Mechanism of K+ channel block by verapamil and related compounds in rat alveolar epithelial cells.

The mechanism by which the phenylalkylamines, verapamil and D600, and related compounds, block inactivating delayed rectifier K+ currents in rat alveolar epithelial cells, was investigated using whole-cell tight-seal recording. Block by phenylalkylamines added to the bath resembles state-dependent block of squid K+ channels by internally applied quarternary ammonium ions (Armstrong, C.M. 1971. Journal of General Physiology. 58:413-437): open channels are blocked preferentially, increased [K+]o accelerates recovery from block, and recovery occurs mainly through the open state. Slow recovery from block is attributed to the existence of a blocked-inactivated state, because recovery was faster in three situations where recovery from inactivation is faster: (a) at high [K+]o, (b) at more negative potentials, and (c) in cells with type l K+ channels, which recover rapidly from inactivation. The block rate was used as a bioassay to reveal the effective concentration of drug at the block site. When external pH, pHo, was varied, block was much faster at pHo 10 than pHo 7.4, and very slow at pHo 4.5. The block rate was directly proportional to the concentration of neutral drug in the bath, suggesting that externally applied drug must enter the membrane in neutral form to reach the block site. High internal pH (pHi 10) reduced the apparent potency of externally applied phenylalkylamines, suggesting that the cationic form of these drugs blocks K+ channels at an internal site. The permanently charged analogue D890 blocked more potently when added to the pipette than to the bath. However, lowering pHi to 5.5 did not enhance block by external drug, and tertiary phenylalkylamines added to the pipette solution blocked weakly. This result can be explained if drug diffuses out of the cell faster than it is delivered from the pipette, the block site is reached preferentially via hydrophobic pathways, or both. Together, the data indicate the neutral membrane-bound drug blocks K+ channels more potently than intracellular cationic drug. Neutral drug has rapid access to the receptor, where block is stabilized by protonation of the drug from the internal solution. In summary, externally applied phenylalkylamines block open or inactivated K+ channels by partitioning into the cell membrane in neutral form and are stabilized at the block site by protonation.

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State-dependent inactivation of K+ currents in rat type II alveolar epithelial cells.

The Journal of General Physiology ◽

10.1085/jgp.95.4.617 ◽

1990 ◽

Vol 95 (4) ◽

pp. 617-646 ◽

Cited By ~ 29

Author(s):

T E DeCoursey

Keyword(s):

Epithelial Cells ◽

Coupled Model ◽

Alveolar Epithelial Cells ◽

Open Channels ◽

Type Ii ◽

K Channels ◽

Tail Current ◽

Alveolar Epithelial ◽

State Dependent ◽

Dependent Inactivation

Inactivation of K+ channels responsible for delayed rectification in rat type II alveolar epithelial cells was studied in Ringer, 160 mM K-Ringer, and 20 mM Ca-Ringer. Inactivation is slower and less complete when the extracellular K+ concentration is increased from 4.5 to 160 mM. Inactivation is faster and more complete when the extracellular Ca2+ concentration is increased from 2 to 20 mM. Several observations suggest that inactivation is state-dependent. In each of these solutions depolarization to potentials near threshold results in slow and partial inactivation, whereas depolarization to potentials at which the K+ conductance, gK, is fully activated results in maximal inactivation, suggesting that open channels inactivate more readily than closed channels. The time constant of current inactivation during depolarizing pulses is clearly voltage-dependent only at potentials where activation is incomplete, a result consistent with coupling of inactivation to activation. Additional evidence for state-dependent inactivation includes cumulative inactivation and nonmonotonic from inactivation. A model like that proposed by C.M. Armstrong (1969. J. Gen. Physiol. 54: 553-575) for K+ channel block by internal quaternary ammonium ions accounts for most of these properties. The fundamental assumptions are: (a) inactivation is strictly coupled to activation (channels must open before inactivating, and recovery from inactivation requires passage through the open state); (b) the rate of inactivation is voltage-independent. Experimental data support this coupled model over models in which inactivation of closed channels is more rapid than that of open channels (e.g., Aldrich, R.W. 1981. Biophys. J. 36:519-532). No inactivation results from repeated depolarizing pulses that are too brief to open K+ channels. Inactivation is proportional to the total time that channels are open during both a depolarizing pulse and the tail current upon repolarization; repolarizing to more negative potentials at which the tail current decays faster results in less inactivation. Implications of the coupled model are discussed, as well as additional states needed to explain some details of inactivation kinetics.

