scholarly journals Temperature Dependence of Voltage-gated H+ Currents in Human Neutrophils, Rat Alveolar Epithelial Cells, and Mammalian Phagocytes

1998 ◽  
Vol 112 (4) ◽  
pp. 503-522 ◽  
Author(s):  
Thomas E. DeCoursey ◽  
Vladimir V. Cherny
Keyword(s):  
Ion Channels ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Alveolar Epithelial Cells ◽  
Human Neutrophils ◽  
Temperature Sensitive ◽  
Whole Cell ◽  
Alveolar Epithelial ◽  
Rate Determining Step ◽  
H Channels

H+ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42°C. Effects of temperature on gating kinetics were distinguished from effects on the H+ current amplitude. The activation and deactivation of H+ currents were both highly temperature sensitive, with a Q10 of 6–9 (activation energy, Ea, ≈ 30–38 kcal/mol), greater than for most other ion channels. The similarity of Ea for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H+ currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant (τact) had similarly high Q10. These results could be explained if H+ channels were composed of several subunits, each of which undergoes a single rate-determining gating transition. H+ current gating in all mammalian cells studied had similarly strong temperature dependences. The H+ conductance increased markedly with temperature, with Q10 ≥ 2 in whole-cell experiments. In excised patches where depletion would affect the measurement less, the Q10 was 2.8 at >20°C and 5.3 at <20°C. This temperature sensitivity is much greater than for most other ion channels and for H+ conduction in aqueous solution, but is in the range reported for H+ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H+ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large Ea of permeation intrinsically limits the conductance of this channel, and appears inconsistent with the channel being a water-filled pore. At physiological temperature, H+ channels provide mammalian cells with an enormous capacity for proton extrusion.

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.

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

1994 ◽  
Vol 103 (5) ◽  
pp. 755-785 ◽  
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.

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.

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.

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.

scholarly journals The Acinetobacter baumannii 19606 OmpA Protein Plays a Role in Biofilm Formation on Abiotic Surfaces and in the Interaction of This Pathogen with Eukaryotic Cells

2009 ◽  
Vol 77 (8) ◽  
pp. 3150-3160 ◽  
Author(s):  
Jennifer A. Gaddy ◽  
Andrew P. Tomaras ◽  
Luis A. Actis
Keyword(s):  
Epithelial Cells ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Protein A ◽  
Bacterial Attachment ◽  
Alveolar Epithelial Cells ◽  
Eukaryotic Cells ◽  
Temperature Sensitive ◽  
Alveolar Epithelial ◽  
Abiotic Surfaces

ABSTRACT The ability of Acinetobacter baumannii to adhere to and persist on surfaces as biofilms could be central to its pathogenicity. The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces. This multistep process also depends on the expression of transcriptional regulatory functions, some of which could sense nutrients available to cells. This report extends previous observations by showing that although outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on plastic, it is essential for bacterial attachment to Candida albicans filaments and A549 human alveolar epithelial cells. In contrast to abiotic surfaces, the interaction with biotic surfaces is independent of the CsuA/BABCDE-mediated pili. The interaction of A. baumannii 19606 with fungal and epithelial cells also results in their apoptotic death, a response that depends on the direct contact of bacteria with these two types of eukaryotic cells. Furthermore, the bacterial adhesion phenotype correlates with the ability of bacteria to invade A549 epithelial cells. Interestingly, the killing activity of cell-free culture supernatants proved to be protease and temperature sensitive, suggesting that its cytotoxic activity is due to secreted proteins, some of which are different from OmpA.

Alveolar epithelial cells type II show a high sensitivity to cigarette smoke extract

Pneumologie ◽  
2014 ◽  
Vol 68 (06) ◽  
Author(s):  
S Seehase ◽  
B Baron-Luehr ◽  
C Kugler ◽  
E Vollmer ◽  
T Goldmann
Keyword(s):  
Epithelial Cells ◽  
Cigarette Smoke ◽  
High Sensitivity ◽  
Cigarette Smoke Extract ◽  
Alveolar Epithelial Cells ◽  
Type Ii ◽  
Alveolar Epithelial
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