Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

1989 ◽  
Vol 110 (3) ◽  
pp. 199-207 ◽  
Author(s):  
Adrian M. Corbett ◽  
Bruce K. Krueger
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Channel Conductance ◽  
Single Channel Conductance

Cation channels from Tetrahymena cilia incorporated into planar lipid bilayers

1985 ◽  
Vol 249 (1) ◽  
pp. C177-C179 ◽  
Author(s):  
Y. Oosawa ◽  
M. Sokabe
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Cation Channel ◽  
Cation Channels ◽  
Planar Lipid Bilayers ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Ciliary Membrane ◽  
Voltage Dependent

A single cation channel from Tetrahymena cilia was incorporated into planar lipid bilayers. This channel selected for K+, Na+, and Li+ over Cl- and gluconate-, and its single channel conductance (at +25 mV) was 211 +/- 8 pS (mean +/- SE) in 100 mM K+-gluconate. The channel was not voltage dependent and may contribute to the resting K+ conductance of ciliary membrane.

scholarly journals Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

1989 ◽  
Vol 94 (5) ◽  
pp. 813-831 ◽  
Author(s):  
D S Duch ◽  
E Recio-Pinto ◽  
C Frenkel ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Reversal Potential ◽  
Channel Structure ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Protein Subunit

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

scholarly journals Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

1992 ◽  
Vol 99 (6) ◽  
pp. 897-930 ◽  
Author(s):  
C Castillo ◽  
R Villegas ◽  
E Recio-Pinto
Keyword(s):  
Binding Affinity ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Slow Process ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Fast Process ◽  
Midpoint Potential ◽  
Gating Charge

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.

Reconstitution of immunopurified alveolar type II cell Na+ channel protein into planar lipid bilayers

1995 ◽  
Vol 268 (5) ◽  
pp. C1148-C1156 ◽  
Author(s):  
O. Senyk ◽  
I. Ismailov ◽  
A. L. Bradford ◽  
R. R. Baker ◽  
S. Matalon ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Na Channel ◽  
Alveolar Type ◽  
Planar Lipid Bilayers ◽  
Type Ii ◽  
Na Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Channel Protein

Low-amiloride-affinity (L-type) Na+ channels have been functionally and immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate. The solubilized proteins were purified by ion-exchange chromatography, followed by immunoaffinity purification over a column to which rabbit polyclonal antibodies raised against purified bovine renal Na+ channel protein were bound. The proteins eluted from the immunoaffinity column were assayed for specific binding of [3H]Br-benzamil and reconstituted into planar lipid bilayers. Sequential purification steps gave a final enrichment in specific [3H]Br-benzamil binding of > 2,000 compared with the homogenate. Single-channel currents of 25 pS were recorded from the immunopurified rabbit ATII cell protein. Addition of the catalytic subunit of protein kinase A (PKA) plus ATP to the presumed cytoplasmic side of the bilayer resulted in a significant increase in the single-channel open probability (Po), from 0.40 +/- 0.14 to 0.8 +/- 0.12, without altering single-channel conductance. The addition of amiloride or ethylisopropyl amiloride (EIPA) to the side opposite that in which PKA acts reduced Po with no change in single-channel conductance. Rabbit ATII Na+ channels in bilayers had an inhibitory constant for amiloride of 8 microM and 1 microM for EIPA. These data confirm the presence of L-type Na+ channels in adult mammalian ATII cells.

scholarly journals Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures.

