scholarly journals Streaming potential measurements in    -rat epithelial Na+ channel in planar lipid bilayers

1997 ◽  
Vol 94 (14) ◽  
pp. 7651-7654 ◽  
Author(s):  
I. I. Ismailov ◽  
V. G. Shlyonsky ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Streaming Potential ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Epithelial Na Channel

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.

scholarly journals Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block

1997 ◽  
Vol 72 (3) ◽  
pp. 1182-1192 ◽  
Author(s):  
I.I. Ismailov ◽  
B.K. Berdiev ◽  
V.G. Shlyonsky ◽  
D.J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Epithelial Na Channel

scholarly journals Protein kinase regulation of a cloned epithelial Na+ channel.

1996 ◽  
Vol 108 (1) ◽  
pp. 49-65 ◽  
Author(s):  
M S Awayda ◽  
I I Ismailov ◽  
B K Berdiev ◽  
C M Fuller ◽  
D J Benos
Keyword(s):  
Protein Kinase ◽  
Lipid Bilayers ◽  
Xenopus Oocytes ◽  
Cytochalasin B ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Alpha Beta ◽  
Epithelial Na Channel ◽  
Stimulation Of

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.

Peptide block of constitutively activated Na+ channels in Liddle's disease

1996 ◽  
Vol 270 (1) ◽  
pp. C214-C223 ◽  
Author(s):  
I. I. Ismailov ◽  
B. K. Berdiev ◽  
C. M. Fuller ◽  
A. L. Bradford ◽  
R. P. Lifton ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Na Channel ◽  
Na Channels ◽  
Increased Risk ◽  
Carboxy Terminal Region ◽  
Stage Renal Disease ◽  
Carboxy Terminal ◽  
End Stage ◽  
Epithelial Na Channel ◽  
Constitutively Active

Hypertension is a multifactorial disorder that results in an increased risk of cardiovascular and end-stage renal disease. Liddle's disease represents a specific hypertensive disease and expresses itself in the human population as an autosomal dominant trait. Recent experimental evidence indicates that patients with Liddle's disease have constitutively active amiloride-sensitive Na+ channels and that these channels are phenotypically expressed in lymphocytes obtained from normal and affected members of the original Liddle's kindred. Linkage analysis indicates that this disease results from a deletion of the carboxy-terminal region of the beta-subunit of a recently cloned epithelial Na+ channel (ENaC). We report the successful immunopurification and reconstitution of both normal and constitutively active lymphocyte Na+ channels into planar lipid bilayers. These channels display all of the characteristics typical of renal Na+ channels, including sensitivity to protein kinase A phosphorylation. We demonstrate that gating of normal Na+ channels is removed by cytoplasmic trypsin digestion and that the constitutively active Liddle's Na+ channels are blocked by a beta- or gamma-ENaC carboxy-terminal peptide in a GTP-dependent fashion.

scholarly journals Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers.

1988 ◽  
Vol 92 (6) ◽  
pp. 747-765 ◽  
Author(s):  
G K Wang
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Lipid Bilayers ◽  
Local Anesthetics ◽  
Direct Interaction ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Na Channels ◽  
Single Class ◽  
Na Ions

Batrachotoxin (BTX)-activated Na+ channels from rabbit skeletal muscle were incorporated into planar lipid bilayers. These channels appear to open most of the time at voltages greater than -60 mV. Local anesthetics, including QX-314, bupivacaine, and cocaine when applied internally, induce different durations of channel closures and can be characterized as "fast" (mean closed duration less than 10 ms at +50 mV), "intermediate" (approximately 80 ms), and "slow" (approximately 400 ms) blockers, respectively. The action of these local anesthetics on the Na+ channel is voltage dependent; larger depolarizations give rise to stronger binding interactions. Both the dose-response curve and the kinetics of the cocaine-induced closures indicate that there is a single class of cocaine-binding site. QX-314, though a quaternary-amine local anesthetic, apparently competes with the same binding site. External cocaine or bupivacaine application is almost as effective as internal application, whereas external QX-314 is ineffective. Interestingly, external Na+ ions reduce the cocaine binding affinity drastically, whereas internal Na+ ions have little effect. Both the cocaine association and dissociation rate constants are altered when external Na+ ion concentrations are raised. We conclude that (a) one cocaine molecule closes one BTX-activated Na+ channel in an all-or-none manner, (b) the binding affinity of cocaine is voltage sensitive, (c) this cocaine binding site can be reached by a hydrophilic pathway through internal surface and by a hydrophobic pathway through bilayer membrane, and (d) that this binding site interacts indirectly with the Na+ ions. A direct interaction between the receptor and Na+ ions seems minimal.

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 pH Alterations “Reset” Ca2+Sensitivity of Brain Na+Channel 2, a Degenerin/Epithelial Na+Ion Channel, in Planar Lipid Bilayers

2001 ◽  
Vol 276 (42) ◽  
pp. 38755-38761 ◽  
Author(s):  
Bakhrom K. Berdiev ◽  
Timothy B. Mapstone ◽  
James M. Markert ◽  
G. Yancey Gillespie ◽  
Jason Lockhart ◽  
...  
Keyword(s):  
Ion Channel ◽  
Lipid Bilayers ◽  
Na Channel ◽  
Planar Lipid Bilayers

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.

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