Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane

2003 ◽  
Vol 284 (6) ◽  
pp. C1614-C1624 ◽  
Author(s):  
So Yeong Lee ◽  
Peter J. Maniak ◽  
David H. Ingbar ◽  
Scott M. O'Grady
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Reversal Potential ◽  
Alveolar Epithelial Cells ◽  
Α Subunit ◽  
Whole Cell ◽  
Voltage Gated ◽  
Alveolar Epithelial ◽  
Kv Channels ◽  
Α Subunits

Whole cell perforated patch-clamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was −60 mV, and depolarizing voltage steps activated voltage-gated K+ (Kv) channels. The voltage-activated currents exhibited a mean reversal potential of −32 mV. Complete activation was achieved at −10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in K+ selectivity, suggesting contributions of multiple Kv α-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between −30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv α-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three β-subunits (Kvβ1.1, Kvβ2.1, and Kvβ3.1), and two K+ channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv α-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv α-, β-, and KChIP subunits explains the variability in inactivation gating and K+ selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal K+ secretion across the alveolar epithelium.

Cl-channel activation is necessary for stimulation of Na transport in adult alveolar epithelial cells

2000 ◽  
Vol 278 (2) ◽  
pp. L239-L244 ◽  
Author(s):  
Scott M. O'Grady ◽  
Xinpo Jiang ◽  
David H. Ingbar
Keyword(s):  
Epithelial Cells ◽  
Adrenergic Receptor ◽  
Apical Membrane ◽  
Receptor Activation ◽  
Alveolar Epithelial Cells ◽  
Na Channels ◽  
Channel Activation ◽  
Alveolar Epithelial ◽  
Cl Channel ◽  
Stimulation Of

In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective β-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.

Voltage-gated proton channels help regulate pHi in rat alveolar epithelium

2005 ◽  
Vol 288 (2) ◽  
pp. L398-L408 ◽  
Author(s):  
Ricardo Murphy ◽  
Vladimir V. Cherny ◽  
Deri Morgan ◽  
Thomas E. DeCoursey
Keyword(s):  
Epithelial Cells ◽  
Ph Regulation ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Proton Channel ◽  
Resting Cells ◽  
Proton Channels ◽  
Voltage Gated ◽  
Alveolar Epithelial

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pHi) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO3−-free solutions [at external pH (pHo) 7.4]. Cells were acid-loaded by the NH4+ prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 μM ZnCl2, a proton channel inhibitor, slowed recovery about twofold. In addition, the pHi minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl2 was observed at pHo 7.4 and pHo 8.0 and in normal and high-K+ Ringer solutions. The observed rate of Zn2+-sensitive pHi recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pHi recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl2 had no effect on pHi in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pHi recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

Adrenergic stimulation of Na+transport across alveolar epithelial cells involves activation of apical Cl−channels

1998 ◽  
Vol 275 (6) ◽  
pp. C1610-C1620 ◽  
Author(s):  
Xinpo Jiang ◽  
David H. Ingbar ◽  
Scott M. O’Grady
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Methanesulfonic Acid ◽  
Reversal Potential ◽  
Short Circuit Current ◽  
Alveolar Epithelial Cells ◽  
Sprague Dawley ◽  
Adrenergic Stimulation ◽  
Alveolar Epithelial ◽  
Stimulation Of

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current ( I sc; 60%) was inhibited by amiloride (IC50 0.96 μM) or benzamil (IC50 0.5 μM). Basolateral addition of terbutaline (2 μM) produced a rapid decrease in I sc, followed by a slow recovery back to its initial amplitude. When Cl− was replaced with methanesulfonic acid, the basal I sc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of −28 mV. Increasing Cl− concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl− conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that β-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl− permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.

scholarly journals The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient.

1995 ◽  
Vol 105 (6) ◽  
pp. 861-896 ◽  
Author(s):  
V V Cherny ◽  
V S Markin ◽  
T E DeCoursey
Keyword(s):  
Epithelial Cells ◽  
Voltage Dependence ◽  
Ph Dependence ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Alveolar Epithelial Cells ◽  
Ph Gradient ◽  
Protonation Site ◽  
Alveolar Epithelial ◽  
H Channels

Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, Vrev, was close to the Nernst potential, EH, varying 52 mV/U pH over four delta pH units (delta pH = pHo - pHi). This result indicates that H+ channels are extremely selective, PH/PTMA > 10(7), and that both internal and external pH, pHi, and pHo, were well controlled. The H+ current amplitude was practically constant at any fixed delta pH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current-voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, gH, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by approximately 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as approximately 20-40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, delta pH. We propose a simple general model to account for the observed delta pH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on delta pH and not on the absolute pH.

scholarly journals α1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase Cζ

2009 ◽  
Vol 29 (13) ◽  
pp. 3455-3464 ◽  
Author(s):  
Galina A. Gusarova ◽  
Laura A. Dada ◽  
Aileen M. Kelly ◽  
Chaya Brodie ◽  
Lee A. Witters ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
A549 Cells ◽  
Dominant Negative ◽  
Alveolar Epithelial Cells ◽  
Ampk Activation ◽  
Α Subunit ◽  
Alveolar Epithelial ◽  
Mitochondrial Ros ◽  
Amp Activated Protein Kinase

ABSTRACT Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient ρ0-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK α was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis.

