Malate-Regulated Channels Permeable to Anions in Vacuoles of Arabidopsis thaliana

1995 ◽  
Vol 22 (1) ◽  
pp. 115 ◽  
Author(s):  
R Cerana ◽  
L Giromini ◽  
R Colombo
Keyword(s):  
Arabidopsis Thaliana ◽  
Patch Clamp ◽  
High Resistance ◽  
Cultured Cells ◽  
Reversal Potential ◽  
Anion Channels ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Inward Currents ◽  
Cytoplasmic Side

Anion channels in isolated vacuoles of Arabidopsis thaliana cultured cells were studied by means of the patch clamp technique in the whole-vacuole configuration. In symmetrical 100 mM KCl, a high resistance of the membrane at positive potentials inside the vacuole was observed. In symmetrical 100 mM K2-malate positive potentials inside the vacuole elicited slowly developing inward currents, due to the opening of channels, which, according to measurements of reversal potential, are selective for malate. The activation potential of the channels shifted as a function of the cytoplasmic malate concentration, but it was always such that the channels opened only to mediate malate influx into the vacuole. The channels were also permeable to succinate, fumarate and, to a lesser extent, oxaloacetate. In vacuoles preincubated with cytoplas- mic malate, inward currents were also elicited in the presence of KCl or KNO3 at the cytoplasmic side of the tonoplast. Malate channels were different from the cation slow vacuolar-type channels with regard to their sensitivity to changes in the cytoplasmic concentrations of Ca2+ and ATP, and in temperature between 10 and 20�C.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

2003 ◽  
Vol 285 (3) ◽  
pp. H1347-H1355 ◽  
Author(s):  
Jin Han ◽  
Nari Kim ◽  
Hyun Joo ◽  
Euiyong Kim
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Clamp Technique ◽  
Arterial Smooth Muscle Cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine conentration value of 83.8 ± 12.9 μM. The Hill coefficient was 1.2 ± 0.3. The slope conductance of the current-voltage relationship was 320.1 ± 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.

Molecular mechanisms of action for CFTR potentiators

2019 ◽  
Author(s):  
◽  
Han-I Yeh
Keyword(s):  
Cystic Fibrosis ◽  
Patch Clamp ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Mechanisms Of Action ◽  
Drug Binding ◽  
Patch Clamp Technique ◽  
Chemical Structures ◽  
Clamp Technique ◽  
Sites Of Action

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] As the culprit behind cystic fibrosis (CF) is the dysfunction of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), pharmacological reagents targeting CFTR may hold the key to the ultimate cure of CF. In this thesis, we present the studies in the mechanisms of action for CFTR potentiators, the small molecules that enhance the functions of CFTR. Using the patch-clamp technique, we demonstrated that the permeant anion nitrate modulates CFTR gating through a mechanism similar to the FDA-approved CFTR potentiator VX-770 (ivacaftor). Via separate sites of action, VX-770 and nitrate stabilize the open channel conformation of CFTR in an energetically additive manner. Next, we investigated the action of a novel CFTR potentiator, GLPG1837, and showed that despite their different chemical structures, GLPG1837 and VX-770 share the same mechanisms of action on CFTR gating and compete for a common binding site in the transmembrane domains of CFTR. An allosteric modulation model is further proposed to explain how the affinity and efficacy of both potentiators are determined by the energetic coupling between drug binding and channel gating. Finally, we combined molecular docking and patch-clamp technique to identify the binding site(s) for GLPG1837 and VX-770.

Understanding Control of Exocytotic Secretion in Single Adenohypophysial Cells

Physiology ◽  
1994 ◽  
Vol 9 (5) ◽  
pp. 219-223
Author(s):  
Robert Zorec ◽  
G. Zupančič ◽  
M. Rupnik ◽  
L. Kocmur ◽  
S. Grilc ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cellular Level ◽  
Membrane Capacitance ◽  
Secretory Cells ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Stimulus Secretion Coupling ◽  
Insight Into

Stimulus-secretion coupling at the cellular level is studied by measuring changes in membrane capacitance in a variety of secretory cells. Attempts to gain new insight into the control of exocytosis in adenohypophysial cells by the patch-clamp technique are briefly outlined.

PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

1998 ◽  
Vol 275 (1) ◽  
pp. C293-C302 ◽  
Author(s):  
Lisa M. Middleton ◽  
Robert D. Harvey
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Perforated Patch Clamp

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

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