scholarly journals A Structural Rearrangement in the Sodium Channel Pore Linked to Slow Inactivation and Use Dependence

2000 ◽  
Vol 116 (5) ◽  
pp. 653-662 ◽  
Author(s):  
Boon-Hooi Ong ◽  
Gordon F. Tomaselli ◽  
Jeffrey R. Balser
Keyword(s):  
Conformational Changes ◽  
Structural Rearrangement ◽  
Side Chain ◽  
Na Channel ◽  
Slow Inactivation ◽  
Na Channels ◽  
Loss Of Function ◽  
Conformational States ◽  
Hek 293 ◽  
Outer Pore

Voltage-gated sodium (Na+) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na+ channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na+ channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (IM; τ ∼ 30 ms) and slower (IS; τ ∼ 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the IM and IS kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na+ channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

scholarly journals Molecular Motions of the Outer Ring of Charge of the Sodium Channel

2003 ◽  
Vol 122 (3) ◽  
pp. 323-332 ◽  
Author(s):  
Wei Xiong ◽  
Ronald A. Li ◽  
Yanli Tian ◽  
Gordon F. Tomaselli
Keyword(s):  
Structural Rearrangement ◽  
Outer Ring ◽  
Na Channel ◽  
Slow Inactivation ◽  
Redox Catalyst ◽  
Cysteine Mutagenesis ◽  
Charged Ring ◽  
Outer Pore ◽  
Inactivation Gating

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain β1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the α carbons of these residues of no more than 7 Å. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.

scholarly journals Ultra-Slow Inactivation in μ1 Na+ Channels Is Produced by a Structural Rearrangement of the Outer Vestibule

1999 ◽  
Vol 76 (3) ◽  
pp. 1335-1345 ◽  
Author(s):  
Hannes Todt ◽  
Samuel. C. Dudley ◽  
John W. Kyle ◽  
Robert J. French ◽  
Harry A. Fozzard
Keyword(s):  
Structural Rearrangement ◽  
Slow Inactivation ◽  
Na Channels ◽  
Outer Vestibule

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 Common Molecular Determinants of Flecainide and Lidocaine Block of Heart Na+ Channels

2003 ◽  
Vol 121 (3) ◽  
pp. 199-214 ◽  
Author(s):  
Huajun Liu ◽  
Joshua Atkins ◽  
Robert S. Kass
Keyword(s):  
Structural Features ◽  
Na Channel ◽  
Na Channels ◽  
Degree Of Ionization ◽  
External Application ◽  
Hek 293 ◽  
Drug Molecules ◽  
Channel Opening ◽  
The Difference ◽  
Charged Form

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6–8.0, ∼50% neutral at pH 7.4) have similar structures but markedly different effects on Na+ channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na+ channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na+ channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na+ channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na+ channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.

scholarly journals Slow Inactivation Does Not Affect Movement of the Fast Inactivation Gate in Voltage-gated Na+ Channels

1998 ◽  
Vol 111 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Vasanth Vedantham ◽  
Stephen C. Cannon
Keyword(s):  
Na Channel ◽  
Slow Inactivation ◽  
Na Channels ◽  
Fast Inactivation ◽  
Voltage Gated ◽  
Cytoplasmic Face ◽  
Voltage Dependent ◽  
Tightly Coupled ◽  
Exclusive Processes ◽  
A Site

Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III–IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (μl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (h∞•) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2–10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

scholarly journals A conserved ring of charge in mammalian Na+channels: a molecular regulator of the outer pore conformation during slow inactivation

2006 ◽  
Vol 576 (3) ◽  
pp. 739-754 ◽  
Author(s):  
Wei Xiong ◽  
Yousaf Z. Farukhi ◽  
Yanli Tian ◽  
Deborah DiSilvestre ◽  
Ronald A. Li ◽  
...  
Keyword(s):  
Slow Inactivation ◽  
Na Channels ◽  
Outer Pore

scholarly journals Block of Tetrodotoxin-resistant Na+ Channel Pore by Multivalent Cations

2004 ◽  
Vol 124 (1) ◽  
pp. 27-42 ◽  
Author(s):  
Chung-Chin Kuo ◽  
Wan-Yu Chen ◽  
Ya-Chin Yang
Keyword(s):  
Conformational Changes ◽  
Selectivity Filter ◽  
Dorsal Root Ganglion Neurons ◽  
Inactivation Kinetics ◽  
Na Channel ◽  
Na Channels ◽  
Na Current ◽  
Single File ◽  
Pore Mouth ◽  
Ganglion Neurons

Tetrodotoxin-resistant (TTX-R) Na+ channels are much less susceptible to external TTX but more susceptible to external Cd2+ block than tetrodotoxin-sensitive (TTX-S) Na+ channels. Both TTX and Cd2+ seem to block the channel near the “DEKA” ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La3+, Zn2+, Ni2+, Co2+, and Mn2+ also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd2+, the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na+ flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na+ currents than those in outward Na+ current, signaling exit of the blocker along with the Na+ flow and a high internal energy barrier for “permeation” of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na+ current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na+ flow–dependent unblocking of the bound La3+ from the open Na+ channel, whereas channel inactivation cannot happen with any discernible speed in the La3+-blocked channel. Thus, the selectivity filter of Na+ channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is “open” to the external solution even if the channel is “closed” (“deactivated”), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.

Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells

1996 ◽  
Vol 270 (4) ◽  
pp. C998-C1010 ◽  
Author(s):  
M. L. Chalfant ◽  
T. G. O'Brien ◽  
M. M. Civan
Keyword(s):  
Collecting Duct ◽  
Cell Permeability ◽  
Na Channel ◽  
Na Channels ◽  
Cortical Collecting Duct ◽  
Ionic Selectivity ◽  
Whole Cell ◽  
Duct Cells ◽  
Excised Patches ◽  
Collecting Duct Cells

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

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