scholarly journals The structural basis for activation of voltage sensor domains in an ion channel TPC1

2018 ◽  
Author(s):  
Alexander F. Kintzert ◽  
Evan M. Green ◽  
Pawel K. Dominik ◽  
Michael Bridges ◽  
Jean-Paul Armache ◽  
...  
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Electrical Potential ◽  
Structural Basis ◽  
Ion Conductance ◽  
Activated State ◽  
Voltage Dependent ◽  
Internal Side ◽  
Transmembrane Electrical Potential ◽  
Gating Charges

AbstractVoltage sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured the first example of a resting-state VSD in an intact ion channel. To generate an activated state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), that shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side, but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side, to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel’s requirement of cytoplasmic Ca2+-ions for activation.

scholarly journals Structural basis for activation of voltage sensor domains in an ion channel TPC1

2018 ◽  
Vol 115 (39) ◽  
pp. E9095-E9104 ◽  
Author(s):  
Alexander F. Kintzer ◽  
Evan M. Green ◽  
Pawel K. Dominik ◽  
Michael Bridges ◽  
Jean-Paul Armache ◽  
...  
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Electrical Potential ◽  
Structural Basis ◽  
Ion Conductance ◽  
Activated State ◽  
Voltage Dependent ◽  
Internal Side ◽  
Transmembrane Electrical Potential ◽  
Gating Charges

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel’s requirement of cytoplasmic Ca2+ ions for activation.

scholarly journals Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

2005 ◽  
Vol 126 (5) ◽  
pp. 419-428 ◽  
Author(s):  
Donato del Camino ◽  
Max Kanevsky ◽  
Gary Yellen
Keyword(s):  
Conformational Changes ◽  
Charge Movement ◽  
Ion Flow ◽  
K Channels ◽  
Closed State ◽  
Channel Opening ◽  
Activated State ◽  
Form Part ◽  
Voltage Dependent ◽  
Flow Through

Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

scholarly journals Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate

2021 ◽  
Author(s):  
Thomas James McCorvie ◽  
Paula M. Loria ◽  
Meihua Tu ◽  
Seungil Han ◽  
Leela Shrestha ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Glycogen Synthase ◽  
Direct Interaction ◽  
Glycogen Storage ◽  
Regulatory Subunit ◽  
Structural Basis ◽  
Phosphate Binding ◽  
Glycogen Storage Diseases ◽  
Activated State ◽  
Synthase Regulation

Glycogen synthase (GYS1), in complex with glycogenin (GYG1), is the central enzyme of muscle glycogen biosynthesis, and its inhibition has been proposed as a therapeutic avenue for various glycogen storage diseases (GSDs). GYS1 activity is inhibited by phosphorylation of its N- and C- termini, which can be relieved by allosteric activation of glucose-6-phosphate. However, the structural basis of GYS1 regulation is unclear. Here, we present the first cryo-EM structures of phosphorylated human GYS1 complexed with a minimal interacting region of GYG1 in the inhibited, activated, and catalytically competent states at resolutions of 3.0-4.0 Å. These structures reveal how phosphorylations of specific N- and C- terminal residues are sensed by different arginine clusters that lock the GYS1 tetramer complex in an inhibited state via inter-subunit interactions. The allosteric activator, glucose-6-phopshate, promotes a conformational change by disrupting these interactions and increases flexibility of GYS1 allowing for a catalytically competent state to occur when bound to the sugar donor UDP-glucose. We also identify an inhibited-like conformation that has not transitioned into the activated state, whereby the locking interaction of phosphorylation with the arginine cluster impedes the subsequent conformational changes due to glucose-6-phosphate binding. Finally, we show that the PP1 phosphatase regulatory subunit PPP1R3C (PTG) is recruited to the GYS1:GYG1 complex through direct interaction with glycogen. Our data provide the first mechanistic insights into human glycogen synthase regulation.

scholarly journals Structural titration of receptor ion channel GLIC gating by HS-AFM

