ATP secretion after hypotonic shock and acute effect on <i>I</i>_<i>sc</i> generated by alveolar epithelial cells.

<p>The time-course of 20% hypotonic shock (20% hypo) in apical ATP accumulation is depicted in A. At T₀, the same volume of liquid was added to the apical and basolateral sides of the cell monolayers to achieve 20% hypotonic shock (▲) with H₂O or an equivalent volume of physiological buffer (■) for the isotonic condition. Aliquots of apical medium were sampled at different time points, and ATP was measured by luciferin/luciferase assay. N≥7, *p<0.05 by Mann-Whitney Test between 20% Hypo at 1 min and time 0. Apical (ATP_ap) or bilateral, but not basolateral (ATP_ba) addition of ATP, decreased total transepithelial current generated by alveolar epithelial cells (<b>B</b>). N≥5, *p<0.05 by Mann-Whitney Test between apical or bilateral addition of ATP and untreated cells. NS, non significant. Bilateral addition of ATP decreased ENaC (1 µM amiloride-sensitive) current (<b>C</b>). N=6, *p<0.05 by Mann-Whitney Test between ATP and untreated controls (Ctrl). To rule out a role of ATP in hypotonic shock modulation of currents, the cells were pretreated with the ATP scavenger apyrase (10 U/ml; Apyr) for 2 min before challenging the monolayers with 20% hypotonic shock (<b>D</b>). Apyrase treatment had no significant effect on the <i>I</i>_<i>sc</i> increase induced by hypotonic shock. N≥4, NS: non significant by Mann-Whitney Test between 20% Hypo in presence of absence of Apyr. *p<0.05 by Mann-Whitney Test between basal (white) or 20% hypo (grey) treated cells.</p

Channels and transporters involved in ionic transport in basal and hypotonic shock conditions.

<p>In the basal condition, a transcellular Cl^- transport via NPPB-sensitive apical channels (Cl^- ch) and basolateral K⁺/Cl^- co-transporter (KCC) generates membrane potential that is optimal for Na⁺ transport via amiloride-sensitive ENaC and NSC. Although alveolar epithelial cells express CFTR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074565#B59" target="_blank">59</a>], in the absence of a cAMP agonist, CFTR is not involved in basal Na⁺ transport. During a 20% hypotonic shock, Ca²⁺/calmodulin and a basolateral Cl^- Influx play a central role in the transient current rise elicited by hypotonicity. NPPB-sensitive and insensitive pathways are involved for this flux. Although basolateral K⁺ channels (K⁺ ch) are not involved in the current rise elicited during hypotonic shock, their inhibition blunts current normalization after hypotonic shock. →: Ionic flux; <b>T</b> : Inhibitor.</p

Role of Na⁺ and K⁺ currents in basal and hypotonic-induced transepithelial current.

<p>Basal transepithelial current (<i>I</i>_<i>sc</i> Basal), hypotonic-induced current (<i>I</i>_<i>sc</i> 20% Hypo) and the current rise elicited by hypotonic shock (Δ <i>I</i>_<i>sc</i> Shock) are depicted in the basal condition (Ctrl), potassium-free condition (K⁺(-)) or after pretreatment with 1µM amiloride (Amil 1), 100 µM amiloride (Amil 100) or 100 µM basolateral clofilium (Clofi). In experiments where the cells were pre-treated with an inhibitor that impacted on <i>I</i>_<i>sc</i>, hypotonic shock was induced when the current was stabilized. For amiloride the current was stable after 5 min. In potassium-free condition (K⁺(-)) and clofilium pre-treatment, a longer incubation was needed (~30 min). N≥4, *p<0.05 by Mann-Whitney Test compared to untreated controls.</p

Inhibition of calmodulin kinase by W7 decreases the hypotonic shock current rise.

<p>Alveolar epithelial cells were pretreated for 20 min with 25 µM W7, a Ca²⁺/calmodulin antagonist, before <i>I</i>_<i>sc</i> recording in Ussing chamber. Total (<i>I</i>_<i>sc</i> Total) and ENaC currents were evaluated in basal condition (Basal; white) or after 20% hypotonic shock (20% Hypo; gray). W7 pretreatment inhibited the total and ENaC <i>I</i>_<i>sc</i> rise after 20% hypotonic shock. N≥4, *p<0.05 by Mann-Whitney Test compared to Basal.</p

Implication of Cl^- channels and KCC in basal and hypotonic-induced transepithelial current.

<p>Basal transepithelial current (<i>I</i>_<i>sc</i> Basal), hypotonic-induced current (<i>I</i>_<i>sc</i> 20% Hypo) and the current rise elicited by hypotonic shock (Δ <i>I</i>_<i>sc</i> Shock) are depicted in the basal conditions (Ctrl), after treatment with 100 µM basolateral bumetanide (Bumet), 100 µM apical (NPPB_a) or basolateral (NPPB_b) NPPB, in bilateral Cl^- reduced buffer (Cl^-(-)) or 100 µM basolateral DIOA. In experiments where the cells were pre-treated with an inhibitor that impacted on <i>I</i>_<i>sc</i>, hypotonic shock was induced when the current was stabilized. For apical and basolateral NPPB, a 5 to 10 min incubation was needed while the current stabilized after ^~30 min for DIOA. Pre-treatment with bumetanide from 10 min to 30 min did not have an impact on <i>I</i>_<i>sc</i> Basal. N≥4, *p<0.05 by Mann-Whitney Test compared to untreated controls. # p<0.05 by Mann-Whitney Test compared to NPPB_a, ¤ p<0.05 by Mann-Whitney Test compared to NPPB_b or DIOA.</p

Presentation1_Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.pdf

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.</p

Additional file 2: of CFTR Knockdown induces proinflammatory changes in intestinal epithelial cells

Bax protein expression in Caco-2/15 cells exposed to the various experimental conditions. Caco-2/15 cells infected or not were stimulated 24 h with either TNF or IL-1β at 25 ng/ml. Bax protein expression was analyzed by Western blotting. Data represent the means ± SEM of n = 3 independent experiments and are reported as the Bax/β-actin ratio. (TIFF 227 kb

Investigating CFTR and KCa3.1 Protein/Protein Interactions

<div><p>In epithelia, Cl^- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl^- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl^- ions and electrolytes needs however to be coupled to an increase in K⁺ conductance in order to recycle K⁺ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K⁺ efflux is ensured by K⁺ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca²⁺ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca²⁺.</p></div

Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells.

<p>Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.</p

CFTR/KCa3.1 interactions modulated by internal Ca²⁺.

<p>A: CFTR and KCa3.1 number densities (molecule/μm²) under control and Ca²⁺ influx conditions. KCa3.1 number density dramatically decreases (4-fold) during Ca²⁺ influx induced by pretreatment with CPA (cyclopiazonic acid) followed by exposure to extracellular Ca²⁺, consistent with either internalization of this channel or its clustering. B: CFTR and KCa3.1 degree of aggregation (DA) under control and Ca²⁺ influx conditions. KCa3.1 degree of aggregation increases significantly (4-fold) during Ca²⁺ influx. The 4-fold decrease in number density is accounted for by the 4-fold increase in KCa3.1 cluster size (DA). C: Significant increase in the fraction of KCa3.1 interacting with CFTR in response to a rise in intracellular Ca²⁺ concentration. Protein/protein interactions occurred on a slow time scale (D) and most interactions involved molecules that were immobilized on the plasma membrane (E). Statistical analysis based on n = 62 cells for control experiments and n = 21 cells for measurements in CPA+ Ca²⁺ conditions.</p