scholarly journals TRPV1 pore turret dictates distinct DkTx and capsaicin gating

2018 ◽  
Vol 115 (50) ◽  
pp. E11837-E11846 ◽  
Author(s):  
Matan Geron ◽  
Rakesh Kumar ◽  
Wenchang Zhou ◽  
José D. Faraldo-Gómez ◽  
Valeria Vásquez ◽  
...  
Keyword(s):  
Physiological Response ◽  
Structure Modeling ◽  
Channel Conductance ◽  
Channel Activation ◽  
Trpv1 Channels ◽  
Gating Mechanism ◽  
Modeling Analysis ◽  
Outer Pore ◽  
Pore Opening ◽  
Unique Capability

Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans, DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel’s outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation.

scholarly journals Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Chloé Habermacher ◽  
Adeline Martz ◽  
Nicolas Calimet ◽  
Damien Lemoine ◽  
Laurie Peverini ◽  
...  
Keyword(s):  
Bound States ◽  
Transmembrane Domain ◽  
P2x Receptors ◽  
Structural Constraints ◽  
Molecular Tweezers ◽  
Gating Mechanism ◽  
Outer Pore ◽  
Pore Opening ◽  
High Resolution Models ◽  
Hinge Bending

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

scholarly journals A General Integrated Method for Design Analysis and Optimization of Missile Structure

Algorithms ◽  
2019 ◽  
Vol 12 (12) ◽  
pp. 257
Author(s):  
Xiaoguang Wang ◽  
Jun Yang ◽  
Jian Guo ◽  
Jun Guo
Keyword(s):  
Research And Development ◽  
Geometric Modeling ◽  
General Structure ◽  
Parametric Method ◽  
Secondary Development ◽  
Structure Modeling ◽  
Shape Transformation ◽  
Modeling And Analysis ◽  
Integrated Method ◽  
Modeling Analysis

In the demonstration phase of a missile scheme, to obtain the optimum proposal, designers need to modify the parameters of the overall structure frequently and significantly, and perform the structural analysis repeatedly. In order to reduce the manual workload and improve the efficiency of research and development, a general integrated method of missile structure modeling, analysis and optimization was proposed. First, CST (Class and Shape transformation functions) parametric method was used to describe the general structure of the missile. The corresponding software geometric modeling and FEM (Finite Element Method) analyzing of the missile were developed in C/C++ language on the basis of the CST parametric method and UG (Unigraphics) secondary development technology. Subsequently, a novel surrogate model-based optimation strategy was proposed to obtain a relatively light mass missile structure under existing shape size. Eventually, different missile models were used to verify the validity of the method. After executing the structure modeling, analysis and optimization modules, satisfactory results can be obtained that demonstrated the stability and adaptability of the proposed method. The method presented saves plenty of time comparing to the traditional manual modeling and analysis method, which provides a valuable technique to improve the efficiency of research and development.

scholarly journals Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

2008 ◽  
Vol 133 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Rebecca A. Falin ◽  
Rebecca Morrison ◽  
Amy-Joan L. Ham ◽  
Kevin Strange
Keyword(s):  
Cell Volume ◽  
Intracellular Signaling ◽  
Anion Channel ◽  
Type I ◽  
Dependent Manner ◽  
Channel Activation ◽  
Gating Mechanism ◽  
Channel Inhibition ◽  
C Terminus ◽  
Regulatory Phosphorylation

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl− cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.

scholarly journals Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid

2017 ◽  
Author(s):  
Ximena Steinberg ◽  
Marina A. Kasimova ◽  
Deny Cabezas-Bratesco ◽  
Jason Galpin ◽  
Ernesto Ladrón-de-Guevara ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Noise Analysis ◽  
Conformational Dynamics ◽  
Selectivity Filter ◽  
Optical Data ◽  
Transient Receptor Potential Vanilloid ◽  
Solvent Exposure ◽  
Channel Activation ◽  
Trpv1 Channels

ABSTRACTTransient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a ‘bundle crossing’ at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

scholarly journals Activity and Ca2+ regulate the mobility of TRPV1 channels in the plasma membrane of sensory neurons

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Eric N Senning ◽  
Sharona E Gordon
Keyword(s):  
Plasma Membrane ◽  
Sensory Neurons ◽  
Dorsal Root ◽  
Optical Recording ◽  
Dorsal Root Ganglion Neurons ◽  
Mechanical Stimuli ◽  
Channel Activation ◽  
Trpv1 Channels ◽  
Ganglion Neurons ◽  
Ca2 Channels

