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.

scholarly journals Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2020 ◽  
Vol 21 (14) ◽  
pp. 4876
Author(s):  
Zbigniew Burdach ◽  
Agnieszka Siemieniuk ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Inward Current ◽  
Bath Solution ◽  
Outward Currents ◽  
Inward Currents

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Apical K+ conductance in maturing rabbit principal cell

1997 ◽  
Vol 272 (3) ◽  
pp. F397-F404 ◽  
Author(s):  
L. M. Satlin ◽  
L. G. Palmer
Keyword(s):  
Reversal Potential ◽  
Principal Cell ◽  
K Channel ◽  
Postnatal Life ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inward Currents ◽  
K Secretion

Net K+ secretion is not detected in cortical collecting ducts (CCDs) isolated from newborn rabbits and perfused in vitro. To establish whether a low apical K+ permeability of the neonatal principal cell limits K+ secretion early in life, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to study the properties and density of conducting K+ channels in principal cells. With KCl in the pipette and a NaCl solution warmed to 37 degrees C in the bath, inward currents with a conductance of approximately 42 pS were observed in 0% (0 out of 13 or 0/13), 10% (2/21), 18% (5/28), 29% (4/14), and 56% (10/18) of cell-attached patches obtained in 1-, 2-, 3-, 4-, and 5-wk-old animals, respectively. The conductance and reversal potential of this channel led us to suspect that it represented the low-conductance K+ channel previously described in the rat CCD by L. G. Palmer, L. Antonian, and G. Frindt (J. Gen. Physiol. 104: 693-710, 1994). The mean number of open channels per patch (NPo) increased progressively (P < 0.05) after birth, from 0 at 1 wk, to 0.06 +/- 0.04 at 2 wk, to 0.40 +/- 0.18 at 3 wk, to 0.74 +/- 0.41 at 4 wk, and to 1.06 +/- 0.28 at 5 wk. The increase in NPo appeared to be due primarily to a developmental increase in N, which is the number of channels; open probability, Po, remained constant at approximately 0.5 for all channels identified after the 2nd wk of life. The increase in number of conducting K+ channels during postnatal life is likely to contribute to the maturational increase in net K+ secretion in the CCDs.

scholarly journals Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block.

1991 ◽  
Vol 98 (1) ◽  
pp. 163-181 ◽  
Author(s):  
W B Ferguson
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Competitive Inhibition ◽  
Reversal Potential ◽  
K Channel ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Single Channel Current

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.

Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells

1986 ◽  
Vol 251 (1) ◽  
pp. C85-C89 ◽  
Author(s):  
N. W. Richards ◽  
D. C. Dawson
Keyword(s):  
Epithelial Cells ◽  
Single Channel ◽  
Reversal Potential ◽  
Cell Swelling ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Colon Cells ◽  
High K ◽  
A Cell ◽  
Single Channel Currents

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry

1993 ◽  
Vol 264 (3) ◽  
pp. F565-F574 ◽  
Author(s):  
G. Frindt ◽  
R. B. Silver ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Homeostasis ◽  
Feedback Regulation ◽  
Feedback Mechanism ◽  
Acute Administration ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Single Channel Currents

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and single-channel currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of amiloride (10 microM) or benzamil (0.5 microM) to the superfusate resulted in a twofold increase in the mean number of open channels (NPo) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NPo reversed within 3 min after removal of amiloride. The increase in NPo was accounted for, at least in part, by an increase in Po. Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NPo. No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel Po by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.

Evidence for KCNQ1 K+ channel expression in rat zymogen granule membranes and involvement in cholecystokinin-induced pancreatic acinar secretion

2008 ◽  
Vol 294 (4) ◽  
pp. C879-C892 ◽  
Author(s):  
Wing-Kee Lee ◽  
Blazej Torchalski ◽  
Eleni Roussa ◽  
Frank Thévenod
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fluid Secretion ◽  
Pancreatic Tissue ◽  
Bk Channel ◽  
K Channel ◽  
Amylase Secretion ◽  
Zymogen Granule ◽  
Open Probability ◽  
Pancreatic Acini

Secretion of enzymes and fluid induced by Ca2+ in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K+ conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K+ permeability (IC50 of ∼10 μM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential ( Erev) of −20.9 ± 0.9 mV, and a single-channel K+ conductance ( gK) of 265.8 ± 44.0 pS ( n = 39). Replacement of cis 500 mM K+ by 500 mM Na+ shifted Erev to −2.4 ± 3.6 mV ( n = 3), indicating K+ selectivity. Single-channel analysis identified several K+ channel groups with distinct channel behaviors. K+ channels with a gK of 651.8 ± 88.0 pS, Erev of −22.9 ± 2.2 mV, and open probability ( Popen) of 0.43 ± 0.06 at 0 mV ( n = 6) and channels with a gK of 155.0 ± 11.4 pS, Erev of −18.3 ± 1.8 mV, and Popen of 0.80 ± 0.03 at 0 mV ( n = 3) were inhibited by 100 μM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca2+-activated K+ channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC50 of ∼10 μM). Thus KCNQ1 may account for ZG K+ conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.

scholarly journals A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2021 ◽  
Vol 22 (23) ◽  
pp. 12621
Author(s):  
Agnieszka Siemieniuk ◽  
Zbigniew Burdach ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Inward Currents ◽  
Sv Channels ◽  
The One ◽  
The Impact

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

scholarly journals Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

2009 ◽  
Vol 134 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Alessandra Abenavoli ◽  
Mattia Lorenzo DiFrancesco ◽  
Indra Schroeder ◽  
Svetlana Epimashko ◽  
Sabrina Gazzarrini ◽  
...  
Keyword(s):  
Single Channel ◽  
Negative Slope ◽  
K Channel ◽  
Open Probability ◽  
Channel Current ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Current Voltage Curve ◽  
Fast Gating ◽  
Basic Features

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.

Development of inwardly rectifying K+ channel family in rat ventricular myocytes

1997 ◽  
Vol 272 (4) ◽  
pp. H1741-H1750 ◽  
Author(s):  
L. H. Xie ◽  
M. Takano ◽  
A. Noma
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
K Current ◽  
Inwardly Rectifying

The ATP-sensitive K+ current (I(K,ATP)), the inward rectifier K+ current (I(K1)), and the acetylcholine-activated K+ current (I(K,ACh)) were recorded in fetal, neonatal, and adult rat ventricular myocytes using the patch-clamp technique. The density (pA/pF) of I(K1) increased from gestation day 10 through neonatal day 1 and then decreased after neonatal day 30. The density of I(K,ATP) activated maximally by metabolic inhibition changed in parallel with the I(K1) density, and the density of I(K,ATP) channel distribution was 1.3 times higher than that of I(K1) throughout the development. We failed to observe developmental changes in the single-channel conductance and the mean open time of I(K1) and I(K,ATP) channels. However, the open probability of the I(K,ATP) channel was lower in fetuses, and the sensitivity to ATP was highest in 1-day neonates. I(K,ACh) were present in the ventricle at all stages of development but at a much lower density than in atrium. The relationship between the resting membrane potential and the development of the inwardly rectifying K-channel family is discussed.

scholarly journals Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

2002 ◽  
Vol 120 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Ion Channels ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Acid Sensing Ion Channels ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetics Of ◽  
Channel Properties

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small.

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