Hong Long Ji

Molecular and Functional Characterization of a Calcium-sensitive Chloride Channel from Mouse Lung

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32–38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32–38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32–38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and byin situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl−current that could be activated by Ca2+ and ionomycin and inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland

The CLCA family of Ca2+-activated Cl- channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.

Ca2+-Activated Cl– Channels: A Newly Emerging Anion Transport Family

Abstract. A new family of chloride transport proteins has recently emerged. These proteins have extensive homology to a protein previously isolated from bovine tracheal epithelium that acts as a Ca2+-sensitive Cl– channel (CaCC) when heterologously expressed or when reconstituted into planar lipid bilayers. Several new members of this family have been identified in human, murine, and bovine epithelia, in addition to some other tissues, and are associated with Ca2+-sensitive conductive chloride transport when heterologously expressed in Xenopus oocytes or HEK 293 cells. The expressed current is also sensitive to inhibitors such as DIDS and niflumic acid. In addition, at least one family member acts as an endothelial cell adhesion molecule. This emerging family may underlie the Ca2+-mediated Cl– conductance responsible for rescue of the cystic fibrosis (CF) knockout mouse from significant airway disease.

δ-subunit confers novel biophysical features to αβγ- human epithelial sodium channel (ENaC) via a physical interaction

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of δ-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the δ-subunit may form multimeric channels with αβγ-ENaC. Reverse transcription-PCR revealed that δ-ENaC is co-expressed with αβγ-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that δ-ENaC is co-expressed with α-, β-, and γ-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for δαβγ-ENaC differed from those of both αβγ- and δβγ-ENaC (n = 6). The contribution of the δ subunit to PLi/PNa ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of δαβγ-ENaC differed from those of αβγ- and δβγ-ENaC. Co-immunoprecipitation of δ-ENaC with α- and γ-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of δ-ENaC with other subunits could account for heterogeneity of native epithelial channels.

Regulation of Epithelial Na+ Channels by Actin in Planar Lipid Bilayers and in the Xenopus Oocyte Expression System

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na+ channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na+ versus K+ permselectivity, and a substantial increase in mean open and closed times of wild-type αβγ-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the α subunit was deleted (αR613 X βγ-rENaC). 2) When αR613 X βγ-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either αR613 X βγ-rENaC or wild-type αβγ-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that α-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

δ ENaC: a novel divergent amiloride-inhibitable sodium channel

The fourth subunit of the epithelial sodium channel, termed delta subunit (δ ENaC), was cloned in human and monkey. Increasing evidence shows that this unique subunit and its splice variants exhibit biophysical and pharmacological properties that are divergent from those of α ENaC channels. The widespread distribution of epithelial sodium channels in both epithelial and nonepithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathological conditions. Genetic studies link SCNN1D deficiency with rare genetic diseases with developmental and functional disorders in the brain, heart, and respiratory systems. Here, we review the progress of research on δ ENaC in genomics, biophysics, proteomics, physiology, pharmacology, and clinical medicine.

Regulation of epithelial sodium channels by cGMP/PKGII

Airway and alveolar fluid clearance is mainly governed by vectorial salt movement via apically located rate‐limiting Na+ channels (ENaC) and basolateral Na+/K+‐ATPases. ENaC is regulated by a spectrum of protein kinases, i.e. protein kinase A (PKA), C (PKC), and G (PKG). However, the molecular mechanisms for the regulation of ENaC by cGMP/PKG remain to be elucidated. In the present study, we studied the pharmacological responses of native epithelial Na+ channels in human Clara cells and human αβγδ ENaCs expressed in oocytes to cGMP. 8‐pCPT‐cGMP increased amiloride‐sensitive short‐circuit current (Isc) across H441 monolayers and heterologously expressed αβγδ ENaC activity in a dose‐dependent manner. Similarly, 8‐pCPT‐cGMP (a PKGII activator) but not 8‐Br‐cGMP (a PKGI activator) increased amiloride‐sensitive whole cell currents in H441 cells in the presence of CFTRinh‐172 and diltiazem. In all cases, the cGMP‐activated Na+ channel activity was inhibited by Rp‐8‐pCPT‐cGMP, a specific PKGII inhibitor. This was substantiated by the evidence that PKGII was the sole isoform expressed in H441 cells at the protein level. Importantly, intratracheal instillation of 8‐pCPT‐cGMP in BALB/c mice increased amiloride‐sensitive alveolar fluid clearance by ∼30%, consistent with the in vitro results. We therefore conclude that PKGII is an activator of lung epithelial Na+ channels, which may expedite the resolution of oedematous fluid in alveolar sacs.

A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating

1 The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide‐binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N‐tail) that also modulate CFTR channel gating by an unknown mechanism. 2 A disease‐associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP ‐activating cocktail than wild‐type CFTR. 3 In excised membrane patches D58N CFTR exhibited a two‐fold reduction in single channel open probability due primarily to shortened open channel bursts. 4 Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5 Conversely, the N‐tail triple mutant exhibited a markedly inhibited response to AMP‐PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild‐type channel. The N‐tail mutant had both a slower response to AMP‐PNP (activation half‐time of 140 ± 20 s vs. 21 ± 4 s for wild type) and a lower steady‐state open probability following AMP‐PNP addition (0.68 ± 0.08 vs. 0.92 ± 0.03 for wild type). 6 Introducing the N‐tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7 We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.

The Role of Pre-H2 Domains of α- and δ-Epithelial Na + Channels in Ion Permeation, Conductance, and Amiloride Sensitivity

Epithelial Na+ channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na+ selectivity, unitary Na+ conductance (γNa), and amiloride sensitivity of δ-ENaC are strikingly different from those of α-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of δ- and α-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li+ and Na+ (PLi/PNa) for the swap α chimeras co-expressed with βγ-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of PLi/PNa for the swap δ constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the γNa of α-ENaC but decreased the γNa of δ-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{i}^{\mathrm{amil}}\) \end{document}) was observed in the α chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{i}^{\mathrm{amil}}\) \end{document} was obtained in the chimeric δ constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the PLi/PNa ratio, single channel Na+ conductance, and amiloride sensitivity of α- and δ-ENaCs.

LOX-1 Deletion Improves Neutrophil Responses, Enhances Bacterial Clearance, and Reduces Lung Injury in a Murine Polymicrobial Sepsis Model

ABSTRACT Inflammatory tissue injury and immunosuppression are the major causes of death in sepsis. Novel therapeutic targets that can prevent excessive inflammation and improve immune responses during sepsis could be critical for treatment of this devastating disease. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a membrane protein expressed in endothelial cells, has been known to mediate vascular inflammation. In the present study, we demonstrated that LOX-1 deletion markedly improved the survival rate in a murine model of polymicrobial sepsis. Wild-type (LOX-1+/+) and LOX-1 knockout (LOX-1−/−) mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. LOX-1 deletion significantly reduced systemic inflammation and inflammatory lung injury during sepsis, together with decreased production of proinflammatory cytokines and reduced lung edema formation. Furthermore, LOX-1 deletion improved host immune responses after the induction of sepsis, as indicated by enhanced bacterial clearance. Interestingly, we were able to demonstrate that LOX-1 is expressed in neutrophils. LOX-1 deletion prevented neutrophil overreaction and increased neutrophil recruitment to infection sites after sepsis induction, contributing at least partly to increased immune responses in LOX-1 knockout mice. Our study results indicate that LOX-1 is an important mediator of inflammation and neutrophil dysfunction in sepsis.