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1057: THE K+ CHANNEL TREK-1 REGULATES CYTOKINE SECRETION FROM ALVEOLAR EPITHELIAL CELLS INDEPENDENTLY OF K

Critical Care Medicine ◽

10.1097/01.ccm.0000509733.49584.c8 ◽

2016 ◽

Vol 44 (12) ◽

pp. 340-340

Author(s):

Andreas Schwingshackl ◽

Bin Teng ◽

Christopher Waters

Keyword(s):

Epithelial Cells ◽

Alveolar Epithelial Cells ◽

Cytokine Secretion ◽

K Channel ◽

Alveolar Epithelial

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K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2012.02.025 ◽

2012 ◽

Vol 1818 (7) ◽

pp. 1682-1690 ◽

Cited By ~ 13

Author(s):

Olivier Bardou ◽

Anik Privé ◽

Francis Migneault ◽

Karl Roy-Camille ◽

André Dagenais ◽

...

Keyword(s):

Epithelial Cells ◽

Promoter Activity ◽

Alveolar Epithelial Cells ◽

K Channels ◽

Alveolar Epithelial ◽

Control Fluid ◽

And Control

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Na(+)-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils.

The Journal of General Physiology ◽

10.1085/jgp.103.5.755 ◽

1994 ◽

Vol 103 (5) ◽

pp. 755-785 ◽

Cited By ~ 26

Author(s):

T E DeCoursey ◽

V V Cherny

Keyword(s):

Epithelial Cells ◽

Voltage Dependence ◽

Single Cells ◽

Alveolar Epithelial Cells ◽

Proton Gradient ◽

Human Neutrophils ◽

Patch Clamp Technique ◽

Simple Diffusion ◽

Pipette Solution ◽

Alveolar Epithelial

Voltage-activated H(+)-selective currents were studied in cultured adult rat alveolar epithelial cells and in human neutrophils using the whole-cell configuration of the patch-clamp technique. The H+ conductance, gH, although highly selective for protons, was modulated by monovalent cations. In Na+ and to a smaller extent in Li+ solutions, H+ currents were depressed substantially and the voltage dependence of activation of the gH shifted to more positive potentials, when compared with the "inert" cation tetramethylammonium (TMA+). The reversal potential of the gH, Vrev, was more positive in Na+ solutions than in inert ion solutions. Amiloride at 100 microM inhibited H+ currents in the presence of all cations studied except Li+ and Na+, in which it increased H+ currents and shifted their voltage-dependence and Vrev to more negative potentials. The more specific Na(+)-H+ exchange inhibitor dimethylamiloride (DMA) at 10 microM similarly reversed most of the suppression of the gH by Na+ and Li+. Neither 500 microM amiloride nor 200 microM DMA added internally via the pipette solution were effective. Distinct inhibition of the gH was observed with 1% [Na+]o, indicating a mechanism with high sensitivity. Finally, the effects of Na+ and their reversal by amiloride were large when the proton gradient was outward (pHo parallel pHi 7 parallel 5.5), smaller when the proton gradient was abolished (pH 7 parallel 7), and absent when the proton gradient was inward (pH 6 parallel 7). We propose that the effects of Na+ and Li+ are due to their transport by the Na(+)-H+ antiporter, which is present in both cell types studied. Electrically silent H+ efflux through the antiporter would increase pHi and possibly decrease local pHo, both of which modulate the gH in a similar manner: reducing the H+ currents at a given potential and shifting their voltage-dependence to more positive potentials. A simple diffusion model suggests that Na(+)-H+ antiport could deplete intracellular protonated buffer to the extent observed. Evidently the Na(+)-H+ antiporter functions in perfused cells, and its operation results in pH changes which can be detected using the gH as a physiological sensor. Thus, the properties of the gH can be exploited to study Na(+)-H+ antiport in single cells under controlled conditions.

