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

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.

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Is there a second external lidocaine binding site on mammalian cardiac cells?

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1989.257.1.h79 ◽

1989 ◽

Vol 257 (1) ◽

pp. H79-H84 ◽

Cited By ~ 5

Author(s):

L. A. Alpert ◽

H. A. Fozzard ◽

D. A. Hanck ◽

J. C. Makielski

Keyword(s):

Binding Site ◽

Cardiac Arrhythmias ◽

Local Anesthetics ◽

Sodium Current ◽

Cardiac Cells ◽

Na Channel ◽

Functional Difference ◽

Na Channels ◽

Internal Perfusion ◽

Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.5.c1148 ◽

1995 ◽

Vol 268 (5) ◽

pp. C1148-C1156 ◽

Cited By ~ 19

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.

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Binding affinity and stereoselectivity of local anesthetics in single batrachotoxin-activated Na+ channels.

The Journal of General Physiology ◽

10.1085/jgp.96.5.1105 ◽

1990 ◽

Vol 96 (5) ◽

pp. 1105-1127 ◽

Cited By ~ 31

Author(s):

G K Wang

Keyword(s):

Binding Site ◽

Binding Affinity ◽

Local Anesthetics ◽

Dwell Time ◽

Hydrophobic Interactions ◽

Na Channels ◽

Structural Determinants ◽

Planar Bilayers ◽

Binding Domains ◽

Hydrophobic Binding

Several local anesthetics (LA) have been previously shown to block muscle batrachotoxin (BTX)-activated Na+ channels in planar bilayers. The mean dwell time of different LA drugs, however, varies widely, from less than 10 ms to longer than several seconds. In this study, we have examined the structural determinants that govern the dwell time, the binding affinity, and the stereoselectivity of LA drugs using cocaine and bupivacaine homologues, RAC compounds, and their available stereoisomers. Our results from the structure-activity experiments reveal that (a) there are two apparent hydrophobic binding domains present in the LA binding site; one interacts with the aromatic moiety of the LA drugs, and the other interacts with the alkyl group attached to the tertiary amine of the LA drugs; (b) the LA mean dwell time and the binding affinity are largely determined by the hydrophobic interactions; (c) the LA binding site is highly stereoselective, with a difference in KD values over 50- and 6-fold for (+/-) cocaine and (+/-) bupivacaine, respectively; (d) the cocaine stereoselectivity is comparable among muscle, brain, and heart BTX-activated Na+ channels; and finally and most unexpectedly, (e) the stereoselectivity of LA drugs in BTX-activated Na+ channels appears greatly different from that reported in normal Na+ channels. Possible explanations for this difference are discussed.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.6.c1489 ◽

1993 ◽

Vol 264 (6) ◽

pp. C1489-C1499 ◽

Cited By ~ 15

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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Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers

Biophysical Journal ◽

10.1016/s0006-3495(84)84082-5 ◽

1984 ◽

Vol 46 (6) ◽

pp. 831-835 ◽

Cited By ~ 34

Author(s):

L. Olans ◽

S. Sariban-Sohraby ◽

D.J. Benos

Keyword(s):

Lipid Bilayers ◽

Planar Lipid Bilayers ◽

Na Channels

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.14.7651 ◽

1997 ◽

Vol 94 (14) ◽

pp. 7651-7654 ◽

Cited By ~ 15

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

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

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.6.c1450 ◽

1995 ◽

Vol 268 (6) ◽

pp. C1450-C1459 ◽

Cited By ~ 92

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.

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

Biophysical Journal ◽

10.1016/s0006-3495(97)78766-6 ◽

1997 ◽

Vol 72 (3) ◽

pp. 1182-1192 ◽

Cited By ~ 48

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

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Effects of luminal Na+ on single Na+ channels in A6 cells, a regulatory role for protein kinase C

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.6.f1094 ◽

1989 ◽

Vol 256 (6) ◽

pp. F1094-F1103 ◽

Cited By ~ 29

Author(s):

B. N. Ling ◽

D. C. Eaton

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Calcium Ionophore ◽

Direct Interaction ◽

Na Channel ◽

Na Channels ◽

Channel Activity ◽

Pipette Solution ◽

Kinase C ◽

Na Permeability

Na+ "self-inhibition" in tight epithelia describes the reduction in apical Na+ permeability observed with increasing luminal Na+ concentration. Patch clamp was used to examine regulation of self-inhibition at the level of single Na+ channels. After cell-attached patches (pipette solution, 129 mM NaCl) were obtained on amphibian distal nephron cells (A6), the 129 mM NaCl (high Na+) apical bath outside of the patch was replaced with 3 mM NaCl (low Na+). Within minutes there was an increase in open channel probability (Po) and the appearance of one to five "new" channels in patch membranes. A similar increase occurred when apical Na+ entry was blocked by luminal amiloride (10 microM). A23187 (1 microM), a calcium ionophore, added after low Na+ exchange, abolished the rise in channel activity. Increased Po and new channels, induced by either luminal Na+ or amiloride, were also reversed by either 4B-phorbol 12-myristate 13-acetate (PMA; 0.1 microM) or 1-oleyl-2-acetyl glycerol (OAG; 10 microM) over 15-30 min. 4 alpha-Phorbol (0.1 microM), an inactive phorbol, did not reduce channel activity. D-Sphingosine (100 microM), a protein kinase C (PKC) inhibitor, increased Po and new channels. Conclusions: 1) modulation of apical Na+ permeability by luminal Na+ does not require direct interaction of Na+ with the channel protein but, rather, appears to involve an intracellular regulatory pathway, 2) relieving self-inhibition alters both the number and kinetics of single Na+ channels, 3) the effect of low Na+ must be modulated via decreased apical Na+ entry and intracellular Na+, since amiloride yielded similar results, 4) changes in intracellular Na+ probably affect Na+ channel activity via cytosolic Ca2+, 5) the effects of decreasing luminal Na+ are reversed by PKC activators and mimicked by PKC inhibitors suggesting a possible role for PKC in Na+ self-inhibition.

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