scholarly journals Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

1991 ◽  
Vol 98 (1) ◽  
pp. 77-93 ◽  
Author(s):  
C K Abrams ◽  
K S Jakes ◽  
A Finkelstein ◽  
S L Slatin
Keyword(s):  
Voltage Dependence ◽  
Ion Selectivity ◽  
Lipid Vesicles ◽  
Voltage Gating ◽  
Site Directed Mutagenesis ◽  
Phospholipid Bilayer ◽  
Voltage Gated ◽  
Colicin E1 ◽  
Gating Charge ◽  
Ion Conducting

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.

scholarly journals Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

2006 ◽  
Vol 127 (3) ◽  
pp. 309-328 ◽  
Author(s):  
Zhongming Ma ◽  
Xing Jian Lou ◽  
Frank T. Horrigan
Keyword(s):  
Voltage Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Voltage Sensing ◽  
Kv Channels ◽  
Gating Charge ◽  
Charged Residues ◽  
Sensor Activation

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

A negatively charged residue in the outer mouth of rat sodium channel determines the gating kinetics of the channel

2003 ◽  
Vol 284 (5) ◽  
pp. C1247-C1254 ◽  
Author(s):  
Zhao Zhang ◽  
Yanfang Xu ◽  
Pei Hong Dong ◽  
Dipika Sharma ◽  
Nipavan Chiamvimonvat
Keyword(s):  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Slow Inactivation ◽  
Voltage Gated ◽  
Charged Residue ◽  
Gating Charge ◽  
Rate Of Recovery ◽  
Domain I ◽  
Pore Lining

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na+ channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the μ1 skeletal muscle Na+channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na+ channel.

scholarly journals Changes in Ion Selectivity Following the Asymmetrical Addition of Charge to the Selectivity Filter of Bacterial Sodium Channels

Entropy ◽  
2020 ◽  
Vol 22 (12) ◽  
pp. 1390
Author(s):  
Olena A. Fedorenko ◽  
Igor A. Khovanov ◽  
Stephen K. Roberts ◽  
Carlo Guardiani
Keyword(s):  
Molecular Dynamics ◽  
Sodium Channels ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Na Channels ◽  
Site Specific ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Gating Charge ◽  
Dynamics Simulations

Voltage-gated sodium channels (NaVs) play fundamental roles in eukaryotes, but their exceptional size hinders their structural resolution. Bacterial NaVs are simplified homologues of their eukaryotic counterparts, but their use as models of eukaryotic Na+ channels is limited by their homotetrameric structure at odds with the asymmetric Selectivity Filter (SF) of eukaryotic NaVs. This work aims at mimicking the SF of eukaryotic NaVs by engineering radial asymmetry into the SF of bacterial channels. This goal was pursued with two approaches: the co-expression of different monomers of the NaChBac bacterial channel to induce the random assembly of heterotetramers, and the concatenation of four bacterial monomers to form a concatemer that can be targeted by site-specific mutagenesis. Patch-clamp measurements and Molecular Dynamics simulations showed that an additional gating charge in the SF leads to a significant increase in Na+ and a modest increase in the Ca2+ conductance in the NavMs concatemer in agreement with the behavior of the population of random heterotetramers with the highest proportion of channels with charge −5e. We thus showed that charge, despite being important, is not the only determinant of conduction and selectivity, and we created new tools extending the use of bacterial channels as models of eukaryotic counterparts.

Ion Selectivity Reversal and Induction of Voltage-Gating by Site-Directed Mutations in theParacoccus denitrificansPorin†

Biochemistry ◽  
1999 ◽  
Vol 38 (7) ◽  
pp. 2206-2212 ◽  
Author(s):  
Krishna Saxena ◽  
Viktoria Drosou ◽  
Elke Maier ◽  
Roland Benz ◽  
Bernd Ludwig
Keyword(s):  
Ion Selectivity ◽  
Voltage Gating

scholarly journals Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

2016 ◽  
Vol 147 (6) ◽  
pp. 437-449 ◽  
Author(s):  
Petronel Tuluc ◽  
Bruno Benedetti ◽  
Pierre Coste de Bagneaux ◽  
Manfred Grabner ◽  
Bernhard E. Flucher
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Voltage Dependence ◽  
Specific Interaction ◽  
Site Directed Mutagenesis ◽  
Control Voltage ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Activation Kinetics ◽  
Transmembrane Segments