1987 ◽  
Vol 89 (6) ◽  
pp. 873-903 ◽  
Author(s):  
W N Green ◽  
L B Weiss ◽  
O S Andersen
Keyword(s):  
Binding Site ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Conformational Changes ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Net Charge ◽  
Toxin Binding ◽  
Wide Range ◽  
Voltage Dependent

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.

scholarly journals Clostridium perfringens Enterotoxin: The Toxin Forms Highly Cation-Selective Channels in Lipid Bilayers

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 341 ◽  
Author(s):  
Roland Benz ◽  
Michel Popoff
Keyword(s):  
Lipid Bilayers ◽  
Clostridium Perfringens ◽  
Propidium Iodide ◽  
Single Channel ◽  
Gastrointestinal Diseases ◽  
Lipid Bilayer Membranes ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Clostridium Perfringens Enterotoxin ◽  
Cation Selectivity

One of the numerous toxins produced by Clostridium perfringens is Clostridium perfringens enterotoxin (CPE), a polypeptide with a molecular mass of 35.5 kDa exhibiting three different domains. Domain one is responsible for receptor binding, domain two is involved in hexamer formation and domain three has to do with channel formation in membranes. CPE is the major virulence factor of this bacterium and acts on the claudin-receptor containing tight junctions between epithelial cells resulting in various gastrointestinal diseases. The activity of CPE on Vero cells was demonstrated by the entry of propidium iodide (PI) in the cells. The entry of propidium iodide caused by CPE was well correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. CPE formed ion-permeable channels in artificial lipid bilayer membranes with a single-channel conductance of 620 pS in 1 M KCl. The single-channel conductance was not a linear function of the bulk aqueous salt concentration indicating that point-negative charges at the CPE channel controlled ion transport. This resulted in the high cation selectivity of the CPE channels, which suggested that anions are presumably not permeable through the CPE channels. The possible role of cation transport by CPE channels in disease caused by C. perfringens is discussed.

scholarly journals Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules.

1986 ◽  
Vol 164 (1) ◽  
pp. 144-155 ◽  
Author(s):  
J D Young ◽  
E R Podack ◽  
Z A Cohn
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Lipid Vesicles ◽  
Cytotoxic T Cell ◽  
Channel Activity ◽  
Planar Bilayer ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Direct Role ◽  
Heterogeneous Size

Histocompatibility-restricted cytotoxic T lymphocytes produce circular lesions on target cell membranes. The pore-forming protein (PFP or perforin 1) that forms these membrane lesions has been purified from lymphocytes. At 37 degrees C, in the presence of Ca2+, this protein polymerizes into a supramolecular tubular complex of Mr greater than 10(6) that partially resists dissociation by SDS and reducing agents. It incorporates spontaneously into planar lipid bilayers during polymerization to form nonselective ion channels, showing heterogeneous size distribution, the smallest conductance per unit being identified as 400 pS in 0.1 M NaCl. PFP/P1 that had been assembled in lipid vesicles before incorporation into planar bilayer show much larger single channel conductance, ranging from 1 to 6 nS in 0.1 M NaCl, suggesting that PFP/P1 may assume multiple functional sizes in proportion to its state of polymerization. The reconstituted channels are relatively voltage-insensitive, with most channels persisting in the open state for seconds to minutes. Nucleated cells are rapidly depolarized by this protein. The purified protein lyses a variety of tumor cells. Polymerization and functional channel activity are absolutely Ca2+-dependent. The activity of this protein may play a direct role in T lymphocyte-mediated cytolysis.

scholarly journals Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin.

1986 ◽  
Vol 88 (1) ◽  
pp. 1-23 ◽  
Author(s):  
B U Keller ◽  
R P Hartshorne ◽  
J A Talvenheimo ◽  
W A Catterall ◽  
M Montal
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Channel Gating ◽  
Neuroblastoma Cells ◽  
Planar Lipid Bilayers ◽  
Transition Rates ◽  
Biophysical Journal ◽  
Single Channel Currents ◽  
Kinetics Of

Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313-322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation.

scholarly journals Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium.

1986 ◽  
Vol 87 (2) ◽  
pp. 327-349 ◽  
Author(s):  
J F Worley ◽  
R J French ◽  
B K Krueger
Keyword(s):  
Rat Brain ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Channel Conductance ◽  
Common Site ◽  
Brain Membrane ◽  
Planar Bilayers ◽  
Toxin Binding

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.

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