GTP modulates run-up of whole-cell Ca2+ channel current in a Ca(2+)-dependent manner

1994 ◽  
Vol 71 (2) ◽  
pp. 814-816 ◽  
Author(s):  
J. J. Wagner ◽  
B. E. Alger
Keyword(s):  
Cell Voltage ◽  
Current Amplitude ◽  
Initial Increase ◽  
Absolute Maximum ◽  
Dependent Manner ◽  
Whole Cell ◽  
Ca1 Neurons ◽  
Channel Current ◽  
Run Up ◽  
Membrane Patch

1. Whole-cell voltage-clamp recordings were obtained from CA1 neurons acutely dissociated from rat hippocampus to study the effects of guanosine 5'-triphosphate (GTP) on the gradual increase in Ca2+ channel current amplitude that takes place over several minutes after breaking in to whole-cell mode ("run-up"). 2. Including GTP (500 microM) in the patch pipette significantly prolonged the duration of run-up of peak Ca2+ channel current to its maximum value compared with controls without GTP when the recording solutions contained Ca2+. On the other hand, GTP significantly enhanced run-up when Mg2+ and Ba2+ were substituted for intracellular and extracellular Ca2+, respectively. 3. The enhancement of run-up of the current in the Mg/Ba condition appeared to be due both to an initial increase in current amplitude that was complete within 30 s after break in and to a more rapid initial rate of run-up when compared with the Ca2+ condition. GTP did not affect the absolute maximum amplitudes of the currents in either Ca2+ or Ba2+ conditions. 4. We conclude that an early GTP-dependent modulation of Ca2+ channel current is qualitatively altered, depending on whether Ca2+ or Ba2+ is used as the charge carrier. Evidence of this modulation is apparent within seconds after rupture of the membrane patch. Conceivably, influences occurring during the period of "equilibration" with electrode contents could alter subsequent regulatory steps.

TXA2 agonists inhibit high-voltage-activated calcium channels in rat hippocampal CA1 neurons

1996 ◽  
Vol 271 (4) ◽  
pp. C1269-C1277 ◽  
Author(s):  
K. S. Hsu ◽  
C. C. Huang ◽  
W. M. Kan ◽  
P. W. Gean
Keyword(s):  
Hippocampal Neurons ◽  
Cell Voltage ◽  
Specific Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Whole Cell ◽  
Ca1 Neurons ◽  
Hippocampal Ca1 Neurons ◽  
Hippocampal Ca1 ◽  
Alpha 7

Whole cell voltage clamp recordings were used to investigate the effects of thromboxane A2 (TXA2) agonists on the voltage-dependent Ca2+ currents in rat hippocampal CA1 neurons. TXA2 agonists [1S-[1 alpha, 2 beta(5Z), 3 alpha(1E, 3S*)4 alpha ]]-7-[3-[3-hydroxy-4-(4'-iodophenoxy)-1-butenyl]-7-oxabicyclo [2,2,1]heptan-2-yl]-5-heptenoic acid (I-BOP) and U-46619, reversibly suppressed the whole cell Ca2+ currents in a concentration-dependent manner. The effect was blocked by specific TXA2 receptor antagonist, SQ-29548. I-BOP as well as U-46619 inhibited both omega-conotoxin GVIA (CgTx)-sensitive and nimodipine sensitive Ca2+ currents but had no effect on CgTx/nimodipine insensitive Ca2+ currents. The I-BOP and U-46619 inhibition of Ca2+ currents was blocked by internal dialysis of hippocampal neurons with specific protein kinase C (PKC) inhibitors, NPC-15437 and PKC inhibitor-(19-36). Pretreatment of hippocampal neurons with either 5 micrograms/ml pertussis toxin (PTX) or 5 micrograms/ml cholera toxin (CTX) did not significantly affect the suppression of the Ca2+ currents by I-BOP and U-46619. Dialyzing with 1 mM guanosine 5'-O-(3-thiotriphosphate) or 1 mM GDP significantly attenuated the I-BOP or U-46619 action. These results demonstrate that TXA2 agonists inhibit both CgTx- and nimodipine-sensitive Ca2+ currents but not CgTx/nimodipine-insensitive currents in rat hippocampal CA1 neurons via a PTX- and CTX-insensitive G protein-coupled activation of the PKC pathway.