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

Synthetic, Sodium-Ion-Conducting Tris(Macrocycle) Channels that Function in a Phospholipid Bilayer Membrane: An Overview

2000 ◽  
Vol 12 (1) ◽  
pp. 13-22
Author(s):  
Stephen L. De Wall ◽  
Eric S. Meadows ◽  
Clare L. Murray ◽  
Hossein Shabany ◽  
George W. Gokel
Keyword(s):  
Bilayer Membrane ◽  
Sodium Ion ◽  
Phospholipid Bilayer ◽  
Phospholipid Bilayer Membrane ◽  
Ion Conducting

Interaction of triblock co-polymer micelles with phospholipid-bilayer: a spectroscopic investigation using a potential chloride channel blocker

2015 ◽  
Vol 17 (9) ◽  
pp. 6597-6605 ◽  
Author(s):  
Aniruddha Ganguly ◽  
Soumen Ghosh ◽  
Nikhil Guchhait
Keyword(s):  
Chloride Channel ◽  
Spectroscopic Investigation ◽  
Channel Blocker ◽  
Lipid Vesicles ◽  
Experimental Results ◽  
Phospholipid Bilayer ◽  
Polymer Micelles ◽  
Binding Interaction ◽  
Chloride Channel Blocker ◽  
Egg Pc

Experimental results reveal that addition of P123 to the drug-bound egg-PC vesicles results in a preferential complexation of the drug with the Pluronic leaving the lipid vesicles aside which indicates a substantially stronger binding interaction of the drug with P123 than that with egg-PC.

scholarly journals Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

1997 ◽  
Vol 109 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Laurent Schild ◽  
Estelle Schneeberger ◽  
Ivan Gautschi ◽  
Dmitri Firsov
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Site Directed Mutagenesis ◽  
Ion Permeation ◽  
Amino Acid Residues ◽  
Α Subunit ◽  
Na Ions ◽  
Membrane Spanning ◽  
A Site ◽  
Epithelial Sodium Channel Enac

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a Ki for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

Characteristics of calcium currents in rabbit portal vein myocytes

1992 ◽  
Vol 263 (2) ◽  
pp. H453-H463 ◽  
Author(s):  
R. H. Cox ◽  
D. Katzka ◽  
M. Morad
Keyword(s):  
Portal Vein ◽  
Concentration Dependence ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Cell Voltage ◽  
Calcium Currents ◽  
Channel Blockers ◽  
Apparent Concentration ◽  
Steady State Inactivation ◽  
Rabbit Portal Vein

The properties of voltage-dependent Ca2+ channels were studied in isolated portal vein myocytes using the whole cell voltage-clamp method. Ca2+ currents (ICa) were identified based on their activation and inactivation potential, their dependence on external Ca2+ ([Ca2+]o), their suppression by organic or inorganic Ca2+ channel blockers, their augmentation by BAY K 8644, and their insensitivity to tetrodotoxin or alterations in external Na+ ([Na+]o). Changing the holding potential from -90 to -40 mV decreased ICa from 4.6 +/- 0.6 to 2.0 +/- 0.3 pA/pF at 0 mV but did not shift its voltage dependence significantly. The voltage dependence of steady-state inactivation and activation was represented by Boltzmann distributions with the following parameters: inactivation, half-maximal voltage (V0.5) = -32 +/- 7 mV and slope factor (k) = 6.1 +/- 0.2 mV; activation, V0.5 = -15 +/- 4 mV and k = 5.6 +/- 0.6 mV. Doubling the [Ca2+]o increased ICa and shifted the voltage dependence of its activation and inactivation by approximately 10 mV toward more positive potentials without altering the window currents. Substituting Na+, Ba2+, or Sr2+ for Ca2+ as the charge carrier through the Ca2+ channel slowed the rate of its inactivation and shifted its voltage dependence toward more negative potentials. Divalent selectivity of the Ca2+ channel showed an apparent concentration dependence: at 2 mMISr less than IBa = ICa, while at 10 mM ICa less than ISr = IBa. Because 50-100 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abolished the apparent concentration dependence of the divalent ion selectivity, this phenomenon was attributed to a high Ca2+ selectivity of the channel. Our data support the presence of only one type of Ca2+ channel in rabbit portal vein myocytes with characteristics similar to the L-type Ca2+ channel described in other cells, but with somewhat different divalent selectivity, holding potential, and [Na+]o dependence.

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