characterization of the epithelial sodium channel delta ... · enac and its isoform (δ2) can be...

Characterization of the epithelial sodium channel delta subunit in

human nasal epithelium

Nadine Bangel-Ruland1*, Katja Sobczak1, Tina Christmann1, Dominik Kentrup2, Hanna

Langhorst1, Kristina Kusche-Vihrog3 & Wolf-Michael Weber1

1Institute of Animal Physiology, University of Muenster, Hindenburgplatz 55, 48143

Muenster, Germany

2Medical Clinic and Policlinic D, University clinics Muenster, Domagkstraße 3A, 48149

Muenster, Germany

3Institute of Physiology II, University clinics Muenster, Robert-Koch-Str. 27b, 48149

Muenster, Germany

*Corresponding author:

Nadine Bangel-Ruland

Institute of Animal Physiology

University of Muenster

Hindenburgplatz 55

48143 Muenster, Germany

Tel: ++49 251 83 21784

Fax: ++49 251 83 21785

Email: [email protected]

This article has an online data supplement, which is accessible from this issue’s table of

content online at www.atsjournals.org

of 34 AJRCMB Articles in Press. Published on June 11, 2009 as doi:10.1165/rcmb.2009-0053OC

Copyright (C) 2009 by the American Thoracic Society.

mailto:[email protected]

2

Abstract

The epithelial sodium channel (ENaC) mediates the first step in Na+ reabsorption in epithelia

cells such as kidney, colon, and airways and may consist of four homologous subunits

(α, β, γ, δ). Predominantly, the α-subunit is expressed in these epithelia and it usually forms

functional channels with the β- and γ-subunits. The δ-subunit was first found in human brain

and kidney but the expression was also detected in human cell lines of lung, pancreatic and

colonic origin. When co-expressed with β and γ accessory subunits in heterologous systems

the two known isoforms of the δ-ENaC subunit (δ1 and δ2) can build amiloride-sensitive Na+

channels. In the present study we demonstrate the expression and function of the δ-subunit in

human nasal epithelium (HNE). We cloned and sequenced the full-length cDNA of the δ-

ENaC subunit and could show that in nasal tissue at least isoform 1 is expressed. Furthermore,

we carried out Western blot analyses and compared the cell surface expression of the δ-

subunit with the classically expressed α-subunit by using immunofluorescence experiments.

Thereby, we could show that the quantity of both subunits is almost similar. Additionally, we

show the functional expression of the δ-ENaC subunit with measurements in modified Ussing

chambers and demonstrate that in HNE a large portion of the Na+ transport is mediated by the

δ-ENaC subunit. Therefore, we suppose that the δ-subunit may possess an important

regulatory function and might interact with other ENaC subunits or members of the

DEG/ENaC family in the human respiratory epithelium.

Keywords: ENaC, human respiratory epithelium, δ-subunit

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Introduction

Members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of non-voltage gated

ion channels are expressed in a huge variety of epithelia and neuronal tissues. The immense

functional diversity includes acid sensing, mechanotransduction and peptide gating mediated

by degenerins and acid-sensitive ion channels (ASICs) as well as Na+ absorption by the

amiloride-sensitive epithelial sodium channel ENaC (1, 2).

Four homologous ENaC subunits named α-, β-, γ- and δ have been identified, which

share sequence homology of ~ 37 % at the amino acid level (3-5). The correlation between

these subunits and the exact stoichiometry of the channel is still a matter of discussion. Over a

long period it was proposed that ENaC is a tetramer (6, 7), while others suggested that ENaC

is formed by nine subunits (8, 9). Since Jasti and colleagues determined the crystal structure

of chicken ASIC1 (10), ENaC was mapped to these structural coordinates and therefore, a

trimeric structure was postulated (11).

