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.

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.

Primary structure and functional properties of an epithelial K channel

1994 ◽  
Vol 266 (3) ◽  
pp. C809-C824 ◽  
Author(s):  
H. Zhou ◽  
S. S. Tate ◽  
L. G. Palmer
Keyword(s):  
Functional Properties ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Rat Kidney ◽  
Reversal Potential ◽  
Ammonium Ion ◽  
Expression Cloning ◽  
K Channel ◽  
Dependent Manner ◽  
Open Probability

Expression cloning in Xenopus oocytes was used to identify a clone for a renal K channel. The clone, named ROMK2, was obtained from a cDNA library constructed in the plasmid vector pSPORT using size-selected poly(A)+ RNA from whole rat kidney. ROMK2 consists of 1,837 nucleotides, with an open reading frame of 1,116 bases predicted to code for a 372-amino acid peptide. The clone appears to be a splice variant of a recently reported K channel (ROMK1) from rat renal outer medulla (Ho, K.H., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362: 31-37, 1993). Northern blot analysis indicates that ROMK2 is expressed in renal cortex, medulla, and papilla. Expression in other tissues appears to be much lower. The functional properties of the channel as measured in Xenopus oocytes indicate its close relationship to ROMK1 and more distant relationship to the inward rectifier K channel (IRK1) (Kubo, Y, T.J. Baldwin, Y. N. Jan, and L. Y. Jan. Nature Lond. 362: 127-133, 1993). The inward conductance of the channel is a saturable function of external K, with a half-maximal conductance at <5 mM. The selectivity sequence for ion permeability based on reversal potential measurements was K > Rb > NH4 > Na, Li. The conductance to Rb was only one-half that to K. Extracellular Ba2+ and Cs+ blocked the channel in a voltage-dependent manner. The high sensitivity of Cs+ block to voltage is consistent with the channel's operating as a multi-ion pore. The channel was blocked by high concentrations (100 microM) of glibenclamide. It did not appear to be blocked by extracellular Na+ or tetraethyl-ammonium ion. Patch-clamp measurements indicated a single-channel conductance of 30 pS in the presence of 110 mM K and high open probability that was weakly dependent on voltage. This channel may be involved in maintaining the membrane potential of renal cells and/or mediating renal K secretion.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

scholarly journals Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 2175
Author(s):  
Adonis Z. Wu ◽  
Tzu-Lun Ohn ◽  
Ren-Jay Shei ◽  
Huei-Fang Wu ◽  
Yong-Cyuan Chen ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Low Dose ◽  
Single Channel ◽  
Cell System ◽  
K Channel ◽  
Lipid Mediator ◽  
Bkca Channel ◽  
High Dose ◽  
Open Probability ◽  
Sphingosine 1 Phosphate

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

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.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule

1995 ◽  
Vol 268 (4) ◽  
pp. F569-F580 ◽  
Author(s):  
L. C. Stoner ◽  
G. E. Morley
Keyword(s):  
Apical Membrane ◽  
Physiological Role ◽  
Reversal Potential ◽  
K Channel ◽  
Apical Surface ◽  
Open Probability ◽  
Volume Regulatory Decrease ◽  
Collecting Tubules ◽  
High Conductance

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P ' 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

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.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

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