scholarly journals Quantitative Analysis of the Voltage-dependent Gating of Mouse Parotid ClC-2 Chloride Channel

2005 ◽  
Vol 126 (6) ◽  
pp. 591-603 ◽  
Author(s):  
Jose Antonio de Santiago ◽  
Keith Nehrke ◽  
Jorge Arreola
Keyword(s):  
Chloride Channel ◽  
Time Course ◽  
Inward Rectification ◽  
Chloride Currents ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
State Models ◽  
New Perspective ◽  
Opening And Closing

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (Vm) and time dependence of the protopore (Pf) and common (Ps) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (ICl) were measured using whole cell patch clamp. WT channels had ICl that showed inward rectification and biexponential time course. Time constants of fast and slow components were ∼10-fold different at negative Vm and corresponded to Pf and Ps, respectively. Pf and Ps were ∼1 at −200 mV, while at Vm ≥ 0 mV, Pf ∼ 0 and Ps ∼ 0.6. Hence, Pf dominated open kinetics at moderately negative Vm, while at very negative Vm both gates contributed to gating. At Vm ≥ 0 mV, mClC-2 closes by shutting off Pf. Three- and two-state models described the open-to-closed transitions of Pf and Ps, respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter Pf or Ps in other ClC channels. Based on the time and Vm dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to Pf, the dominant regulator of gating, while the C258 residue alters the Vm dependence of Pf, probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

Functional and pharmacological properties of canine ERG potassium channels

2003 ◽  
Vol 284 (1) ◽  
pp. H256-H267 ◽  
Author(s):  
Jixin Wang ◽  
Kimberly Della Penna ◽  
Hao Wang ◽  
Jerzy Karczewski ◽  
Thomas M. Connolly ◽  
...  
Keyword(s):  
Inward Rectification ◽  
Delayed Rectifier ◽  
Pharmacological Properties ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Channel Opening ◽  
Maximum Activation ◽  
Voltage Dependent ◽  
Cardiac Delayed Rectifier ◽  
Action Potential Repolarization

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K+ channels and examined their biophysical and pharmacological properties with whole cell patch clamp and35S-labeled MK-499 ([35S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K+ current ( I Kr). Channel opening was time- and voltage dependent with threshold near −40 mV. The half-maximum activation voltage was −7.8 ± 2.4 mV at 23°C, shifting to −31.9 ± 1.2 mV at 36°C. Channels activated with a time constant of 13 ± 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K+ selective (Na+-to-K+permeability ratio = 0.007), and were potently inhibited by I Kr blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC50 values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [35S]MK-499 binding from cERG and hERG with IC50 values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

scholarly journals Characterization of α3 Glycine Receptors with Ginkgolide B and Picrotoxin

2018 ◽  
Author(s):  
Sampurna Chakrabarti ◽  
Anil Neelakantan ◽  
Malcolm M. Slaughter
Keyword(s):  
Beta Subunits ◽  
Ginkgolide B ◽  
Glycine Receptors ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Voltage Dependent ◽  
The Central Nervous System ◽  
Subunit Isoforms

AbstractGinkgolide B (GB) and picrotoxin (PTX) are antagonists of the major inhibitory receptors of the central nervous system: GABA and glycine receptors (GlyRs). GlyRs contain one or more of the four alpha subunit isoforms of which α1 and α2 have been extensively studied. This report compares GB and PTX block of α3 GlyRs expressed in HEK 293 cells, using whole-cell patch clamp techniques. In CNS, α3 exists as a heteropentamer in conjunction with beta subunits in a 2α:3β ratio. Thus, the nature of block was also tested in α3β heteromeric glycine receptors. GB and PTX blocked α3 GlyRs both in the presence (liganded state) and absence of glycine (unliganded state). This property is unique to α3 subunits; α1 and α2 subunits are only blocked in the liganded state. The GB block of α3 GlyRs is voltage-dependent (more effective when the cell is depolarized) and non-competitive, while the PTX block is competitive and not voltage-dependent. The heteromeric and homomeric α3 GlyRs recovered significantly faster from unliganded GB block compared to liganded GB block, but no such distinction was found for PTX block suggesting more than one binding site for GB. This study sheds light on features of the α3 GlyR that distinguish it from the more widely studied α1 and α2 subunits. Understanding these properties can help decipher the physiological functioning of GlyRs in the CNS and may permit development of subunit specific drugs.