Predominantly, the α-subunit is expressed in epithelia such as kidney, lung, and colon

and there it usually forms functional channels with the β- and γ-subunits (3). It is involved in

the control of Na+ balance, blood volume and pressure. Beside the α-ENaC the δ-subunit is

characterized as a pore-forming subunit that when expressed together with β and γ produces

amiloride-sensitive Na+ currents that are quiet different to those that are generated by the

classical ENaC channel composition. It was reported that δ-ENaC generates a constitutive

current that is 2 magnitudes larger when associated with β- and γ-subunits than that of

homomeric δ-ENaC channels (5). While co-expression of α- with γ-ENaC increases the

current by one order of magnitude (3, 12), it could be demonstrated that none of the α-, β- or,

γ- subunits alone altered or increased the δ-ENaC current when co-expressed with this subunit

(5). These experiments were carried out in the Xenopus laevis oocyte expression system that

allows characterizing functional properties of the particular subunit under defined conditions.

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Up to now it is not known whether the δ-ENaC subunit associates with other subunits

in vivo to play a role in physiological functions. However, the δ-subunit has a widespread

distribution in different tissues (5, 13-15) and therefore, is often co-expressed with the other

ENaC subunits and/or ASICs (16, 17). When expressed in human brain, it was shown that δ-

ENaC and its isoform (δ2) can be activated by protons, indicating that they may contribute to

pH sensitivity in this tissue (13, 18). New data show that δ-ENaC is also a candidate molecule

for pH sensing in the gastrointestinal system in humans (19) as well as in human skin (15).

In general, it is known that changes in the stoichiometry of heteromultimeric proteins,

e.g. ENaC, lead to different functional properties (20). Since ENaC and ASIC subunits are co-

expressed in multiple tissues and cell types it could be demonstrated that combinations of

ASIC1 with ENaC subunits exhibit novel electrophysiological properties (17). It was shown

that ion channel properties, including cation selectivity and pharmacological inhibition, are

affected by the subunit composition of the expressed channel.

These observations prompted us to investigate if the δ-subunit is expressed in human

respiratory epithelium and whether this subunit is also functionally active. Previously, we

have already demonstrated the expression of α-, β-, and γ-ENaC in HNE (21). Since cells

derived from HNE have been reliable and useful tools for the study of ion transport in the

respiratory tract (22, 23), we used that technique to study the expression and function of δ-

ENaC.

First of all we investigated the expression of the δ-subunit in HNE. Thereby, we (i)

detected the δ-subunit via RT-PCR and (ii) showed its presence in Western blot experiments.

Additionally, we (iii) compared the cell surface expression of the δ-subunit with the

classically expressed α-subunit by using immunofluorescence experiments and showed that

the quantity of both subunits is almost similar. Thereafter, we were interested in the portion of

the δ-ENaC mediated Na+ current, which (iv) could be distinguished from the amiloride-

sensitive current by the specific blocker Evans Blue. It was shown that the application of

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Evans Blue inhibits the activity of δ-ENaC while the α-subunit was not affected (24).

Therefore, Evans Blue seems to be a δ-subunit specific inhibitor and allows distinguishing

between the functional activity of the α- and δ-subunits, respectively. Our data represent the

first functional characterization of the ENaC δ-subunit in HNE.

Material and Methods

Patients

We obtained nasal specimens from patients undergoing nasal surgery. The samples were nasal

polyps or turbinates of patients suffering from chronic sinusitis. The study was approved by

the committees for human studies of the University of Muenster (Ethik Kommission

Muenster) and the University of Giessen (Ethik Kommission der Justus-Liebig-Universitaet

Giessen). All patients gave informed consent.

Extraction of RNA

Total RNA was extracted from primary cultured human nasal cells following the standard

protocol of RNeasy Mini Kit plus (Qiagen, Hilden, Germany). The concentration of RNA was

determined by absorbance measurement at 260 nm (A260) in a spectrophotometer using UV

cuvettes. The integrity and size distribution of total RNA was analyzed via denaturing agarose

gel electrophoresis and ethidium bromide staining.

cDNA synthesis

Total RNA (1 µg) was used to generate first-strand cDNA by using the SuperScript II reverse

transcriptase kit (Invitrogen, Karlsruhe, Germany) and oligo (dT) primers.

