scholarly journals The intracellular domain of homomeric glycine receptors modulates agonist efficacy

2020 ◽  
pp. jbc.RA119.012358 ◽  
Author(s):  
Josip Ivica ◽  
Remigijus Lape ◽  
Vid Jazbec ◽  
Jie Yu ◽  
Hongtao Zhu ◽  
...  
Keyword(s):  
Single Channel ◽  
Transmembrane Helix ◽  
Glycine Receptors ◽  
Transmembrane Helices ◽  
Structural Comparison ◽  
Open Probability ◽  
Partial Agonists ◽  
C Terminus ◽  
Intracellular Domains ◽  
Limited Sequence

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for ICD in pentameric ligand-gated channels.

scholarly journals Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

scholarly journals Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

2014 ◽  
Vol 143 (2) ◽  
pp. 289-307 ◽  
Author(s):  
Line Garneau ◽  
Hélène Klein ◽  
Marie-France Lavoie ◽  
Emmanuelle Brochiero ◽  
Lucie Parent ◽  
...  
Keyword(s):  
Selectivity Filter ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Channel Pore ◽  
Transmembrane Segments ◽  
Aromatic Interactions ◽  
C Terminus ◽  
Helical Segment ◽  
Channel Opening ◽  
Dynamics Simulations

The Ca2+-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca2+ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca2+ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca2+-dependent gating of KCa3.1 originates from the binding of Ca2+ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.

scholarly journals Different agonists induce distinct single-channel conductance states in TRPV1 channels

2018 ◽  
Vol 150 (12) ◽  
pp. 1735-1746 ◽  
Author(s):  
Jesús Aldair Canul-Sánchez ◽  
Ileana Hernández-Araiza ◽  
Enrique Hernández-García ◽  
Itzel Llorente ◽  
Sara L. Morales-Lázaro ◽  
...  
Keyword(s):  
Lysophosphatidic Acid ◽  
Single Channel ◽  
Membrane Properties ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Trpv1 Channels ◽  
C Terminus ◽  
Conductance States ◽  
Positively Charged

The TRPV1 ion channel is a membrane protein that is expressed in primary afferent nociceptors, where it is activated by a diverse array of stimuli. Our prior work has shown that this channel is activated by lysophosphatidic acid (LPA), an unsaturated lysophospholipid that is produced endogenously and released under certain pathophysiological conditions, resulting in the sensation of pain. Macroscopic currents activated by saturating concentrations of LPA applied to excised membrane patches are larger in magnitude than those activated by saturating concentrations of capsaicin, which causes near-maximal TRPV1 open probability. Here we show that activation of TRPV1 by LPA is associated with a higher single-channel conductance than activation by capsaicin. We also observe that the effects of LPA on TRPV1 are not caused by an increase in the surface charge nor are they mimicked by a structurally similar lipid, ruling out the contribution of change in membrane properties. Finally, we demonstrate that the effects of LPA on the unitary conductance of TRPV1 depend upon the presence of a positively charged residue in the C terminus of the channel, suggesting that LPA induces a distinct conformational change.

scholarly journals Regulating G protein-coupled receptors by topological inversion

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Bray Denard ◽  
Sungwon Han ◽  
JungYeon Kim ◽  
Elliott M Ross ◽  
Jin Ye
Keyword(s):  
G Protein ◽  
Transmembrane Helix ◽  
Peritoneal Macrophages ◽  
G Protein Coupled Receptors ◽  
Editorial Note ◽  
Transmembrane Helices ◽  
Ccr5 Chemokine Receptor ◽  
C Terminus ◽  
G Protein Coupled ◽  
Mouse Peritoneal Macrophages

G protein-coupled receptors (GPCRs) are a family of proteins containing seven transmembrane helices, with the N- and C-terminus of the protein located at the extracellular space and cytosol, respectively. Here, we report that ceramide or related sphingolipids might invert the topology of many GPCRs that contain a GXXXN motif in their first transmembrane helix. The functional significance of this topological regulation is illustrated by the CCR5 chemokine receptor. In the absence of lipopolysaccharide (LPS), CCR5 adopts a topology consistent with that of GPCR, allowing mouse peritoneal macrophages to migrate toward its ligand CCL5. LPS stimulation results in increased production of dihydroceramide, which inverts the topology of CCR5, preventing macrophages from migrating toward CCL5. These results suggest that GPCRs may not always adopt the same topology and can be regulated through topological inversion.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells

1992 ◽  
Vol 263 (5) ◽  
pp. C1081-C1087 ◽  
Author(s):  
A. M. Frace ◽  
D. C. Eaton
Keyword(s):  
Single Channel ◽  
Gh3 Cells ◽  
Disulfonic Acid ◽  
State Transitions ◽  
Trinitrobenzene Sulfonic Acid ◽  
Voltage Sensitivity ◽  
Pituitary Cells ◽  
Amino Groups ◽  
Open Probability ◽  
Long Duration

