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 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.

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.

Differential Ca2+ sensitivities of BK(Ca) isochannels in bovine mesenteric vascular smooth muscle

1994 ◽  
Vol 266 (5) ◽  
pp. C1182-C1189 ◽  
Author(s):  
S. C. Sansom ◽  
J. D. Stockand
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Mesenteric Arteries ◽  
Ca Channels ◽  
Planar Bilayer ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Gating Charge ◽  
Gating Charges

The planar bilayer method was used to characterize the properties of large Ca(2+)-activated K+ [BK(Ca)] channels of smooth muscle from bovine mesenteric arteries. We found two isochannels of BK(Ca), differing in sensitivity to Ca2+ on the intracellular side of the channel. The first isochannel, Kc1, had a single-channel conductance of 287 +/- 8 pS and required a potential of -33 mV to activate to an open probability (Po) of 0.5 with 1 microM Ca2+. The single-channel conductance of the second isochannel, Kc2 (282 +/- 8 pS), was not statistically different from that of Kc1 but required a potential of 41 mV to activate to a Po of 0.5 with 1.0 microM Ca2+. At a channel voltage of 0 mV, the Ca2+ concentrations for activating Po to 0.5 were 0.2 and 10 microM for Kc1 and Kc2, respectively. The equivalent gating charges, estimated from the Boltzmann equation, were 2.4 and 2.2 for Kc1 and Kc2, respectively. The K/Cl selectivity of Kc1 was > 40 and not significantly different from Kc2. The Po of either isochannel did not change when protein kinase A or alkaline phosphatase was added to the intracellular side. We conclude that bovine mesenteric arteries contain two distinct isochannels of BK(Ca) that differ in Ca2+ sensitivity but are identical with respect to single-channel conductance, equivalent gating charge, and K+/Cl- selectivities.

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.

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 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 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.

scholarly journals Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

2013 ◽  
Vol 141 (4) ◽  
pp. 493-497 ◽  
Author(s):  
Yanyan Geng ◽  
Xiaoyu Wang ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Negative Slope ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Linear Current ◽  
Joint Application ◽  
Current Voltage ◽  
Α Subunits

Large-conductance, voltage- and Ca2+-activated K+ (BK) channels display near linear current–voltage (I-V) plots for voltages between −100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca2+ and Mg2+. This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca2+ and Mg2+ in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus β1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus β1 subunits with joint application of 2.5 mM Ca2+ plus 2.5 mM Mg2+, as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca2+/Mg2+, as they can reduce conductance and induce negative slopes.

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