Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ

Glibenclamide binding sites were identified in a membrane preparation from canine aortic smooth muscle. The dissociation constant for [3H]glibenclamide binding was 10 +/- 2 nM, with a density of 420 +/- 108 fmol/mg protein. The properties of ATP-sensitive potassium (KATP) channels from the same membrane preparation incorporated into planar lipid bilayers were investigated. ATP was a potent inhibitor of the channels with half-maximal inhibition of channel activity by 41 microM ATP. Glibenclamide inhibited channel activity, and cromakalim activated the channel in the presence of ATP. Blockers of Ca(2+)-activated K+ (KCa) channels (charybdotoxin and tetraethylammonium ions) did not affect KATP channels in concentrations that caused significant block of KCa channels in bilayers. This membrane preparation should allow further biochemical and functional characterization of KATP channels and glibenclamide receptors in arterial smooth muscle.

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A consensus segment in the M2 domain of the hP2X7 receptor shows ion channel activity in planar lipid bilayers and in biological membranes

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2011.09.010 ◽

2012 ◽

Vol 1818 (1) ◽

pp. 64-71 ◽

Cited By ~ 3

Author(s):

Cristina Alves Magalhães de Souza ◽

Pedro Celso Nogueira Teixeira ◽

Robson Xavier Faria ◽

Oxana Krylova ◽

Peter Pohl ◽

...

Keyword(s):

Ion Channel ◽

Lipid Bilayers ◽

Biological Membranes ◽

Planar Lipid Bilayers ◽

Channel Activity

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Channel activity of deamidated isoforms of prion protein fragment 106-126 in planar lipid bilayers

Journal of Neuroscience Research ◽

10.1002/jnr.1213 ◽

2001 ◽

Vol 66 (2) ◽

pp. 214-220 ◽

Cited By ~ 25

Author(s):

Joseph I. Kourie ◽

Peter V. Farrelly ◽

Christine L. Henry

Keyword(s):

Prion Protein ◽

Lipid Bilayers ◽

Planar Lipid Bilayers ◽

Channel Activity ◽

Protein Fragment

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Phosphorylation of Nodulin 26 on Serine 262 Affects Its Voltage-sensitive Channel Activity in Planar Lipid Bilayers

Journal of Biological Chemistry ◽

10.1074/jbc.270.45.27051 ◽

1995 ◽

Vol 270 (45) ◽

pp. 27051-27057 ◽

Cited By ~ 43

Author(s):

Jung Weon Lee ◽

Yuxin Zhang ◽

C. David Weaver ◽

Nirah H. Shomer ◽

Charles F. Louis ◽

...

Keyword(s):

Lipid Bilayers ◽

Planar Lipid Bilayers ◽

Channel Activity

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Regulation of cardiac L-type Ca channels in planar lipid bilayers by G proteins and protein phosphorylation

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.6.c1473 ◽

1993 ◽

Vol 264 (6) ◽

pp. C1473-C1479 ◽

Cited By ~ 69

Author(s):

Y. Wang ◽

C. Townsend ◽

R. L. Rosenberg

Keyword(s):