2018 ◽  
Vol 115 (41) ◽  
pp. 10333-10338 ◽  
Author(s):  
Yi Ruan ◽  
Kevin Kao ◽  
Solène Lefebvre ◽  
Arin Marchesi ◽  
Pierre-Jean Corringer ◽  
...  
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
High Speed ◽  
Conformational Dynamics ◽  
Protein Protein Interactions ◽  
Ion Conductance ◽  
Single Molecule Level ◽  
Selective Channel

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein–protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

Structure and Pharmacology of Voltage-Gated Sodium and Calcium Channels

2020 ◽  
Vol 60 (1) ◽  
pp. 133-154 ◽  
Author(s):  
William A. Catterall ◽  
Michael J. Lenaeus ◽  
Tamer M. Gamal El-Din
Keyword(s):  
Calcium Channels ◽  
Structural Basis ◽  
Signaling Proteins ◽  
Ion Conductance ◽  
Voltage Gated ◽  
X Ray Crystallography ◽  
Physiological Processes ◽  
Cryo Electron Microscopy ◽  
Voltage Dependent ◽  
Structural Principles

Voltage-gated sodium and calcium channels are evolutionarily related transmembrane signaling proteins that initiate action potentials, neurotransmission, excitation-contraction coupling, and other physiological processes. Genetic or acquired dysfunction of these proteins causes numerous diseases, termed channelopathies, and sodium and calcium channels are the molecular targets for several major classes of drugs. Recent advances in the structural biology of these proteins using X-ray crystallography and cryo-electron microscopy have given new insights into the molecular basis for their function and pharmacology. Here we review this recent literature and integrate findings on sodium and calcium channels to reveal the structural basis for their voltage-dependent activation, fast and slow inactivation, ion conductance and selectivity, and complex pharmacology at the atomic level. We conclude with the theme that new understanding of the diseases and therapeutics of these channels will be derived from application of the emerging structural principles from these recent structural analyses.

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

2020 ◽  
Vol 6 (50) ◽  
pp. eabd6798
Author(s):  
Po Wei Kang ◽  
Annie M. Westerlund ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Alex K. Dou ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Conformational Changes ◽  
State Dependent ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Activated State ◽  
Voltage Dependent ◽  
Functional Consequences ◽  
Open States ◽  
Voltage Sensing Domain

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the cardiac IKs current. Although cryo–electron microscopy 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. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. 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 physiological function.

scholarly journals Gating currents

2018 ◽  
Vol 150 (7) ◽  
pp. 911-932 ◽  
Author(s):  
Francisco Bezanilla
Keyword(s):  
Membrane Proteins ◽  
Conformational Changes ◽  
Structural Changes ◽  
Ionic Currents ◽  
Gating Current ◽  
Gating Currents ◽  
Basic Principles ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels ◽  
Gating Charges

Many membrane proteins sense the voltage across the membrane where they are inserted, and their function is affected by voltage changes. The voltage sensor consists of charges or dipoles that move in response to changes in the electric field, and their movement produces an electric current that has been called gating current. In the case of voltage-gated ion channels, the kinetic and steady-state properties of the gating charges provide information of conformational changes between closed states that are not visible when observing ionic currents only. In this Journal of General Physiology Milestone, the basic principles of voltage sensing and gating currents are presented, followed by a historical description of the recording of gating currents. The results of gating current recordings are then discussed in the context of structural changes in voltage-dependent membrane proteins and how these studies have provided new insights on gating mechanisms.

scholarly journals Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels

2005 ◽  
Vol 126 (1) ◽  
pp. 55-69 ◽  
Author(s):  
Guangyu Wang ◽  
Mohammad Shahidullah ◽  
Carmen A. Rocha ◽  
Candace Strang ◽  
Paul J. Pfaffinger ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Α Subunit ◽  
Irreversible Inhibition ◽  
Tetramerization Domain ◽  
Activated State ◽  
Kv4 Channels ◽  
Voltage Dependent ◽  
Membrane Spanning ◽  
Modification Rate

Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ∼200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact α-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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