Hypothesis: do voltage-gated H+channels in alveolar epithelial cells contribute to CO2 elimination by the lung?

2000 ◽  
Vol 278 (1) ◽  
pp. C1-C10 ◽  
Author(s):  
Thomas E. DeCoursey
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Facilitated Diffusion ◽  
Electrochemical Gradient ◽  
Voltage Gated ◽  
Alveolar Epithelial ◽  
H Channels ◽  
Apical Membranes

Although alveolar epithelial cells were the first mammalian cells in which voltage-gated H+ currents were recorded, no specific function has yet been proposed. Here we consider whether H+ channels contribute to one of the main functions of the lung: CO2 elimination. This idea builds on several observations: 1) some cell membranes have low CO2permeability, 2) carbonic anhydrase is present in alveolar epithelium and contributes to CO2 extrusion by facilitating diffusion, 3) the transepithelial potential difference favors selective activation of H+ channels in apical membranes, and 4) the properties of H+ channels are ideally suited to the proposed role. H+channels open only when the electrochemical gradient for H+ is outward, imparting directionality to the diffusion process. Unlike previous facilitated diffusion models, [Formula: see text] and H+ recombine to form CO2 in the alveolar subphase. Rough quantitative considerations indicate that the proposed mechanism is plausible and indicate a significant capacity for CO2 elimination by the lung by this route. Fully activated alveolar H+ channels extrude acid equivalents at three times the resting rate of CO2 production.

Modeling the effect of stretch and plasma membrane tension on Na+-K+-ATPase activity in alveolar epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. L40-L53 ◽  
Author(s):  
Jacob L. Fisher ◽  
Susan S. Margulies
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Alveolar Epithelial Cells ◽  
Cyclic Stretch ◽  
Model Parameters ◽  
Membrane Tension ◽  
Membrane Area ◽  
Whole Cell ◽  
Alveolar Epithelial

While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na+-K+-ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na+-K+-ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na+-K+-ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretch-induced responses, including stretch-induced cell death and stretch-induced modulation of Na+-K+-ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na+-K+-ATPase stimulation for an assortment of traditional and novel ventilation techniques.

Regulation of the voltage-gated K+ channel KCNA10 by KCNA4B, a novel β-subunit

2002 ◽  
Vol 283 (1) ◽  
pp. F142-F149 ◽  
Author(s):  
Shulan Tian ◽  
Weimin Liu ◽  
Yanling Wu ◽  
Hamid Rafi ◽  
Alan S. Segal ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
K Channel ◽  
Kinetic Properties ◽  
Functional Assay ◽  
Accessory Proteins ◽  
Amino Acid Homology ◽  
Α Subunit ◽  
Voltage Gated ◽  
Kv Channels ◽  
Α Subunits

Voltage-gated K+ (Kv) channels are heteromultimeric complexes consisting of pore-forming α-subunits and accessory β-subunits. Several β-subunits have been identified and shown to interact with specific α-subunits to modify their levels of expression or some of their kinetic properties. The aim of the present study was to isolate accessory proteins for KCNA10, a novel Kv channel α-subunit functionally related to Kv and cyclic nucleotide-gated cation channels. Because one distinguishing feature of KCNA10 is a putative cyclic nucleotide-binding domain located at the COOH terminus, the entire COOH-terminal region was used to probe a human cardiac cDNA library using the yeast two-hybrid system. Interacting clones were then rescreened in a functional assay by coinjection with KCNA10 in Xenopus oocytes. One of these clones (KCNA4B), when injected alone in oocytes, produced no detectable current. However, when coinjected with KCNA10, it increased KCNA10 current expression by nearly threefold. In addition, the current became more sensitive to activation by cAMP. KCNA4B can be coimmunoprecipitated with the COOH terminus of KCNA10 and full-length KCNA10. It encodes a soluble protein (141 aa) with no amino acid homology to known β-subunits but with limited structural similarity to the NAD(P)H-dependent oxidoreductase superfamily. KCNA4Bis located on chromosome 13 and spans ∼16 kb, and its coding region is made up of five exons. In conclusion, KCNA4B represents the first member of a new class of accessory proteins that modify the properties of Kv channels.

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

1999 ◽  
Vol 114 (6) ◽  
pp. 819-838 ◽  
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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