Acute reduction in whole cell conductance in anoxic turtle brain

1999 ◽  
Vol 277 (3) ◽  
pp. R887-R893 ◽  
Author(s):  
H. S. Ghai ◽  
L. T. Buck
Keyword(s):  
Brain Slices ◽  
Cell Voltage ◽  
Dependent Manner ◽  
Recording Electrode ◽  
Whole Cell ◽  
Artificial Cerebrospinal Fluid ◽  
Intracellular Ca ◽  
Specific Antagonist ◽  
Acute Changes ◽  
Dose Dependent

We tested the effect of anoxia, a “mimic” turtle artificial cerebrospinal fluid (aCSF) consisting of high Ca2+ and Mg2+ concentrations and low pH and adenosine perfusions, on whole cell conductance ( G w) in turtle brain slices using a whole cell voltage-clamp technique. With EGTA in the recording electrode, anoxic or adenosine perfusions did not change G w significantly (values range between 2.15 ± 0.24 and 3.24 ± 0.56 nS). However, perfusion with normoxic or anoxic mimic aCSF significantly decreased G w. High [Ca2+] (4.0 or 7.8 mM) perfusions alone could reproduce the changes in G w found with the mimic perfusions. With the removal of EGTA from the recording electrode, G wdecreased significantly during both anoxic and adenosine perfusions. The A1-receptor agonist N 6-cyclopentyladenosine reduced G w in a dose-dependent manner, whereas the A1-receptor specific antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked both the adenosine- and anoxic-mediated changes in G w. These data suggest a mechanism involving A1-receptor-mediated changes in intracellular [Ca2+] that result in acute changes in G w with the onset of anoxia.

scholarly journals Human Ether-à-go-go–related Gene K+ Channel Gating Probed with Extracellular Ca2+

1999 ◽  
Vol 113 (4) ◽  
pp. 565-580 ◽  
Author(s):  
J.P. Johnson ◽  
Franklin M. Mullins ◽  
Paul B. Bennett
Keyword(s):  
Voltage Dependence ◽  
Membrane Surface ◽  
Chinese Hamster ◽  
Cell Voltage ◽  
Current Amplitude ◽  
K Channel ◽  
Related Gene ◽  
Surface Charges ◽  
Whole Cell ◽  
Inactivation Gating

Human ether-à-go-go–related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 ± 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go–related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.

HERG-Like Potassium Current Regulates the Resting Membrane Potential in Glomus Cells of the Rabbit Carotid Body

2000 ◽  
Vol 83 (3) ◽  
pp. 1150-1157 ◽  
Author(s):  
Jeffrey L. Overholt ◽  
Eckhard Ficker ◽  
Tianen Yang ◽  
Hashim Shams ◽  
Gary R. Bright ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Carotid Body ◽  
Cell Voltage ◽  
Resting Membrane Potential ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Whole Cell ◽  
Glomus Cells ◽  
Tail Current

Direct evidence for a specific K+ channel underlying the resting membrane potential in glomus cells of the carotid body has been absent. The product of the human ether-a-go-go–related gene (HERG) produces inward rectifier currents that are known to contribute to the resting membrane potential in other neuronal cells. The goal of the present study was to determine whether carotid body glomus cells express HERG-like K+ current, and if so, to determine whether a HERG-like current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20–30 mV positive to the resting membrane potential. Raising extracellular K+revealed a slowly deactivating inward tail current indicative of HERG-like K+ current. HERG-like currents were not found in cells resembling type II cells. The HERG-like current was blocked by dofetilide (DOF) in a concentration-dependent manner (IC50 = 13 ± 4 nM, mean ± SE) and high concentrations of Ba2+ (1 and 10 mM). The biophysical and pharmacological characteristics of this inward tail current suggest that it is conducted by a HERG-like channel. The steady-state activation properties of the HERG-like current ( V h = −44 ± 2 mV) suggest that it is active at the resting membrane potential in glomus cells. In whole cell, current-clamped glomus cells (average resting membrane potential, − 48 ± 4 mV), DOF, but not tetraethylammonium, caused a significant (13 mV) depolarizing shift in the resting membrane potential. Using fluorescence imaging, DOF increased [Ca2+]i in isolated glomus cells. In an in-vitro carotid body preparation, DOF increased basal sensory discharge in the carotid sinus nerve in a concentration-dependent manner. These results demonstrate that glomus cells express a HERG-like current that is active at, and responsible for controlling the resting membrane potential.

scholarly journals Dose-dependent and Isoform-specific Modulation of Ca2+ Channels by RGK GTPases

2006 ◽  
Vol 128 (5) ◽  
pp. 605-613 ◽  
Author(s):  
Lillian Seu ◽  
Geoffrey S. Pitt
Keyword(s):  
Current Amplitude ◽  
Dependent Manner ◽  
Β Subunit ◽  
Excitable Cells ◽  
Gating Kinetics ◽  
Channel Current ◽  
Mechanism Of Inhibition ◽  
High Affinity Binding Site ◽  
Dose Dependent ◽  
Ca2 Channels

