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.

scholarly journals Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

2013 ◽  
Vol 142 (4) ◽  
pp. 351-366 ◽  
Author(s):  
Elena C. Gianulis ◽  
Qiangni Liu ◽  
Matthew C. Trudeau
Keyword(s):  
Cyclic Nucleotide ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Specific Interaction ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Nucleotide Binding ◽  
Cyclic Nucleotide Binding ◽  
Partial Restoration ◽  
Herg Channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.

scholarly journals Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

2011 ◽  
Vol 300 (4) ◽  
pp. C792-C802 ◽  
Author(s):  
Joanne E. Millership ◽  
Daniel C. Devor ◽  
Kirk L. Hamilton ◽  
Corina M. Balut ◽  
Jason I. E. Bruce ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Direct Interaction ◽  
Cell Types ◽  
P38 Map Kinase ◽  
Hek293 Cells ◽  
Jnk Signaling ◽  
Whole Cell Patch Clamp ◽  
Numerous Cell ◽  
Patch Clamp Electrophysiology ◽  
Calcium Activated Potassium Channel

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K+ ions nor promotes Ca2+ entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca2+ entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K+ efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca2+ entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.

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 Effect of Geraniol and Citronellol Essential Oils on the Biophysical Gating Properties of AMPA Receptors

2019 ◽  
Vol 9 (21) ◽  
pp. 4693 ◽  
Author(s):  
Mohammad Qneibi ◽  
Nidal Jaradat ◽  
Nour Emwas
Keyword(s):  
Essential Oils ◽  
Ampa Receptors ◽  
Neurological Diseases ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Future Drug ◽  
Drug Synthesis ◽  
Patch Clamp Electrophysiology ◽  
Calming Effect ◽  
Natural Template

Essential oils have been advertised endlessly to be very beneficial for the health of humans, and an extensive amount of research examines the validity of such claims. In contribution, the current study evaluates the neuroprotective properties of Citronellol and Geraniol essential oils (EOs). In relationship to the biophysical gating properties of different the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits, the EOs were administered to HEK293 (Human embryonic kidney 293) cells and examined for any inhibition and effect on desensitization or deactivation rates, using whole-cell patch-clamp electrophysiology. Our results demonstrated the highest levels of inhibition from Citronellol oil by four-fold on all AMPARs subunits. Likewise, Geraniol oil had a similar inhibiting impact on the receptors, and both oils decreased the desensitization and deactivation rates of the inhibited receptors. Thus, the examined EOs of this study portray neuroprotective qualities by targeting AMPARs activation and reducing desensitization and deactivation rates. Finally, the results of the current study entail a better understanding of AMPARs, provides a natural template for future drug synthesis to treat neurological diseases associated with excessive AMPAR activation, and offers a possible mechanism by which these essential oils deploy their ‘calming’ effect.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

scholarly journals Nucleus–Vacuole Junctions in Saccharomyces cerevisiae Are Formed Through the Direct Interaction of Vac8p with Nvj1p

2000 ◽  
Vol 11 (7) ◽  
pp. 2445-2457 ◽  
Author(s):  
Xiaozhou Pan ◽  
Paul Roberts ◽  
Yan Chen ◽  
Erik Kvam ◽  
Natalyia Shulga ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescent Protein ◽  
Close Contact ◽  
Direct Interaction ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Importin Α ◽  
Green Fluorescent ◽  
Pore Complexes

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including β-catenin and importin α, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus–vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40–60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in “orphan” rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Δ cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Δ andvac8-Δ cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

scholarly journals Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons

2016 ◽  
Vol 115 (3) ◽  
pp. 1307-1313 ◽  
Author(s):  
Hilary E. Wakefield ◽  
Ralph F. Fregosi ◽  
Andrew J. Fuglevand
Keyword(s):  
Time Course ◽  
Input Resistance ◽  
Current Injection ◽  
Synaptic Activity ◽  
High Concentration ◽  
Membrane Potential Change ◽  
Firing Rates ◽  
Whole Cell Patch Clamp ◽  
Patch Clamp Electrophysiology ◽  
High Firing

The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

scholarly journals Molecular Coupling of S4 to a K+ Channel's Slow Inactivation Gate

2000 ◽  
Vol 116 (5) ◽  
pp. 623-636 ◽  
Author(s):  
Eli Loots ◽  
Ehud Y. Isacoff
Keyword(s):  
Transmembrane Domain ◽  
Direct Interaction ◽  
K Channel ◽  
Slow Inactivation ◽  
Domain Interaction ◽  
Voltage Sensing ◽  
Molecular Gates ◽  
Voltage Sensing Domain ◽  
Pore Domain ◽  
Bond Disruption

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K+ channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4–pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.

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