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

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.

Download Full-text

Regulation and function of the two-pore-domain (K2P) potassium channel Trek-1 in alveolar epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00078.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. L93-L102 ◽

Cited By ~ 21

Author(s):

Andreas Schwingshackl ◽

Bin Teng ◽

Manik Ghosh ◽

Alina Nico West ◽

Patrudu Makena ◽

...

Keyword(s):

Cell Proliferation ◽

Epithelial Cells ◽

Cyclin D1 ◽

Alveolar Epithelial Cells ◽

Lung Epithelial Cells ◽

Cytokine Secretion ◽

Nuclear Antigen ◽

Alveolar Epithelial ◽

Mediator Secretion ◽

Pore Domain

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K+ channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K+ channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.

Download Full-text

The 2-Pore Domain Potassium Channel TREK-1 Regulates Cytokine Secretion from Human Alveolar Epithelial Cells Independently of Potassium Currents

Biophysical Journal ◽

10.1016/j.bpj.2015.11.2414 ◽

2016 ◽

Vol 110 (3) ◽

pp. 449a

Author(s):

Andreas Schwingshackl ◽

Bin Teng ◽

Marc Borsotto ◽

Christopher M. Waters

Keyword(s):

Epithelial Cells ◽

Potassium Channel ◽

Potassium Currents ◽

Alveolar Epithelial Cells ◽

Cytokine Secretion ◽

Alveolar Epithelial ◽

Pore Domain ◽

Human Alveolar Epithelial Cells

Download Full-text

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.

Download Full-text