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

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.

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TRPV1 pore turret dictates distinct DkTx and capsaicin gating

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809662115 ◽

2018 ◽

Vol 115 (50) ◽

pp. E11837-E11846 ◽

Cited By ~ 18

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.

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The evolved divergence of γ-secretase-susceptibility of homologous proteins Ngfrb and Nradd in zebrafish

BMC Research Notes ◽

10.1186/s13104-021-05876-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Tanya Jayne ◽

Morgan Newman ◽

Lachlan Baer ◽

Michael Lardelli

Keyword(s):

Transmembrane Domain ◽

Chimeric Protein ◽

Differential Susceptibility ◽

Duplication Event ◽

Zebrafish Embryos ◽

Structural Constraints ◽

Homologous Proteins ◽

Western Immunoblotting ◽

Secretase Activity ◽

The Stability

Abstract Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.

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

eLife ◽

10.7554/elife.48576 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

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.

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

The Journal of General Physiology ◽

10.1085/jgp.20028672 ◽

2002 ◽

Vol 120 (4) ◽

pp. 509-516 ◽

Cited By ~ 29

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.

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Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

The Journal of General Physiology ◽

10.1085/jgp.201110623 ◽

2011 ◽

Vol 138 (2) ◽

pp. 179-194 ◽

Cited By ~ 31

Author(s):

Iehab Talukder ◽

Lonnie P. Wollmuth

Keyword(s):

Nmda Receptor ◽

Ligand Binding ◽

Ion Channel ◽

Nmda Receptors ◽

Transmembrane Domain ◽

Conformational Dynamics ◽

Functional Feature ◽

Channel Pore ◽

Activation Gating ◽

Pore Opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel–forming domain, composed of M1–M4 segments, are connected by three linkers: S1–M1, M3–S2, and S2–M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3–S2 and S2–M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.

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

10.1101/624478 ◽

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.

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Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

10.1101/2020.08.27.270991 ◽

2020 ◽

Cited By ~ 1

Author(s):

Fei Jin ◽

Minxuan Sun ◽

Takashi Fujii ◽

Yurika Yamada ◽

Jin Wang ◽

...

Keyword(s):

Transmembrane Domain ◽

Channel Activity ◽

State Structure ◽

Channel Pore ◽

Closed State ◽

Channel Inactivation ◽

Pore Opening ◽

Functional Analyses ◽

Pore Domain ◽

Cytoplasmic Side

ABSTRACTMgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear.Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

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

10.1101/2020.07.04.187161 ◽

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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Transmembrane Domain 3 (TM3) Governs Orai1 and Orai3 Pore Opening in an Isoform-Specific Manner

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.635705 ◽

2021 ◽

Vol 9 ◽

Author(s):

Adéla Tiffner ◽

Lena Maltan ◽

Marc Fahrner ◽

Matthias Sallinger ◽

Sarah Weiß ◽

...

Keyword(s):

Transmembrane Domain ◽

Constitutive Activity ◽

Channel Activation ◽

Conserved Residues ◽

Closed State ◽

Specific Manner ◽

Pore Opening ◽

Domain 3 ◽

Orai Channels

Graphical AbstractOrai1 and Orai3 channel activation depends in an isoform-specific manner on two non-conserved residues in TM3 (Orai1: V181, L185, Orai3: A156, F160). Mutation of these residues to alanine leads in the absence of STIM1 to small constitutive activity of the respective Orai1 mutants, however, to huge constitutive currents of the respective Orai3 mutants. Overall, two non-conserved residues in TM3 control the maintenance of the closed state as well as an opening permissive conformation of Orai channels in an isoform-specific manner.

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