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Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00376.2005 ◽

2006 ◽

Vol 291 (6) ◽

pp. L1207-L1219 ◽

Cited By ~ 35

Author(s):

Claudie Leroy ◽

Anik Privé ◽

Jean-Charles Bourret ◽

Yves Berthiaume ◽

Pasquale Ferraro ◽

...

Keyword(s):

Epithelial Cells ◽

Fluid Transport ◽

Alveolar Epithelial Cells ◽

Prolonged Treatment ◽

K Channel ◽

Sulfonylurea Receptor ◽

Fluid Absorption ◽

Alveolar Epithelial ◽

Voltage Dependent ◽

The Impact

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027–L1037, 2004), we identified an ATP-sensitive K+ (KATP) channel in alveolar epithelial cells, formed by inwardly rectifying K+ channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of KATP, voltage-dependent K+ channel KvLQT1, and calcium-activated K+ (KCa) channel modulators modified Na+ and Cl− currents in alveolar monolayers. In addition, it was shown previously that a KATP opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K+ channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of KATP, KvLQT1, and KCa channels identified by PCR. Glibenclamide and clofilium (KATP and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na+ current, and forskolin-activated Cl− currents, whereas pinacidil, a KATP activator, increased them. Interestingly, K+ inhibitors or membrane depolarization (induced by valinomycin in high-K+ medium) decreased α-, β-, and γ-ENaC and CFTR mRNA. α-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na+ transport through ENaC, we tested the impact of K+ channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K+ channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.

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Ph-Dependent Inhibition of Voltage-Gated H+ Currents in Rat Alveolar Epithelial Cells by Zn2+ and Other Divalent Cations

The Journal of General Physiology ◽

10.1085/jgp.114.6.819 ◽

1999 ◽

Vol 114 (6) ◽

pp. 819-838 ◽

Cited By ~ 118

Author(s):

Vladimir V. Cherny ◽

Thomas E. DeCoursey

Keyword(s):

Epithelial Cells ◽

Divalent Cations ◽

Active Species ◽

Alveolar Epithelial Cells ◽

Protonation Site ◽

Pipette Solution ◽

Voltage Gated ◽

Alveolar Epithelial ◽

High Concentrations ◽

H Channels

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl2 reduced the H+ current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H+ current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn2+ were inconsistent with classical voltage-dependent block in which Zn2+ binds within the membrane voltage field. Instead, Zn2+ binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn2+ were strongly pHo dependent but were insensitive to pHi, suggesting that protons and Zn2+ compete for external sites on H+ channels. The apparent potency of Zn2+ in slowing activation was ∼10× greater at pHo 7 than at pHo 6, and ∼100× greater at pHo 6 than at pHo 5. The pHo dependence suggests that Zn2+, not ZnOH+, is the active species. Evidently, the Zn2+ receptor is formed by multiple groups, protonation of any of which inhibits Zn2+ binding. The external receptor bound H+ and Zn2+ with pKa 6.2–6.6 and pKM 6.5, as described by several models. Zn2+ effects on the proton chord conductance–voltage (gH–V) relationship indicated higher affinities, pKa 7 and pKM 8. CdCl2 had similar effects as ZnCl2 and competed with H+, but had lower affinity. Zn2+ applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H+ currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn2+ receptor may be the same modulatory protonation site(s) at which pHo regulates H+ channel gating.

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