scholarly journals Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

2017 ◽  
Vol 114 (10) ◽  
pp. E1857-E1865 ◽  
Author(s):  
Tomoya Kubota ◽  
Thomas Durek ◽  
Bobo Dang ◽  
Rocio K. Finol-Urdaneta ◽  
David J. Craik ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Structural Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Voltage Sensor ◽  
Structural Basis ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Voltage Dependent

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed inXenopusoocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

scholarly journals Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

2014 ◽  
Vol 144 (2) ◽  
pp. 147-157 ◽  
Author(s):  
Tamer M. Gamal El-Din ◽  
Todd Scheuer ◽  
William A. Catterall
Keyword(s):  
Membrane Potential ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Voltage Dependence ◽  
Periodic Paralysis ◽  
Membrane Potentials ◽  
Structural Basis ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Gating Charges

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.

scholarly journals Structural basis for the modulation of voltage-gated sodium channels by animal toxins

Science ◽  
2018 ◽  
Vol 362 (6412) ◽  
pp. eaau2596 ◽  
Author(s):  
Huaizong Shen ◽  
Zhangqiang Li ◽  
Yan Jiang ◽  
Xiaojing Pan ◽  
Jianping Wu ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Structural Basis ◽  
Shellfish Poisoning ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Puffer Fish ◽  
Gating Modifier ◽  
Extracellular Side ◽  
Fish And Shellfish ◽  
Shed Light

Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories—pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Navchannel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDIIand the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Navchannel drugs.

scholarly journals Understanding the State Dependence of Voltage Sensor Toxin Action on Voltage Gated Sodium Channels

2015 ◽  
Vol 108 (2) ◽  
pp. 574a
Author(s):  
Phuong T. Nguyen ◽  
Ian H. Kimball ◽  
Kenneth S. Eum ◽  
Bruce E. Cohen ◽  
Jon T. Sack ◽  
...  
Keyword(s):  
Sodium Channels ◽  
State Dependence ◽  
The State ◽  
Voltage Sensor ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

scholarly journals Valproic acid interactions with the NavMs voltage-gated sodium channel

2019 ◽  
Vol 116 (52) ◽  
pp. 26549-26554 ◽  
Author(s):  
Geancarlo Zanatta ◽  
Altin Sula ◽  
Andrew J. Miles ◽  
Leo C. T. Ng ◽  
Rubben Torella ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Anticonvulsant Drug ◽  
Drug Binding ◽  
Voltage Sensor ◽  
Full Length ◽  
Binding Studies ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

Valproic acid (VPA) is an anticonvulsant drug that is also used to treat migraines and bipolar disorder. Its proposed biological targets include human voltage-gated sodium channels, among other membrane proteins. We used the prokaryotic NavMs sodium channel, which has been shown to be a good exemplar for drug binding to human sodium channels, to examine the structural and functional interactions of VPA. Thermal melt synchrotron radiation circular dichroism spectroscopic binding studies of the full-length NavMs channel (which includes both pore and voltage sensor domains), and a pore-only construct, undertaken in the presence and absence of VPA, indicated that the drug binds to and destabilizes the channel, but not the pore-only construct. This is in contrast to other antiepileptic compounds that have previously been shown to bind in the central hydrophobic core of the pore region of the channel, and that tend to increase the thermal stability of both pore-only constructs and full-length channels. Molecular docking studies also indicated that the VPA binding site is associated with the voltage sensor, rather than the hydrophobic cavity of the pore domain. Electrophysiological studies show that VPA influences the block and inactivation rates of the NavMs channel, although with lower efficacy than classical channel-blocking compounds. It thus appears that, while VPA is capable of binding to these voltage-gated sodium channels, it has a very different mode and site of action than other anticonvulsant compounds.

scholarly journals Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ

2016 ◽  
Vol 291 (13) ◽  
pp. 7205-7220 ◽  
Author(s):  
Brad R. Green ◽  
Joanna Gajewiak ◽  
Sandeep Chhabra ◽  
Jack J. Skalicky ◽  
Min-Min Zhang ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Structural Basis ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

scholarly journals An unconventional conductance is required for pacemaking of nigral dopamine neurons

2020 ◽  
Author(s):  
Kevin Jehasse ◽  
Laurent Massotte ◽  
Sebastian Hartmann ◽  
Romain Vitello ◽  
Sofian Ringlet ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Sodium Channels ◽  
Dopamine Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Voltage Gated ◽  
Molecular Identity ◽  
Voltage Gated Sodium Channels ◽  
Voltage Dependent ◽  
Ionic Mechanisms ◽  
Voltage Gated Channels

ABSTRACTAlthough several ionic mechanisms are known to control rate and regularity of the pacemaker in dopamine (DA) neurons from the substantia nigra pars compacta (SNc), a conductance essential for pacing has yet to be defined. Here we provide pharmacological evidence that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidine (XG), an established blocker of gating pore currents in mutant voltage gated sodium channels, selectively stops pacemaking of DA SNc neurons and is without effect on the main pore of their voltage-gated channels. We isolated a voltage-dependent, non-inactivating XG-sensitive current of 20-25 pA which operates in the relevant subthreshold range and is carried by both Na+ and Cl- ions. While the molecular identity of this conductance remains to be determined, we show that this XG-sensitive current is crucial to sustain pacemaking in these neurons.

scholarly journals SCN5A mutation G615E results in NaV1.5 voltage-gated sodium channels with normal voltage-dependent function yet loss of mechanosensitivity

Channels ◽  
2019 ◽  
Vol 13 (1) ◽  
pp. 287-298 ◽  
Author(s):  
Peter R. Strege ◽  
Arnaldo Mercado-Perez ◽  
Amelia Mazzone ◽  
Yuri A. Saito ◽  
Cheryl E. Bernard ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Voltage Gated ◽  
Dependent Function ◽  
Voltage Gated Sodium Channels ◽  
Voltage Dependent ◽  
Scn5a Mutation

cAMP measurements with FRET-based sensors in excitable cells

2012 ◽  
Vol 40 (1) ◽  
pp. 179-183 ◽  
Author(s):  
Katy L. Everett ◽  
Dermot M.F. Cooper
Keyword(s):  
Energy Transfer ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Populations ◽  
Limited Information ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Distinct Cell ◽  
Ca2 Channels

The development of FRET (fluorescence resonance energy transfer)-based sensors for measuring cAMP has opened the door to sophisticated insights into single-cell cAMP dynamics. cAMP can be measured in distinct cell populations and even in distinct microdomains within cells. However, there is still only limited information on cAMP dynamics in excitable cells, particularly as a function of the activity of voltage-gated Ca2+ channels. A major reason for this is the pH shifts that can occur in excitable cells and their effects on fluorescent proteins.

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