Eicosanoids modulate apical Ca(2+)-dependent K+ channels in cultured rabbit principal cells

1992 ◽  
Vol 263 (1) ◽  
pp. F116-F126 ◽  
Author(s):  
B. N. Ling ◽  
C. L. Webster ◽  
D. C. Eaton
Keyword(s):  
Single Channel ◽  
Primary Cultures ◽  
Cell Volume Regulation ◽  
Principal Cell ◽  
Resting Membrane Potential ◽  
Open Probability ◽  
Channel Activation ◽  
K Channels ◽  
Pg E2

Patch clamp technology was utilized to study the effects of apical phospholipase A2 (PLA2) metabolites on “maxi K” channels in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures (B. N. Ling, C. F. Hinton, and D. C. Eaton. Kidney Int. 40: 441–452, 1991). At resting membrane potential, this channel is quiescent in the cell-attached configuration. Apical application of the PLA2 agonist melittin (1 microgram/ml) for 10 min increased single-channel open probability (Po) from 0.0004 +/- 0.0010 to 0.11 +/- 0.05. Similarly, apical exposure to 50 microM arachidonic acid (AA) or 0.5 microM prostaglandin (PG) E2, but not 0.5 microM PGF2 alpha, also increased channel activity. Conversely, 10 microM of the PLA2 antagonist quinacrine applied apically decreased Po. Removal of apical bath Ca2+ did not prevent melittin-, AA-, or PGE2-induced channel activation. We then examined the role of maxi K channels and eicosanoids in principal cell volume regulation. Within seconds of reducing basolateral bath tonicity (285 to 214 mosmol/kgH2O), NPo (i.e., no. of channels x Po) initially increased approximately 200%, followed by a delayed but prolonged activation phase that was attenuated by removal of apical bath Ca2+. Pretreatment with 10 microM quinacrine, 100 microM indomethacin (cyclooxygenase inhibitor), or 0.25 microM thapsigargin (to deplete intracellular Ca2+ stores) abolished the initial phase of swelling-induced channel activation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The role of Mg2+ in the inactivation of inwardly rectifying K+ channels in aortic endothelial cells.

1995 ◽  
Vol 105 (4) ◽  
pp. 463-484 ◽  
Author(s):  
T R Elam ◽  
J B Lansman
Keyword(s):  
Endothelial Cells ◽  
Open Channel ◽  
Single Channel ◽  
Vascular Endothelial Cells ◽  
Membrane Surface ◽  
Potential Drop ◽  
Open Probability ◽  
K Channels ◽  
Inwardly Rectifying

We have studied the role of Mg2+ in the inactivation of inwardly rectifying K+ channels in vascular endothelial cells. Inactivation was largely eliminated in Mg(2+)-free external solutions and the extent of inactivation was increased by raising Mg2+o. The dose-response relation for the reduction of channel open probability showed that Mg2+o binds to a site (KD = approximately 25 microM at -160 mV) that senses approximately 38% of the potential drop from the external membrane surface. Analysis of the single-channel kinetics showed that Mg2+ produced a class of long-lived closures that separated bursts of openings. Raising Mg2+o reduced the burst duration, but less than expected for an open-channel blocking mechanism. The effects of Mg2+o are antagonized by K+o in manner which suggests that K+ competes with Mg2+ for the inactivation site. Mg2+o also reduced the amplitude of the single-channel current at millimolar concentrations by a rapid block of the open channel. A mechanism is proposed in which Mg2+ binds to the closed channel during hyperpolarization and prevents it from opening until it is occupied by K+.

Amiloride-sensitive sodium channels in rabbit cortical collecting tubule primary cultures

1991 ◽  
Vol 261 (6) ◽  
pp. F933-F944 ◽  
Author(s):  
B. N. Ling ◽  
C. F. Hinton ◽  
D. C. Eaton
Keyword(s):  
High Selectivity ◽  
Primary Cultures ◽  
Principal Cell ◽  
Na Channel ◽  
Open Probability ◽  
Inward Current ◽  
Collecting Tubule ◽  
Apical Membranes ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

Patch-clamp methodology was applied to principal cell apical membranes of rabbit cortical collecting tubule (CCT) primary cultures grown on collagen supports in the presence of aldosterone (1.5 microM). The most frequently observed channel had a unit conductance of 3-5 pS, nonlinear current-voltage (I-V) relationship, Na permeability (PNa)-to-K permeability (PK) ratio greater than 19:1, and inward current at all applied potentials (Vapp) less than +80 mV (n = 41). Less frequently, an 8- to 10-pS channel with a linear I-V curve, PNa/PK less than 5:1, and inward current at Vapp less than +40 mV was also observed (n = 7). Luminal amiloride (0.75 microM) decreased the open probability (Po) for both of these channels. Mean open time for the high-selectivity Na+ channel was 2.1 +/- 0.5 s and for the low-selectivity Na+ channel was 50 +/- 12 ms. In primary cultures grown without aldosterone the high-selectivity Na+ channel was rarely observed (1 of 32 patches). Lastly, a 26- to 35-pS channel, nonselective for Na+ over K+, was not activated by cytoplasmic Ca2+ or voltage nor inhibited by amiloride (n = 17). We conclude that under specific growth conditions, namely permeable transporting supports and chronic mineralocorticoid hormone exposure, principal cell apical membranes of rabbit CCT primary cultures contain 1) both high-selectivity and low-selectivity, amiloride-inhibitable Na+ channels and 2) amiloride-insensitive, nonselective cation channels.

