A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule

The Na+ channel is the primary target of anticonvulsants carbamazepine, phenytoin, and lamotrigine. These drugs modify Na+ channel gating as they have much higher binding affinity to the inactivated state than to the resting state of the channel. It has been proposed that these drugs bind to the Na+ channel pore with a common diphenyl structural motif. Diclofenac is a widely prescribed anti-inflammatory agent that has a similar diphenyl motif in its structure. In this study, we found that diclofenac modifies Na+ channel gating in a way similar to the foregoing anticonvulsants. The dissociation constants of diclofenac binding to the resting, activated, and inactivated Na+ channels are ∼880 μM, ∼88 μM, and ∼7 μM, respectively. The changing affinity well depicts the gradual shaping of a use-dependent receptor along the gating process. Most interestingly, diclofenac does not show the pore-blocking effect of carbamazepine on the Na+ channel when the external solution contains 150 mM Na+, but is turned into an effective Na+ channel pore blocker if the extracellular solution contains no Na+. In contrast, internal Na+ has only negligible effect on the functional consequences of diclofenac binding. Diclofenac thus acts as an “opportunistic” pore blocker modulated by external but not internal Na+, indicating that the diclofenac binding site is located at the junction of a widened part and an acutely narrowed part of the ion conduction pathway, and faces the extracellular rather than the intracellular solution. The diclofenac binding site thus is most likely located at the external pore mouth, and undergoes delicate conformational changes modulated by external Na+ along the gating process of the Na+ channel.

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On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+Channel Binding Site

Journal of Biological Chemistry ◽

10.1074/jbc.m503500200 ◽

2005 ◽

Vol 280 (28) ◽

pp. 26206-26215 ◽

Cited By ~ 26

Author(s):

Ossama B. Kashlan ◽

Shaohu Sheng ◽

Thomas R. Kleyman

Keyword(s):

Binding Site ◽

Na Channel ◽

Α Subunit ◽

Epithelial Na Channel

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Is there a second external lidocaine binding site on mammalian cardiac cells?

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1989.257.1.h79 ◽

1989 ◽

Vol 257 (1) ◽

pp. H79-H84 ◽

Cited By ~ 5

Author(s):

L. A. Alpert ◽

H. A. Fozzard ◽

D. A. Hanck ◽

J. C. Makielski

Keyword(s):

Binding Site ◽

Cardiac Arrhythmias ◽

Local Anesthetics ◽

Sodium Current ◽

Cardiac Cells ◽

Na Channel ◽

Functional Difference ◽

Na Channels ◽

Internal Perfusion ◽

Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

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A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel

Biophysical Journal ◽

10.1016/s0006-3495(94)80746-5 ◽

1994 ◽

Vol 66 (1) ◽

pp. 1-13 ◽

Cited By ~ 169

Author(s):

G.M. Lipkind ◽

H.A. Fozzard

Keyword(s):

Binding Site ◽

Structural Model ◽

Na Channel

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Specific Neosaxitoxin Interactions with the Na+ Channel Outer Vestibule Determined by Mutant Cycle Analysis

Biophysical Journal ◽

10.1016/s0006-3495(01)76049-3 ◽

2001 ◽

Vol 80 (2) ◽

pp. 698-706 ◽

Cited By ~ 29

Author(s):

Jennifer L. Penzotti ◽

Gregory Lipkind ◽

Harry A. Fozzard ◽

Samuel C. Dudley

Keyword(s):

Na Channel ◽

Outer Vestibule

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Inhibition of the Collapse of the Shaker K+ Conductance by Specific Scorpion Toxins

The Journal of General Physiology ◽

10.1085/jgp.200308871 ◽

2004 ◽

Vol 123 (3) ◽

pp. 265-279 ◽

Cited By ~ 4

Author(s):

Froylan Gómez-Lagunas ◽

Cesar V.F. Batista ◽

Timoteo Olamendi-Portugal ◽

Martha E. Ramírez-Domínguez ◽

Lourival D. Possani

Keyword(s):