Low-voltage activated calcium currents increase in basal forebrain neurons from aged rats

1995 ◽  
Vol 74 (2) ◽  
pp. 876-887 ◽  
Author(s):  
D. Murchison ◽  
W. H. Griffith
Keyword(s):  
Time Course ◽  
Low Voltage ◽  
Age Groups ◽  
Medial Septum ◽  
Calcium Currents ◽  
Fischer 344 ◽  
Diagonal Band ◽  
Whole Cell Patch Clamp ◽  
Age Related ◽  
Voltage Dependent

1. Whole cell patch-clamp recordings were made of low-voltage-activated (LVA) calcium (Ca2+) currents using 2 mM barium (Ba2+) as charge carrier. Acutely dissociated neurons from medial septum (MS) and the nucleus of the diagonal band (nDB) were examined in young adult (1–3 mo) and aged (24–26 mo) Fischer 344 rats. 2. Most neurons in both age groups displayed LVA currents: 84% of young cells (110/131) and 87% in aged cells (62/71). Using cell capacitance as an indication of cell size, aged cells were significantly smaller (P < 0.05; 15.4 +/- 0.6 pF; mean +/- SE) than young cells (18.0 +/- 0.5 pF), although a single distribution of cell sizes was present in both populations. 3. The LVA currents were enhanced in cells from aged animals. When LVA currents were studied without activation of high voltage activated currents, the current density (pA/pF) was significantly (P < 0.05) increased at negative potentials in aged neurons (young: 4.92 +/- 0.35 pA/pF; Aged: 5.92 +/- 0.45 pA/pF, at a prepulse potential of -110 mV). No change in voltage-dependent activation or inactivation was seen. The time course of recovery from inactivation also was unchanged. 4. Kinetic parameters of LVA currents were compared in both age groups. No age-related difference in time-dependent activation or inactivation was observed. A single distribution of decay time constants of LVA currents was present in both age groups. 5. These results show that MS/nDB cells maintain robust LVA currents and have increased current densities in very old rats. An increased LVA current in the aged neurons suggests that their ability to fire rhythmically or in bursts is retained or enhanced and that the resulting increase in intracellular Ca2+ may contribute to an altered Ca2+ homeostasis.

Cl- current in IMCD cells activated by hypotonicity: time course, ATP dependence, and inhibitors

1996 ◽  
Vol 271 (3) ◽  
pp. F552-F559 ◽  
Author(s):  
K. A. Volk ◽  
C. Zhang ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Time Course ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Renal Medulla ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The hypertonic environment of the renal medulla can change rapidly according to the state of hydration of the animal. We used primary cultures of rat inner medullary collecting duct (IMCD) cells to investigate the characteristics of Cl- currents activated by an acute reduction in osmolarity (ICl(osm)). Using the whole cell patch-clamp technique, we identified an outwardly rectifying current that decayed slowly at strongly depolarizing voltages. The onset of ICl(osm) began 6.7 min after the fall in bath osmolarity, a delay longer than reported in other cell types. Hypotonicity did not induce an increase in intracellular Ca2+ concentration, and activation of ICl(osm) did not require the presence of Ca2+. Intracellular ATP was needed to evoke ICl(osm) when the hypotonic stimulus was modest (50 mosmol/l or less) but was not necessary when the stimulus was stronger (100 mosmol/ l). ICl(osm) was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by tamoxifen or glibenclamide. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid produced a voltage-dependent block. Acute reduction in osmolarity using cells grown on filters did not induce a Cl- secretory current. The ICl(osm) of IMCD cells appears to be on the basolateral membrane and displays some unique features.

Preferential potentiation by hypotonic cell swelling of muscarinic cation current in guinea pig ileum

1997 ◽  
Vol 272 (1) ◽  
pp. C240-C253 ◽  
Author(s):  
Y. Waniishi ◽  
R. Inoue ◽  
Y. Ito
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Muscarinic Receptor ◽  
Time Course ◽  
Cell Swelling ◽  
Patch Clamp Technique ◽  
Cationic Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Hypotonic Cell Swelling

The effects of hypotonic cell swelling (HCS) on muscarinic receptor-activated cationic current in guinea pig ileal smooth muscle were investigated by the whole cell patch-clamp technique. With nystatin-perforated recording, reduced external tonicity from 312 to 262 mosM caused cell swelling but hardly affected the membrane currents activated by depolarization, such as outward-rectifying K and voltage-dependent Ca currents. In contrast, the inward current evoked by carbachol at -60 mV was greatly increased (approximately 50%) by the same extent of hypotonicity. This effect is likely to occur through potentiation of nonselective cation channels coupled to the muscarinic receptor (mNSCCs) and probably does not involve elevated intracellular Ca2+ concentration ([Ca2+]i), since neither removal of external Ca2+ nor [Ca2+]i buffering with 10 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid significantly affected the results. Furthermore, the time course and degree of this potentiation closely matched those of video-microscopically monitored HCS. These results support the view that mechanosensitive modulation may be a powerful mechanism to regulate mNSCCs activity in gut smooth muscle, together with membrane potential and [Ca2+]i.

scholarly journals Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

1995 ◽  
Vol 105 (2) ◽  
pp. 209-226 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
Time Course ◽  
Measurement Techniques ◽  
Fast Inactivation ◽  
Patch Clamp Recording ◽  
Crac Channels ◽  
Whole Cell Patch Clamp ◽  
Close Proximity ◽  
Rapid Inactivation ◽  
Voltage Dependent ◽  
Crac Channel

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

Potassium and chloride currents in rat gastric enterochromaffin-like cells

1996 ◽  
Vol 270 (5) ◽  
pp. G739-G745 ◽  
Author(s):  
Donald D. F. Loo ◽  
George Sachs ◽  
Christian Prinz
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Gradient Centrifugation ◽  
Reversal Potential ◽  
Resting Potential ◽  
Chloride Currents ◽  
Whole Cell Patch Clamp ◽  
Ecl Cell ◽  
Steady State Current ◽  
Stimulation Of