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.

Molecular physiology of cardiac potassium channels

1996 ◽  
Vol 76 (1) ◽  
pp. 49-67 ◽  
Author(s):  
K. K. Deal ◽  
S. K. England ◽  
M. M. Tamkun
Keyword(s):  
Potassium Channels ◽  
Action Potential ◽  
Single Channel ◽  
Resting Membrane Potential ◽  
Molecular Physiology ◽  
K Channels ◽  
Relative Contribution ◽  
Outward Currents ◽  
Important Challenge ◽  
Heterologous Expression Systems

The cardiac action potential results from the complex, but precisely regulated, movement of ions across the sarcolemmal membrane. Potassium channels represent the most diverse class of ion channels in heart and are the targets of several antiarrhythmic drugs. Potassium currents in the myocardium can be classified into one of two general categories: 1) inward rectifying currents such as IK1, IKACh, and IKATP; and 2) primarily voltage-gated currents such as IKs, IKr, IKp, IKur, and Ito. The inward rectifier currents regulate the resting membrane potential, whereas the voltage-activated currents control action potential duration. The presence of these multiple, often overlapping, outward currents in native cardiac myocytes has complicated the study of individual K+ channels; however, the application of molecular cloning technology to these cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of the function and pharmacology of a single channel type. This review addresses the progress made toward understanding the complex molecular physiology of K+ channels in mammalian myocardium. An important challenge for the future is to determine the relative contribution of each of these cloned channels to cardiac function.

Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I K,ACh Channel Activity in Atrial Tachycardia Remodeling

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Niels Voigt ◽  
Ange Maguy ◽  
Yung-Hsin Yeh ◽  
Xiao-Yan Qi ◽  
Ursula Ravens ◽  
...  
Keyword(s):  
Atrial Tachycardia ◽  
Single Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Direct Demonstration ◽  
Open Time ◽  
Therapy Target ◽  
Channel Properties ◽  
Constitutively Active

Background: Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I K,ACh that increases susceptibility to AF, direct demonstration of dysregulated I K,ACh channel function is lacking. We studied AT effects on single I K,ACh channel activity in dog atria. Methods: I K,ACh channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min −1 ) dogs. Results : AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification were observed in CTL and AT (Figure ). AT produced prominent agonist-independent I K,ACh activity due to 7-fold increased opening frequency (f o ) and 10-fold increased open probability (P o ) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I K,ACh activation (10 μm carbachol, CCh), f o was 38% lower, open time constant 25% higher, and P o and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f o and P o by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of I K,ACh. Conclusions : AT produces prominent agonist-independent constitutive single-channel I K,ACh activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I K,ACh , as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I K,ACh channels in AT-remodeling and support their interest as a potential novel AF-therapy target.

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

2005 ◽  
Vol 289 (3) ◽  
pp. C717-C726 ◽  
Author(s):  
My N. Helms ◽  
Ling Yu ◽  
Bela Malik ◽  
Dean J. Kleinhenz ◽  
C. Michael Hart ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Single Channel ◽  
Short Circuit ◽  
Primary Cultures ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
No Production ◽  
Open Probability ◽  
No Donor

Several studies have shown that nitric oxide (NO) inhibits Na+ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na+ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 μM dexamethasone and 10% bovine serum. After 1.5 μM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound ( P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability ( Po) from 0.186 ± 0.043 to 0.045 ± 0.009 ( n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na+ transport is by altering iNOS production of NO.

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.

Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells

1992 ◽  
Vol 262 (2) ◽  
pp. C453-C460 ◽  
Author(s):  
K. Naruse ◽  
D. S. McGehee ◽  
G. S. Oxford
Keyword(s):  
Intracellular Signaling ◽  
Single Channel ◽  
Outward Current ◽  
Transient Outward Current ◽  
Ca Channel ◽  
Ca Channels ◽  
Drg Neurons ◽  
Open Probability ◽  
K Channels ◽  
Drg Neuron

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

scholarly journals Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules

2003 ◽  
Vol 122 (2) ◽  
pp. 177-190 ◽  
Author(s):  
Herve Barriere ◽  
Radia Belfodil ◽  
Isabelle Rubera ◽  
Michel Tauc ◽  
Florian Lesage ◽  
...  
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Primary Cultures ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Proximal Tubules ◽  
Wild Type ◽  
Convoluted Tubules ◽  
K Currents

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.

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