scholarly journals On the Structural Basis for Size-selective Permeation of Organic Cations through the Voltage-gated Sodium Channel

1997 ◽  
Vol 110 (6) ◽  
pp. 693-715 ◽  
Author(s):  
Ye-Ming Sun ◽  
Isabelle Favre ◽  
Laurent Schild ◽  
Edward Moczydlowski
Keyword(s):  
Complex Function ◽  
Hek293 Cells ◽  
Structural Basis ◽  
Organic Cations ◽  
Na Channel ◽  
Ionic Selectivity ◽  
Alkali Cations ◽  
Voltage Gated ◽  
Charged Residues ◽  
Molecular Sieving

Recent evidence indicates that ionic selectivity in voltage-gated Na+ channels is mediated by a small number of residues in P-region segments that link transmembrane elements S5 and S6 in each of four homologous domains denoted I, II, III, and IV. Important determinants for this function appear to be a set of conserved charged residues in the first three homologous domains, Asp(I), Glu(II), and Lys(III), located in a region of the pore called the DEKA locus. In this study, we examined several Ala-substitution mutations of these residues for alterations in ionic selectivity, inhibition of macroscopic current by external Ca2+ and H+, and molecular sieving behavior using a series of organic cations ranging in size from ammonium to tetraethylammonium. Whole-cell recording of wild-type and mutant channels of the rat muscle μ1 Na+ channel stably expressed in HEK293 cells was used to compare macroscopic current–voltage behavior in the presence of various external cations and an intracellular reference solution containing Cs+ and very low Ca2+. In particular, we tested the hypothesis that the Lys residue in domain III of the DEKA locus is responsible for restricting the permeation of large organic cations. Mutation of Lys(III) to Ala largely eliminated selectivity among the group IA monovalent alkali cations (Li+, Na+, K+, Rb+, Cs+) and permitted inward current of group IIA divalent cations (Mg2+, Ca2+, Sr2+, Ba2+). This same mutation also resulted in the acquisition of permeability to many large organic cations such as methylammonium, tetramethylammonium, and tetraethylammonium, all of which are impermeant in the native channel. The results lead to the conclusion that charged residues of the DEKA locus play an important role in molecular sieving behavior of the Na+ channel pore, a function that has been previously attributed to a hypothetical region of the channel called the “selectivity filter.” A detailed examination of individual contributions of the Asp(I), Glu(II), and Lys(III) residues and the dependence on molecular size suggests that relative permeability of organic cations is a complex function of the size, charge, and polarity of these residues and cation substrates. As judged by effects on macroscopic conductance, charged residues of the DEKA locus also appear to play a role in the mechanisms of block by external Ca2+ and H+, but are not essential for the positive shift in activation voltage that is produced by these ions.

scholarly journals Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Paul G DeCaen ◽  
Yuka Takahashi ◽  
Terry A Krulwich ◽  
Masahiro Ito ◽  
David E Clapham
Keyword(s):  
Divalent Cations ◽  
Ion Selectivity ◽  
Resting Membrane Potential ◽  
Na Channel ◽  
Ph Homeostasis ◽  
Ionic Selectivity ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Alkaliphilic Bacillus

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na+ to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na+ channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na+, Ca2+ and K+ ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na+ or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

Structural Basis for the Voltage-gated Na+ Channel Selectivity of the Scorpion α-Like Toxin BmK M1

2005 ◽  
Vol 353 (4) ◽  
pp. 788-803 ◽  
Author(s):  
Xiang Ye ◽  
Frank Bosmans ◽  
Chong Li ◽  
Ying Zhang ◽  
Da-Cheng Wang ◽  
...  
Keyword(s):  
Structural Basis ◽  
Na Channel ◽  
Voltage Gated ◽  
Channel Selectivity

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

2012 ◽  
Vol 140 (4) ◽  
pp. 435-454 ◽  
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.

scholarly journals Permeation of Large Tetra-Alkylammonium Cations through Mutant and Wild-Type Voltage-Gated Sodium Channels as Revealed by Relief of Block at High Voltage

2000 ◽  
Vol 115 (4) ◽  
pp. 435-454 ◽  
Author(s):  
Chien-Jung Huang ◽  
Isabelle Favre ◽  
Edward Moczydlowski
Keyword(s):  
Important Determinant ◽  
Organic Cations ◽  
Inward Rectification ◽  
Na Channel ◽  
Na Channels ◽  
Wild Type ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Positive Voltage ◽  
Internal Block

Many large organic cations are potent blockers of K+ channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na+ channels is an important determinant of Na+/K+ discrimination, exclusion of Ca2+, and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA+) that block Na+ channels in a voltage-dependent fashion. We investigated block by a series of TAA+ cations of the wild-type rat muscle Na+ channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA+ and larger TAA+ cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA+ cations as revealed by a positive inflection in the macroscopic I–V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA+) symmetrical TAA+ cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA+ cations with a molecular diameter as large as 15 Å can permeate Na+ channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na+ channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA+ cations and potentially other kinds of local anesthetic molecules.

scholarly journals Statistical Approach to Incorporating Experimental Variability into a Mathematical Model of the Voltage-Gated Na+ Channel and Human Atrial Action Potential

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1516
Author(s):  
Daniel Gratz ◽  
Alexander J Winkle ◽  
Seth H Weinberg ◽  
Thomas J Hund
Keyword(s):  
Action Potential ◽  
Mathematical Models ◽  
Cardiac Myocyte ◽  
Population Level ◽  
Na Channel ◽  
Model Parameters ◽  
Healthy Population ◽  
Experimental Measurements ◽  
Voltage Gated ◽  
Experimental Variability

The voltage-gated Na+ channel Nav1.5 is critical for normal cardiac myocyte excitability. Mathematical models have been widely used to study Nav1.5 function and link to a range of cardiac arrhythmias. There is growing appreciation for the importance of incorporating physiological heterogeneity observed even in a healthy population into mathematical models of the cardiac action potential. Here, we apply methods from Bayesian statistics to capture the variability in experimental measurements on human atrial Nav1.5 across experimental protocols and labs. This variability was used to define a physiological distribution for model parameters in a novel model formulation of Nav1.5, which was then incorporated into an existing human atrial action potential model. Model validation was performed by comparing the simulated distribution of action potential upstroke velocity measurements to experimental measurements from several different sources. Going forward, we hope to apply this approach to other major atrial ion channels to create a comprehensive model of the human atrial AP. We anticipate that such a model will be useful for understanding excitability at the population level, including variable drug response and penetrance of variants linked to inherited cardiac arrhythmia syndromes.

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.

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