The effects of amino group specific reagents were examined on single, large-conductance, Ca(2+)-activated, K+ channels in excised membrane patches from GH3 cells. The reagents used include trinitrobenzene sulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido derivative, and sulfophenyl-isothiocyanate. These reagents react covalently with peptide terminal amino groups and the epsilon amino groups of lysine residues, thereby removing positive charge. Internal application of 0.1-1.0 mM reagent to inside-out patches irreversibly increases channel open probability. Single-channel conductance and voltage sensitivity are not affected by modification. Analysis of channel openings and closures shows that the increase in open probability is predominantly due to the loss of long-duration closures of the channel; however, the lengths of long-duration openings are increased. After the modification in the presence of Ca2+ was performed, the channel open probability remains large, regardless of the internal Ca2+ concentration. Transitions among several open and closed states of the modified channel are present in the absence of Ca2+, suggesting that many state transitions are not directly dependent on Ca2+ binding or dissociation.

scholarly journals The inhibitory effect of Gβγ and Gβ isoform specificity on ENaC activity

2013 ◽  
Vol 305 (9) ◽  
pp. F1365-F1373 ◽  
Author(s):  
Ling Yu ◽  
Otor Al-Khalili ◽  
Billie Jeanne Duke ◽  
James D. Stockand ◽  
Douglas C. Eaton ◽  
...  
Keyword(s):  
Single Channel ◽  
Inhibitory Effect ◽  
G Protein Coupled Receptors ◽  
Open Probability ◽  
A6 Cells ◽  
Kinase C ◽  
Downstream Effectors ◽  
Isoform Specificity ◽  
Low Levels ◽  
G Protein Coupled

Epithelial Na+ channel (ENaC) activity, which determines the rate of renal Na+ reabsorption, can be regulated by G protein-coupled receptors. Regulation of ENaC by Gα-mediated downstream effectors has been studied extensively, but the effect of Gβγ dimers on ENaC is unclear. A6 cells endogenously contain high levels of Gβ1 but low levels of Gβ3, Gβ4, and Gβ5 were detected by Q-PCR. We tested Gγ2 combined individually with Gβ1 through Gβ5 expressed in A6 cells, after which we recorded single-channel ENaC activity. Among the five β and γ2 combinations, β1γ2 strongly inhibits ENaC activity by reducing both ENaC channel number ( N) and open probability ( Po) compared with control cells. In contrast, the other four β-isoforms combined with γ2 have no significant effect on ENaC activity. By using various inhibitors to probe Gβ1γ2 effects on ENaC regulation, we found that Gβ1γ2-mediated ENaC inhibition involved activation of phospholipase C-β and its enzymatic products that induce protein kinase C and ERK1/2 signaling pathways.

scholarly journals Potentiation of TRPC5 by Protons

2007 ◽  
Vol 282 (46) ◽  
pp. 33868-33878 ◽  
Author(s):  
Marcus Semtner ◽  
Michael Schaefer ◽  
Olaf Pinkenburg ◽  
Tim D. Plant
Keyword(s):  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
High Sensitivity ◽  
Transient Receptor Potential Channel ◽  
Extracellular Ph ◽  
Dependent Manner ◽  
Open Probability ◽  
Transient Receptor

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca2+-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La3+ and Gd3+. This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC50 of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H+ on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H+ or Gd3+ that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H+ indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca2+ entry and depolarization.

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

scholarly journals P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation

2021 ◽  
Vol 22 (12) ◽  
pp. 6542
Author(s):  
Kate Dunning ◽  
Adeline Martz ◽  
Francisco Andrés Peralta ◽  
Federico Cevoli ◽  
Eric Boué-Grabot ◽  
...  
Keyword(s):  
Single Channel ◽  
Cell Membrane Permeability ◽  
Large Molecule ◽  
Cholesterol Depletion ◽  
Open Probability ◽  
P2x7 Receptors ◽  
Biochemical Assays ◽  
Functional Complex ◽  
Macropore Formation ◽  
Current Facilitation

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

scholarly journals Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 2175
Author(s):  
Adonis Z. Wu ◽  
Tzu-Lun Ohn ◽  
Ren-Jay Shei ◽  
Huei-Fang Wu ◽  
Yong-Cyuan Chen ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Low Dose ◽  
Single Channel ◽  
Cell System ◽  
K Channel ◽  
Lipid Mediator ◽  
Bkca Channel ◽  
High Dose ◽  
Open Probability ◽  
Sphingosine 1 Phosphate

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

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