TRPV1 channels are gated by a variety of thermal, chemical, and mechanical stimuli. We used optical recording of Ca2+ influx through TRPV1 to measure activity and mobility of single TRPV1 molecules in isolated dorsal root ganglion neurons and cell lines. The opening of single TRPV1 channels produced sparklets, representing localized regions of elevated Ca2+. Unlike sparklets reported for L-type Ca2+ channels, TRPV4 channels, and AchR channels, TRPV1 channels diffused laterally in the plasma membrane as they gated. Mobility was highly variable from channel-to-channel and, to a smaller extent, from cell to cell. Most surprisingly, we found that mobility decreased upon channel activation by capsaicin, but only in the presence of extracellular Ca2+. We propose that decreased mobility of open TRPV1 could act as a diffusion trap to concentrate channels in cell regions with high activity.

scholarly journals ML277 specifically enhances the fully activated open state of KCNQ1 by modulating VSD-pore coupling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Panpan Hou ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Yuan Gao ◽  
Jianmin Cui
Keyword(s):  
Long Qt Syndrome ◽  
Xenopus Oocytes ◽  
Heart Rhythm ◽  
Long Qt ◽  
Gating Mechanism ◽  
New Strategy ◽  
Pore Opening ◽  
Qt Syndrome ◽  
Voltage Sensing Domain ◽  
Iks Channel

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form IKs channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in Xenopus oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native IKs currents.

scholarly journals Slow Inactivation Does Not Block the Aqueous Accessibility to the Outer Pore of Voltage-gated Na Channels

2002 ◽  
Vol 120 (4) ◽  
pp. 509-516 ◽  
Author(s):  
Arie F. Struyk ◽  
Stephen C. Cannon
Keyword(s):  
Reaction Rates ◽  
Slow Inactivation ◽  
Na Channels ◽  
Fast Inactivation ◽  
Voltage Gated ◽  
State Dependent ◽  
Gating Mechanism ◽  
Dependent Change ◽  
Outer Pore ◽  
Inactivation Gating

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.

scholarly journals Mechanism of pore opening in the calcium-activated chloride channel TMEM16A

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andy K. M. Lam ◽  
Raimund Dutzler
Keyword(s):  
Conformational Changes ◽  
Anion Channel ◽  
Initial Step ◽  
Ion Permeation ◽  
Autocorrelation Analysis ◽  
Channel Activation ◽  
Steady State Current ◽  
Cooperative Process ◽  
Intracellular Ca ◽  
Pore Opening

AbstractThe anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.

scholarly journals ML277 specifically enhances pore opening of KCNQ1 with VSD at the activated state by modulating VSD-pore coupling

2019 ◽  
Author(s):  
Panpan Hou ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Yuan Gao ◽  
Jianmin Cui
Keyword(s):  
Voltage Dependence ◽  
Heart Rhythm ◽  
Inactivation Kinetics ◽  
Loss Of Function ◽  
Auxiliary Subunit ◽  
Gating Mechanism ◽  
Activated State ◽  
New Strategy ◽  
Pore Opening ◽  
Voltage Sensing Domain

AbstractIn response to membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states that correspond to the stepwise activation of the voltage sensing domain (VSD) to the intermediate (I) and activated (A) states. In the heart, KCNQ1 associates with the auxiliary subunit KCNE1 to form the IKs channel that regulates heart rhythm. More than 300 of loss-of-function KCNQ1 mutations cause long QT syndrome (LQTS). KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Thus, enhancing AO state presents a potential therapy for anti-LQTS. Here, we systematically tested modulations of KCNQ1 channels by a KCNQ1 activator, ML277. It enhances the current amplitude, slows down activation, deactivation and inactivation kinetics, shifts the voltage dependence of activation to more positive voltages, decreases the Rb+/K+ permeability ratio, and selectively increases currents of mutant KCNQ1 channels that open only to the AO state. All these observations are consistent with the mechanism that ML277 specifically potentiates the AO state. On the other hand, ML277 does not affect the VSD activation, suggesting that it potentiates the AO state by enhancing the electromechanical (E-M) coupling when the VSD moves to the activated state. Our results suggest that ML277 provides a unique tool to investigate the gating mechanism of KCNQ1 and IKs channels. The specificity of ML277 to increase the AO state of native IKs currents also suggests a new strategy for anti-LQTS therapy.

scholarly journals Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

2020 ◽  
Author(s):  
Po Wei Kang ◽  
Annie M. Westerlund ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Alex K. Dou ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Conformational Change ◽  
State Dependent ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Activated State ◽  
Voltage Dependent ◽  
Functional Consequences ◽  
Pore Opening ◽  
Voltage Sensing Domain

AbstractCalmodulin (CaM) and PIP2 are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the IKs current important for repolarization of cardiac action potentials. Although cryo-EM structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully-activated state allows PIP2 to compete with CaM for binding to VSD, leading to the conformational change that alters the VSD-pore coupling. We identify a motif in the KCNQ1 cytosolic domain which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its function in the heart.

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