation, 11.1 microM; maximal stimulation, 235.1% of basal), an effect that was blocked by the DA2 antagonists domperidone and (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)-methyl] benzamine. Inhibition of intracellular calcium release with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (100 microM) blocked the dopamine response, whereas voltage-dependent calcium-channel blockers had no effect. Inhibition of phospholipase A2 (PLA2) activity with quinacrine (100 microM) completely blocked the dopamine-stimulated PGE2 production, whereas inhibition of polyphosphoinositol hydrolysis with neomycin (100 microM) or inhibition of protein kinase C with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 microM) did not. Pertussis toxin (PT) treatment completely blocked the dopamine-stimulated PGE2 production but not the arachidonic acid-stimulated PGE2 production. These results suggest that dopamine, acting through the DA2K receptor, may be an important regulator of PGE2 production in IMCD cells. Furthermore, our results are most consistent with either a direct interaction of the DA2K receptor with PLA2 through a PT-sensitive G protein or an indirect interaction with PLA2 through mobilization of intracellular calcium.

scholarly journals Idiosyncratic Gating of HERG-like K+ Channels in Microglia

1998 ◽  
Vol 111 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Peter S. Pennefather ◽  
Wei Zhou ◽  
Thomas E. DeCoursey
Keyword(s):  
Slow Component ◽  
Outward Current ◽  
Data Availability ◽  
Transient Outward Current ◽  
Maximal Conductance ◽  
Voltage Dependent ◽  
Simple Kinetic Model ◽  
Window Current ◽  
Herg Channels ◽  
Deactivation Process

A simple kinetic model is presented to explain the gating of a HERG-like voltage-gated K+ conductance described in the accompanying paper (Zhou, W., F.S. Cayabyab, P.S. Pennefather, L.C. Schlichter, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:781–794). The model proposes two kinetically distinct closing pathways, a rapid one favored by depolarization (deactivation) and a slow one favored by hyperpolarization (inactivation). The overlap of these two processes leads to a window current between −50 and +20 mV with a peak at −36 mV of ∼12% maximal conductance. The near absence of depolarization-activated outward current in microglia, compared with HERG channels expressed in oocytes or cardiac myocytes, can be explained if activation is shifted negatively in microglia. As seen with experimental data, availability predicted by the model was more steeply voltage dependent, and the midpoint more positive when determined by making the holding potential progressively more positive at intervals of 20 s (starting at −120 mV), rather than progressively more negative (starting at 40 mV). In the model, this hysteresis was generated by postulating slow and ultra-slow components of inactivation. The ultra-slow component takes minutes to equilibrate at −40 mV but is steeply voltage dependent, leading to protocol-dependent modulation of the HERG-like current. The data suggest that “deactivation” and “inactivation” are coupled through the open state. This is particularly evident in isotonic Cs+, where a delayed and transient outward current develops on depolarization with a decay time constant more voltage dependent and slower than the deactivation process observed at the same potential after a brief hyperpolarization.

Structural Requirements of Human Ether-a-go-go–  related Gene Channels for Block by Bupivacaine

2007 ◽  
Vol 106 (3) ◽  
pp. 523-531 ◽  
Author(s):  
Cornelia C. Siebrands ◽  
Patrick Friederich
Keyword(s):  
Aromatic Amino Acids ◽  
Selectivity Filter ◽  
Site Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Related Gene ◽  
Wild Type ◽  
Aromatic Residues ◽  
Ic50 Value ◽  
Herg Channels

Background Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. Methods Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. Results Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001). Conclusions The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.

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