Binding Site ◽

Selectivity Filter ◽

Channel Block ◽

Scorpion Toxins ◽

K Channels ◽

Outer Vestibule ◽

Plausible Model ◽

K Current ◽

Do So ◽

External K

The Shaker B K+ conductance (GK) collapses when the channels are closed (deactivated) in Na+ solutions that lack K+ ions. Also, it is known that external TEA (TEAo) impedes the collapse of GK (Gómez-Lagunas, F. 1997. J. Physiol. 499:3–15; Gómez-Lagunas, F. 2001. J. Gen. Physiol. 118:639–648), and that channel block by TEAo and scorpion toxins are two mutually exclusive events (Goldstein, S.A.N., and C. Miller. 1993. Biophys. J. 65:1613–1619). Therefore, we tested the ability of scorpion toxins to inhibit the collapse of GK in 0 K+. We have found that these toxins are not uniform regarding the capacity to protect GK. Those toxins, whose binding to the channels is destabilized by external K+, are also effective inhibitors of the collapse of GK. In addition to K+, other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K+ channels. The inhibition of the drop of GK follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K+ current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect GK in 0 K+ do so by interacting either with the most external K+ binding site of the selectivity filter (suggesting that the K+ occupancy of only that site of the pore may be enough to preserve GK) or with sites capable of binding K+ located in the outer vestibule of the pore, above the selectivity filter.

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Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

The Journal of General Physiology ◽

10.1085/jgp.201210853 ◽

2012 ◽

Vol 140 (4) ◽

pp. 435-454 ◽

Cited By ~ 23

Author(s):

Chien-Jung Huang ◽

Laurent Schild ◽

Edward G. Moczydlowski

Keyword(s):

Na Channel ◽

Ionic Selectivity ◽

Voltage Gated ◽

Trapped Ion ◽

Toxin Binding ◽

Outer Vestibule ◽

Nav Channels ◽

Na Ions ◽

Gene Isoforms ◽

Conformational Coupling

Voltage-gated Na+ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca2+ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca2+ or Na+ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca2+ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

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Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers.

The Journal of General Physiology ◽

10.1085/jgp.92.6.747 ◽

1988 ◽

Vol 92 (6) ◽

pp. 747-765 ◽

Cited By ~ 35

Author(s):

G K Wang

Keyword(s):

Binding Site ◽

Binding Affinity ◽

Lipid Bilayers ◽

Local Anesthetics ◽

Direct Interaction ◽

Na Channel ◽

Planar Lipid Bilayers ◽

Na Channels ◽

Single Class ◽

Na Ions

Batrachotoxin (BTX)-activated Na+ channels from rabbit skeletal muscle were incorporated into planar lipid bilayers. These channels appear to open most of the time at voltages greater than -60 mV. Local anesthetics, including QX-314, bupivacaine, and cocaine when applied internally, induce different durations of channel closures and can be characterized as "fast" (mean closed duration less than 10 ms at +50 mV), "intermediate" (approximately 80 ms), and "slow" (approximately 400 ms) blockers, respectively. The action of these local anesthetics on the Na+ channel is voltage dependent; larger depolarizations give rise to stronger binding interactions. Both the dose-response curve and the kinetics of the cocaine-induced closures indicate that there is a single class of cocaine-binding site. QX-314, though a quaternary-amine local anesthetic, apparently competes with the same binding site. External cocaine or bupivacaine application is almost as effective as internal application, whereas external QX-314 is ineffective. Interestingly, external Na+ ions reduce the cocaine binding affinity drastically, whereas internal Na+ ions have little effect. Both the cocaine association and dissociation rate constants are altered when external Na+ ion concentrations are raised. We conclude that (a) one cocaine molecule closes one BTX-activated Na+ channel in an all-or-none manner, (b) the binding affinity of cocaine is voltage sensitive, (c) this cocaine binding site can be reached by a hydrophilic pathway through internal surface and by a hydrophobic pathway through bilayer membrane, and (d) that this binding site interacts indirectly with the Na+ ions. A direct interaction between the receptor and Na+ ions seems minimal.

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