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca2+ influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs—Gem, Rem, and Rem2—decreased CaV1.2 Ca2+ current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca2+ currents and affect Ca2+ influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca2+ channel accessory β subunit's interaction with its high affinity binding site in the pore-forming α1C subunit (AID) from its other putative contact sites by utilizing an α1C•β3 concatemer in which the AID was mutated to prevent β subunit interaction. This mutant concatemer generated currents with all the hallmarks of β subunit modulation, demonstrating that AID-β–independent interactions are sufficient for β subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca2+ channel current modulation.

scholarly journals Inhibition of ROMK channels by low extracellular K+ and oxidative stress

2013 ◽  
Vol 305 (2) ◽  
pp. F208-F215 ◽  
Author(s):  
Gustavo Frindt ◽  
Hui Li ◽  
Henry Sackin ◽  
Lawrence G. Palmer
Keyword(s):  
Oxidative Stress ◽  
Collecting Duct ◽  
Cell Voltage ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Pipette Solution ◽  
Whole Cell ◽  
Tert Butyl Hydroperoxide ◽  
Low K ◽  
And Oxidative Stress

We tested the hypothesis that low luminal K+ inhibits the activity of ROMK channels in the rat cortical collecting duct. Whole-cell voltage-clamp measurements of the component of outward K+ current inhibited by the bee toxin Tertiapin-Q ( ISK) showed that reducing the bath concentration ([K+]o) to 1 mM resulted in a decline of current over 2 min compared with that observed at 10 mM [K+]o. However, maintaining tubules in 1 mM [K+]o without establishing whole-cell clamp conditions did not affect ISK. The [K+]o-dependent decline was not prevented by increasing cytoplasmic-side pH or by inhibition of phosphatase activity. It was, however, abolished by the inclusion of 0.5 mM DTT in the pipette solution to prevent oxidation of the intracellular environment. Conversely, treatment of intact tubules with the oxidant H2O2 (100 μM) decreased ISK in a [K+]o-dependent manner. Treatment of the tubules with the phospholipase C inhibitor U73122 prevented the effect of low [K+]o, suggesting the involvement of this enzyme in the process. We examined these effects further using Xenopus oocytes expressing ROMK2 channels. A 50-min exposure to the permeant oxidizing agent tert-butyl hydroperoxide (t-BHP; 500 μM) did not affect outward K+ currents with [K+]o = 10 mM but reduced currents by 50% with [K+]o = 1 mM and by 75% with [K+]o = 0.1 mM. Pretreatment of the oocytes with U73122 prevented the effects of t-BHP. Under conditions of low dietary K intake, K+ secretion by distal nephron segments may be suppressed by a combination of low luminal [K+]o and oxidative stress.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2020 ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

AbstractThe patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

scholarly journals Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts

2016 ◽  
Vol 310 (2) ◽  
pp. H262-H268 ◽  
Author(s):  
Hanne C. Gadeberg ◽  
Simon M. Bryant ◽  
Andrew F. James ◽  
Clive H. Orchard
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Coronary Artery Ligation ◽  
Sham Operation ◽  
Ca Current ◽  
Artery Ligation ◽  
Whole Cell ◽  
Rat Hearts ◽  
T Tubules

In mammalian cardiac ventricular myocytes, Ca efflux via Na/Ca exchange (NCX) occurs predominantly at T tubules. Heart failure is associated with disrupted t-tubular structure, but its effect on t-tubular function is less clear. We therefore investigated t-tubular NCX activity in ventricular myocytes isolated from rat hearts ∼18 wk after coronary artery ligation (CAL) or corresponding sham operation (Sham). NCX current ( INCX) and l-type Ca current ( ICa) were recorded using the whole cell, voltage-clamp technique in intact and detubulated (DT) myocytes; intracellular free Ca concentration ([Ca]i) was monitored simultaneously using fluo-4. INCX was activated and measured during application of caffeine to release Ca from sarcoplasmic reticulum (SR). Whole cell INCX was not significantly different in Sham and CAL myocytes and occurred predominantly in the T tubules in Sham myocytes. CAL was associated with redistribution of INCX and ICa away from the T tubules to the cell surface and an increase in t-tubular INCX/ ICa density from 0.12 in Sham to 0.30 in CAL myocytes. The decrease in t-tubular INCX in CAL myocytes was accompanied by an increase in the fraction of Ca sequestered by SR. However, SR Ca content was not significantly different in Sham, Sham DT, and CAL myocytes but was significantly increased by DT of CAL myocytes. In Sham myocytes, there was hysteresis between INCX and [Ca]i, which was absent in DT Sham but present in CAL and DT CAL myocytes. These data suggest altered distribution of NCX in CAL myocytes.

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