scholarly journals Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+.

1984 ◽  
Vol 84 (5) ◽  
pp. 665-686 ◽  
Author(s):  
E Moczydlowski ◽  
S S Garber ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
Membrane Vesicles ◽  
Receptor Site ◽  
Single Channels ◽  
Planar Lipid Bilayers ◽  
Na Channels ◽  
Plasma Membrane Vesicles ◽  
Voltage Range ◽  
Conduction Pathway ◽  
Almost All

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to -80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.

Anion channels in rat liver canalicular plasma membranes reconstituted into planar lipid bilayers

1992 ◽  
Vol 262 (6) ◽  
pp. G1027-G1032 ◽  
Author(s):  
M. Sellinger ◽  
S. A. Weinman ◽  
R. M. Henderson ◽  
A. Zweifach ◽  
J. L. Boyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Rat Liver ◽  
Membrane Vesicles ◽  
Planar Lipid Bilayers ◽  
Anion Channels ◽  
Plasma Membrane Vesicles ◽  
Patch Clamp Technique ◽  
Bile Formation ◽  
Cl Conductance

Previous studies from this laboratory have demonstrated a Cl(-)-HCO3- exchanger and have provided evidence for a Cl- conductance in rat liver canalicular plasma membrane vesicles. To further investigate the apical Cl- conductance, we performed single-channel analysis after incorporation of canalicular liver plasma membrane vesicles into planar lipid bilayers. This was necessary, because the canalicular membrane is not accessible for the patch-clamp technique. Two types of anion channels could be identified (30- and 90-pS conductance) corresponding to the class of small and intermediate channels, respectively. The kinetics of the small channel were found to be voltage dependent with a maximum for the open probability at -20 mV. In contrast, intermediate channel kinetics were voltage independent. The anion channels described above could allow electrogenic Cl- efflux, to compensate Cl- influx via the electroneutral Cl(-)-HCO3- exchanger. Further studies will be required to prove their functional importance in bile formation.

scholarly journals Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.

1995 ◽  
Vol 106 (3) ◽  
pp. 445-466 ◽  
Author(s):  
I I Ismailov ◽  
B K Berdiev ◽  
D J Benos
Keyword(s):  
Protein Kinase ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Single Channels ◽  
Planar Lipid Bilayers ◽  
Na Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Epithelial Na Channels ◽  
Cis And Trans

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.

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

1984 ◽  
Vol 46 (6) ◽  
pp. 831-835 ◽  
Author(s):  
L. Olans ◽  
S. Sariban-Sohraby ◽  
D.J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Na Channels

Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers

1989 ◽  
Vol 256 (4) ◽  
pp. C902-C912 ◽  
Author(s):  
R. J. Bridges ◽  
R. T. Worrell ◽  
R. A. Frizzell ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Kinetic Scheme ◽  
Disulfonic Acid ◽  
Lipid Bilayer Membranes ◽  
Plasma Membrane Vesicles ◽  
Open Probability ◽  
Cl Channel ◽  
Cl Channels

We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.

Ca(2+)-activated K+ channels in pregnant rat myometrium: modulation by a beta-adrenergic agent

1992 ◽  
Vol 263 (5) ◽  
pp. C1049-C1056 ◽  
Author(s):  
K. Anwer ◽  
L. Toro ◽  
C. Oberti ◽  
E. Stefani ◽  
B. M. Sanborn
Keyword(s):  
Lipid Bilayers ◽  
External Solution ◽  
Membrane Vesicles ◽  
K Channel ◽  
Plasma Membrane Vesicles ◽  
Beta Adrenergic ◽  
Pregnant Rat ◽  
Whole Cell ◽  
Control Value ◽  
Adrenergic Agent