G Proteins ◽

Lipid Bilayers ◽

Okadaic Acid ◽

Initial Level ◽

Planar Lipid Bilayers ◽

Ca Channel ◽

Ca Channels ◽

Channel Activity ◽

Open Probability ◽

Gs Alpha

We have studied the effects of activated G proteins (Gs alpha and Gi1 alpha), adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA), and okadaic acid on L-type Ca channels incorporated from porcine ventricular sarcolemma into planar lipid bilayers. Channel activity evoked by membrane depolarizations diminished to extremely low levels within 2 min of incorporation (channel "rundown"). When Gs alpha [activated with guanosine 5'-O-(3-thiotriphosphate)] was present in the intracellular chamber, the initial level of channel activity was increased and rundown was delayed, so that channel activity was sustained for longer times after incorporation. The effect was specific for activated Gs alpha; activated Gi1 alpha, heat-denatured, activated Gs alpha, and unactivated Gs alpha did not augment channel activity. Activated Gi1 alpha inhibited the stimulation of Ca channel activity by Gs alpha. Treatment of the sarcolemmal membranes with PKA and Mg-ATP also increased the initial channel open probability and delayed their rundown. Addition of intracellular Gs alpha to PKA-treated channels increased the initial level of activity above that seen with PKA or Gs alpha alone, suggesting different nonocclusive pathways for the channel stimulation. This was also supported by the observation that activated Gi1 alpha had no effect on PKA-treated channels. Okadaic acid (100 nM) increased the level of Ca channel activity, suggesting that dephosphorylation by endogenous phosphatases participated in the downregulation of the channels in cell-free membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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The heterogeneity of ion channels in chromaffin granule membranes

Cellular & Molecular Biology Letters ◽

10.2478/s11658-006-0027-1 ◽

2006 ◽

Vol 11 (3) ◽

Cited By ~ 3

Author(s):

Renata Hordejuk ◽

Adam Szewczyk ◽

Krzysztof Dołowy

Keyword(s):

Lipid Bilayers ◽

Adrenal Glands ◽

Single Channel ◽

Ion Homeostasis ◽

Planar Lipid Bilayers ◽

Chromaffin Granules ◽

Channel Activity ◽

Chromaffin Granule ◽

Catecholamine Synthesis ◽

Single Channel Activity

AbstractChromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic 86Rb+ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.

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Volume Flux Across Red Cell AQP1 and E. Coli AQPZ Water Channel Proteins Reconstituted into Planar Lipid Bilayers

Molecular Biology and Physiology of Water and Solute Transport ◽

10.1007/978-1-4615-1203-5_6 ◽

2000 ◽

pp. 41-48 ◽

Cited By ~ 1

Author(s):

Sapar M. Saparov ◽

Ulrich Rothe ◽

Mario J. Borgnia ◽

Peter Agre ◽

Peter Pohl

Keyword(s):

Lipid Bilayers ◽

Water Channel ◽

Planar Lipid Bilayers ◽

Red Cell ◽

Volume Flux ◽

E Coli ◽

Channel Proteins

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Effects of Carbohydrates on the Ion Conductance of the Hemocyanin Channel

Zeitschrift für Naturforschung C ◽

10.1515/znc-1985-1-217 ◽

1985 ◽

Vol 40 (1-2) ◽

pp. 85-91

Author(s):

Gianfranco Menestrina ◽

Flavia Pasquali

Keyword(s):

Voltage Clamp ◽

Lipid Bilayers ◽

Planar Lipid Bilayers ◽

Ionic Conductance ◽

Ion Conductance ◽

Time Constants ◽

Voltage Curve ◽

High Concentrations ◽

Hemocyanin Channel

Abstract The effects of glucose and sucrose on the ionic conductance properties of the channel formed by Megathura crenulata hemocyanin in planar lipid bilayers have been studied using membranes of different compositions. It was found that glucose at high concentrations strongly affects the time constants of the current relaxations observed in membranes containing many channels after a step in the voltage clamp from ground to a positive value. At much lower concentrations both sucrose and glucose strengthened the binding of Ba2+ to the protein, what in turn has the effect to shift the conductance voltage curve of the pore towards negative potentials. The possible mechanism underlying these effects and the analogies with other studies on the interaction of sugars and alcohols with proteins have been discussed.

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The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

Biochemical Journal ◽

10.1042/bj3300559 ◽

1998 ◽

Vol 330 (1) ◽

pp. 559-564 ◽

Cited By ~ 18

Author(s):

C. Edwin THROWER ◽

J. A. Edward LEA ◽

P. Alan DAWSON

Keyword(s):

Lipid Bilayers ◽

Single Channel ◽

Planar Lipid Bilayers ◽

Channel Activity ◽

Open Probability ◽

Channel Current ◽

Single Channel Current ◽

Inositol 1,4,5 Trisphosphate ◽

Trisphosphate Receptor ◽

Artificial Bilayers

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

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