The gastric enterochromaffin-like (ECL) cell se cretes histamine in response to secretagogues (gastrin, acetylcholine) by calcium signaling-dependent exocytosis of intracellular vacuoles containing the hormone. ECL cells were isolated from rat fundic gastric mucosa by elutriation and density-gradient centrifugation. Currents across the plasma membrane were measured using whole cell patch-clamp methods. These cells had a low conductance of 0.5 nS and resting potential of -50 mV Depolarization activated a K+ current that was blocked by Ba2+. Steady-state current in absence of K+ was due to Cl- because of the magnitude of the reversal potential and the effects of Cl- removal. Stimulation of secretion by gastrin, cholecystokinin octapeptide (CCK-8), and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate activated the Cl- conductance with a time course similar to that of histamine release. Therefore the ECL cell maintains a high resting potential, largely due to K+ currents, and stimulation of secretion activates a Cl- current, perhaps deriving from the membrane of the secretory granule that fuses with the plasma membrane. The depolarization that ensues may activate the K+ current to maintain the membrane potential during exocytosis. exocytosis; acid secretion Submitted on July 31, 1995 Accepted on October 2, 1995

Single delayed rectifier potassium channels from rabbit coronary artery myocytes

1993 ◽  
Vol 264 (4) ◽  
pp. H1146-H1153 ◽  
Author(s):  
K. A. Volk ◽  
E. F. Shibata
Keyword(s):  
Coronary Artery ◽  
Potassium Channels ◽  
Potassium Channel ◽  
Time Course ◽  
Delayed Rectifier ◽  
Voltage Dependent ◽  
Rabbit Coronary Artery ◽  
State Models ◽  
Calcium Activated Potassium Channel ◽  
Ik Channel

Cell-attached patches from rabbit coronary artery single smooth muscle cells contained two distinct potassium channel types, namely a large conductance calcium-activated potassium channel and a smaller voltage-activated potassium channel representing the delayed rectifier (IK). When a physiological potassium ion gradient was used, the average slope conductance of single IK channels was 7.26 pS. The time course of activation measured from ensemble averages was well fit by a single exponential raised to the power of 2 and was voltage dependent. Experiments were then performed with potassium (140 mM) on both sides of the membrane to resolve single IK channel currents during deactivation. Ensemble averages of this activity were well described by a two-component exponential, and the time constants were voltage dependent. Mean open times were significantly shorter during deactivation than during activation. Closed time distributions typically had two components. These kinetic characteristics were used in testing various state models for voltage-dependent potassium channels.

scholarly journals Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent.

1989 ◽  
Vol 94 (2) ◽  
pp. 213-232 ◽  
Author(s):  
G Cota ◽  
C M Armstrong
Keyword(s):  
Time Course ◽  
Inactivation Rate ◽  
Gh3 Cells ◽  
Na Channel ◽  
Pituitary Cells ◽  
Channel Inactivation ◽  
Internal Medium ◽  
Whole Cell Patch Clamp ◽  
Before And After ◽  
Voltage Dependent

We have determined the time course of Na channel inactivation in clonal pituitary (GH3) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near -40 mV), and a much smaller increase for large ones (approximately 1.5x at +40 mV). For technical reasons it was sometimes convenient to study outward INa recorded with no Na+ outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNa-V curve after papain, obtained by dividing the INa-V curve by the IIV curve, was left-shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papain. The rate of the inactivation step was found to vary only slightly from -100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from +20 to +60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by less than 10% from -100 to +60 mV. The data are considered in terms of existing models of the Na channel.

Glibenclamide blocks volume-sensitive Cl− channels by dual mechanisms

1998 ◽  
Vol 275 (2) ◽  
pp. C343-C351 ◽  
Author(s):  
Yan Liu ◽  
Shigetoshi Oiki ◽  
Takehiko Tsumura ◽  
Takahiro Shimizu ◽  
Yasunobu Okada
Keyword(s):  
Time Course ◽  
Weak Acid ◽  
Independent Manner ◽  
Inactivation Curve ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Extracellular Side ◽  
Charged Form ◽  
Almost All

To study the mechanisms of glibenclamide actions on volume-sensitive Cl−channels, whole cell patch-clamp studies were performed at various pH levels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl− currents in the entire range of voltage examined (−100 to +100 mV) and accelerated the depolarization-induced inactivation at pH 7.5. When glibenclamide was applied from the intracellular side, in contrast, no effect was observed. At acidic pH, at which the weak acid glibenclamide exists largely in the uncharged form, the instantaneous current was, in a voltage-independent manner, suppressed by the extracellular drug at micromolar concentrations without significantly affecting the depolarization-induced inactivation. At alkaline pH, at which almost all of the drug is in the charged form, glibenclamide speeded the inactivation time course and induced a leftward shift of the steady-state inactivation curve at much higher concentrations. Thus it is concluded that glibenclamide exerts inhibiting actions on swelling-activated Cl−channels from the extracellular side and that the uncharged form is mainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.

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