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.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

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.

Single-channel characteristics of a purified bovine renal amiloride-sensitive Na+ channel in planar lipid bilayers

1993 ◽  
Vol 264 (6) ◽  
pp. C1489-C1499 ◽  
Author(s):  
Y. Oh ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Dependent Manner ◽  
Na Channels ◽  
Channel Characteristics ◽  
Large Channel ◽  
Small Channels ◽  
Small Conductance

We have purified an amiloride-inhibitable Na+ channel protein from bovine renal papillae using ion-exchange and immunoaffinity chromatography. In the present study, these purified Na+ channels were reconstituted into planar lipid bilayers, and their single-channel characteristics were studied. We observed both large- and small-conductance Na(+)-selective ion channels in planar lipid bilayers. Single-channel conductance for the large- and small-conductance channels saturated as a function of Na+ concentration. These relations could be fitted by a simple Langmuir isotherm with a Michaelis constant of 55 and 45 mM and a maximum open-state conductance of 56 or 8.4 pS, respectively. Both channels were perfectly cation selective, with a Na(+)-to-K+ permeability ratio of 6.7:1 for the large channel and 7.8:1 for the small channel, and their open single-channel current-voltage relations were linear when bathed with symmetrical Na+ solutions. The percent open time of the reconstituted large or small channels varied between 10 and 50% or 1 and 20%, respectively. After application of amiloride, both the large- and small-conductance Na+ channels were inhibited in a dose-dependent manner.

A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers

1995 ◽  
Vol 268 (6) ◽  
pp. C1450-C1459 ◽  
Author(s):  
M. S. Awayda ◽  
I. I. Ismailov ◽  
B. K. Berdiev ◽  
D. J. Benos
Keyword(s):  
Hydrostatic Pressure ◽  
Pressure Gradient ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Stretch Activation ◽  
Hydrostatic Pressure Gradient ◽  
Epithelial Na Channel

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.

Regulation of low-amiloride-affinity sodium channels in alveolar type II cells

1994 ◽  
Vol 267 (1) ◽  
pp. L94-L100 ◽  
Author(s):  
G. Yue ◽  
R. L. Shoemaker ◽  
S. Matalon
Keyword(s):  
Single Channel ◽  
Alveolar Type ◽  
Type Ii ◽  
Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Ionic Substitution ◽  
Beta Agonists ◽  
Single Channel Currents ◽  
Channel Currents

We determined the mechanisms by which beta-agonists increase sodium (Na+) currents across rat alveolar type II (ATII) cells grown in primary culture. When ATII cells were patched in the cell-attached mode using symmetrical Na+ solutions (150 mM Na(+)-glutamate), single-channel currents were observed for holding potentials between -80 and 30 mV (referenced to the pipette solution) with a single-channel conductance of 27 +/- 3 pS, a mean open time (tau 1) of 3.3 +/- 0.15 ms and an open probability (Po) of 0.36 +/- 0.06 (n = 7). Addition of 10 microM terbutaline into the bath increased tau 1 to 6.43 +/- 0.5 ms and Po to 0.62 +/- 0.06 (n = 7) without affecting channel conductance. Single-channel currents with a conductance of 25 +/- 2 pS were also recorded across ATII cells patched in the inside-out mode. Addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 in the bath solution (150 mM Na(+)-glutamate) increased the single channel tau 1 from 3.26 +/- 0.15 to 7.38 +/- 0.38 and Po from 0.41 +/- 0.06 to 0.72 +/- 0.07 (n = 6) without altering conductance. Addition of 1 microM amiloride or ethylisopropylamiloride (EIPA) in the pipette solution (150 mM Na(+)-glutamate) blocked single-channel activity almost completely. Ionic substitution experiments showed the relative permeability of Na+ to K+ and Na+ to Cl- to be 7:1 and 8:1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells

1998 ◽  
Vol 111 (6) ◽  
pp. 825-846 ◽  
Author(s):  
Toru Ishikawa ◽  
Yoshinori Marunaka ◽  
Daniela Rotin
Keyword(s):  
Single Channel ◽  
Mdck Cells ◽  
Na Channel ◽  
Channel Activity ◽  
Channel Conductance ◽  
Na Current ◽  
Single Channel Conductance ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
A Charge

The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (Po), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nPo) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.

scholarly journals Single Na+ channels activated by veratridine and batrachotoxin.

1987 ◽  
Vol 89 (3) ◽  
pp. 459-480 ◽  
Author(s):  
S S Garber ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
Open Channel ◽  
Voltage Dependence ◽  
Single Channel ◽  
Reversal Potential ◽  
Na Channel ◽  
Single Channels ◽  
Na Channels ◽  
Ionic Selectivity ◽  
Symmetrical Solution

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.

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.

Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers

1993 ◽  
Vol 265 (1) ◽  
pp. C85-C91 ◽  
Author(s):  
Y. Oh ◽  
P. R. Smith ◽  
A. L. Bradford ◽  
D. Keeton ◽  
D. J. Benos
Keyword(s):  
Protein Kinase ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Apical Membrane ◽  
Polyclonal Antibodies ◽  
Na Channel ◽  
Na Channels ◽  
Lipid Bilayer Membranes ◽  
Single Channel Activity ◽  
Phosphorylation Mechanism

To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.

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