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.

scholarly journals Single calcium channel behavior in native skeletal muscle.

1995 ◽  
Vol 105 (2) ◽  
pp. 227-247 ◽  
Author(s):  
R T Dirksen ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channels ◽  
Single Channel ◽  
Bay K 8644 ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Whole Cell ◽  
Macroscopic Properties ◽  
Voltage Dependent

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.

scholarly journals Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties.

1989 ◽  
Vol 93 (1) ◽  
pp. 23-41 ◽  
Author(s):  
M I Behrens ◽  
A Oberhauser ◽  
F Bezanilla ◽  
R Latorre
Keyword(s):  
Optic Nerve ◽  
Dissociation Constant ◽  
Sodium Channels ◽  
Single Channel ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Apparent Dissociation Constant ◽  
Planar Bilayers ◽  
Voltage Range ◽  
Voltage Dependent

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.

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 Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

1987 ◽  
Vol 90 (3) ◽  
pp. 375-395 ◽  
Author(s):  
E Recio-Pinto ◽  
D S Duch ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Bilayers ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Single Channel Currents ◽  
Channel Currents

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

scholarly journals Deuterium oxide and temperature effects on the properties of endplate channels at the frog neuromuscular junction.

1985 ◽  
Vol 85 (2) ◽  
pp. 137-156 ◽  
Author(s):  
C A Lewis
Keyword(s):  
Temperature Dependence ◽  
Voltage Dependence ◽  
Single Channel ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Reversal Potential ◽  
The Other ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
The Mean

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).

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.

Voltage-dependent aminoglycoside blockade of the sarcoplasmic reticulum K+ channel

1986 ◽  
Vol 250 (3) ◽  
pp. C361-C364 ◽  
Author(s):  
Y. Oosawa ◽  
M. Sokabe
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Voltage Dependence ◽  
Single Channel ◽  
Phospholipid Bilayer ◽  
K Channel ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Voltage Dependent ◽  
The Way

Single-channel conductance of the K+ channel from sarcoplasmic reticulum (SR) was reduced by aminoglycoside antibiotics such as neomycin and ribostamycin and also by n-hexylamine from either side of the membrane in a dose- and voltage-dependent manner. K+ channels were incorporated into an artificial phospholipid bilayer. This inhibition follows a single-site titration curve. The voltage dependence of the inhibition is explained by assuming that these drugs bind to the open state of a single channel on one site located approximately 40% of the way through the membrane from the cis side (the side to which SR vesicles are added) when drugs are added to the cis side and bind on another site located approximately 40% of the way through the membrane from the trans side (the opposite side to the cis side) when drugs are added to the trans side.

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