scholarly journals Electron and proton transport by NADPH oxidases

2005 ◽  
Vol 360 (1464) ◽  
pp. 2315-2325 ◽  
Author(s):  
Nicolas Demaurex ◽  
Gábor L Petheö
Keyword(s):  
Patch Clamp ◽  
Nadph Oxidase ◽  
White Blood Cells ◽  
Proton Channel ◽  
Redox Activity ◽  
Patch Clamp Technique ◽  
Proton Channels ◽  
Clamp Technique ◽  
Electron And Proton Transport ◽  
Excised Patches

The NADPH oxidase is the main weapon of phagocytic white blood cells that are the first line of defence of our body against invading pathogens, and patients lacking a functional oxidase suffer from severe and recurrent infections. The oxidase is a multisubunit enzyme complex that transports electrons from cytoplasmic NADPH to molecular oxygen in order to generate superoxide free radicals. Electron transport across the plasma membrane is electrogenic and is associated with the flux of protons through voltage-activated proton channels. Both proton and electron currents can be recorded with the patch-clamp technique, but whether the oxidase is a proton channel or a proton channel modulator remains controversial. Recently, we have used the inside–out configuration of the patch-clamp technique to record proton and electron currents in excised patches. This approach allows us to measure the oxidase activity under very controlled conditions, and has provided new information about the enzymatic activity of the oxidase and its coupling to proton channels. In this chapter I will discuss how the unique characteristics of the electron and proton currents associated with the redox activity of the NADPH oxidase have extended our knowledge about the thermodynamics and the physiological regulation of this remarkable enzyme.

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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