The properties of Ca(2+)-activated K+ currents and channels were characterized in pregnant rat myometrium in whole cell and cell-attached patches and in lipid bilayers. Membrane depolarization of cultured myometrial cells from a holding potential of -50 to +70 mV in 10-mV steps under voltage-clamp conditions (whole cell mode) activated K+ outward currents (IK). At +70 mV, in the presence of 0.2 mM external Ca2+, the amplitude and activation time constant of IK were 15.0 +/- 2.1 microA/microF and 1.5 +/- 0.2 ms, respectively. Addition of 1 microM A23187 to the external solution increased the current from a control value of 16.0 +/- 2.0 to 67.9 +/- 9.1 microA/microF. Charybdotoxin, a blocker of Ca(2+)-activated K (KCa) channels, and a low concentration of tetraethylammonium chloride (TEA; 1 mM) decreased the amplitude of IK by 47 and 62%, respectively. In cell-attached patches from these cells, 1 microM A23187 increased the open time probability of a 143 +/- 6.0 pS K+ channel. Incorporation of plasma membrane vesicles from pregnant myometrium into lipid bilayers resulted in one predominant type of K+ channel. The unitary conductance of the K+ channel was 326 +/- 9.0 pS in symmetrical 450 mM KCl. The channel activation was both voltage and Ca2+ dependent. TEA inhibited the channel activity with a dissociation constant (Kd) of 378 +/- 10 microM at -60 mV or 1,477 +/- 80 microM at +60 mV. The whole cell currents were found to be stimulated by isoproterenol, a beta-adrenergic agent.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins.

1984 ◽  
Vol 84 (5) ◽  
pp. 687-704 ◽  
Author(s):  
E Moczydlowski ◽  
S Hall ◽  
S S Garber ◽  
G S Strichartz ◽  
C Miller
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Hydroxyl Groups ◽  
Binding Affinities ◽  
Na Channels ◽  
Plasma Membrane Vesicles ◽  
Toxin Binding ◽  
Voltage Dependent ◽  
Toxin Receptor

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field.

scholarly journals Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel.

1987 ◽  
Vol 90 (3) ◽  
pp. 427-449 ◽  
Author(s):  
C Miller ◽  
R Latorre ◽  
I Reisin
Keyword(s):  
Lipid Bilayers ◽  
Dissociation Rate ◽  
K Channel ◽  
Single Channels ◽  
Closed Channel ◽  
Association Rate ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
High Conductance

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.

scholarly journals Two P domain potassium channels (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Douglas A. Bayliss ◽  
Gábor Czirják ◽  
Péter Enyedi ◽  
Steve A.N. Goldstein ◽  
Florian Lesage ◽  
...  
Keyword(s):  
Single Channels ◽  
Primary Sequence ◽  
Α Subunit ◽  
Voltage Range ◽  
K Channels ◽  
Structural And Functional Properties ◽  
Conduction Pathway ◽  
K2p Channels ◽  
The Common ◽  
P Domain

The 4TM family of K channels mediate many of the background potassium currents observed in native cells. They are open across the physiological voltage-range and are regulated by a wide array of neurotransmitters and biochemical mediators. The pore-forming α-subunit contains two pore loop (P) domains and two subunits assemble to form one ion conduction pathway lined by four P domains. It is important to note that single channels do not have two pores but that each subunit has two P domains in its primary sequence; hence the name two P domain, or K2P channels (and not two-pore channels). Some of the K2P subunits can form heterodimers across subfamilies (e.g. K2P3.1 with K2P9.1). The nomenclature of 4TM K channels in the literature is still a mixture of IUPHAR and common names. The suggested division into subfamilies, described in the More detailed introduction, is based on similarities in both structural and functional properties within subfamilies and this explains the "common abbreviation" nomenclature in the tables below.

scholarly journals Two P domain potassium channels in GtoPdb v.2021.2

2021 ◽  
Vol 2021 (2) ◽  
Author(s):  
Austin M. Baggetta ◽  
Douglas A. Bayliss ◽  
Gábor Czirják ◽  
Péter Enyedi ◽  
Steve A.N. Goldstein ◽  
...  
Keyword(s):  
Single Channels ◽  
Primary Sequence ◽  
Α Subunit ◽  
Voltage Range ◽  
K Channels ◽  
Structural And Functional Properties ◽  
Conduction Pathway ◽  
K2p Channels ◽  
The Common ◽  
P Domain

The 4TM family of K channels mediate many of the background potassium currents observed in native cells. They are open across the physiological voltage-range and are regulated by a wide array of neurotransmitters and biochemical mediators. The pore-forming α-subunit contains two pore loop (P) domains and two subunits assemble to form one ion conduction pathway lined by four P domains. It is important to note that single channels do not have two pores but that each subunit has two P domains in its primary sequence; hence the name two P domain, or K2P channels (and not two-pore channels). Some of the K2P subunits can form heterodimers across subfamilies (e.g. K2P3.1 with K2P9.1). The nomenclature of 4TM K channels in the literature is still a mixture of IUPHAR and common names. The suggested division into subfamilies, described in the More detailed introduction, is based on similarities in both structural and functional properties within subfamilies and this explains the "common abbreviation" nomenclature in the tables below.

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