Molecular cloning of δ-ENaC cDNA

After PCR amplification with specific oligonucleotide primers according to the published

sequences of human ENaC (http://www.ncbi.nih.gov), all expected distinct bands were

excised using the QIAquick Gel Extraction Kit (Qiagen) and cloned into the pSC-A vector

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(StrataCloneTM PCR Cloning Kit, Stratagene, Heidelberg, Germany). The nucleotide sequence

of δ-ENaC cDNA was submitted to GenBank, accession number EU489064.

Sequence data analysis

For cDNA sequence analysis we used the tools provided by the ExPasy Molecular Biology

Server of the Swiss Institute of Bioinformatics (http://www.expasy.ch) and the National

Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov). All clones were

commercially sequenced by GENterprise (GENterprise GmbH, Mainz, Germany).

Cell culture

Primary cell culture of the HNE was performed as described before (25). The nasal epithelial

cells were isolated by enzymatic digestion for 24-48 h and afterwards seeded on permeable

collagen filters with a diameter of 14 mm (Cellagen TM dics CD 24, MP Biomedicals,

Heidelberg, Germany). The cells were cultured with serum-free F-12 Nutrient Mixture (Ham)

(Invitrogen/Gibco, Karlsruhe, Germany) supplemented with the following agents: insulin

(2 µg/ml) (Invitrogen/Gibco), epidermal growth factor (13 ng/ml) (Sigma, Deisenhofen,

Germany), endothelial cell growth supplement (7.5 µg/ml) (Becton Dickinson GmbH,

Heidelberg, Germany), triiodo-thyronine (3 nM) (Sigma), hydrocortisone (100 nM) (Sigma),

gentamycin (10 µg/ml) (Biochrom AG, Berlin, Germany), penicillin/streptomycin (100 U/ml)

(Invitrogen/Gibco), L-Glutamin (2 mM) (Invitrogen/Gibco) and transferrin (4 µg/ml)

(Invitrogen/Gibco). Previously, it was shown that these supplements had no effect on the

electrical parameters of the HNE (25). Cells were incubated in 95 % air and 5 % CO2 at

37 °C. 7-9 days after seeding the cells on the membrane we normally obtained confluent

monolayers and proceeded with Ussing chamber measurements.

For immunofluorescence experiments cells were grown in culture dishes (Ø = 3.5 cm) coated

with collagen (0.15 mg/ml, Collagen Typ I, Sigma). After reaching confluence, cells were

split using trypsin (0.05 %) and then cultured on 8-well diagnostic microscope slides (Menzel

GmbH, Braunschweig, Germany) also coated with collagen. These slides were placed in Petri

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dishes filled with culture medium. For immunofluorescence experiments the cells were fixed

in glutaraldehyde at 37 °C. Briefly, glutaraldehyde was added to a final concentration of

0.05 % to the cell culture medium. After a period of 15 min the fixative was removed and

cells were stored at 4 °C.

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Protein biochemistry

Membrane proteins from primary cultured HNE cells were isolated using lysis buffer (1 mM

Tris ,15 mM NaCl, 0.2 mM EDTA, 2 % Triton X-100) and a protease inhibitor cocktail

(Sigma) was added to this detergent mixture. It was shown that 2 % Triton-X 100 is sufficient

to solve all of the intracellular and cell surface pools of the sodium channel ENaC (26). The

extracts were homogenized with a Sonifier®ultrasonic cell disrupter (Branson, Danbury,

USA) and centrifuged for 30 min at 3220 g at 4 °C to remove insoluble material. The

concentration of the proteins in the supernatant was measured photometrically using the BCA

test (Pierce, Rockford, USA). For the detection of the α- and δ-ENaC subunits 30 µg of total

membrane proteins were separated via SDS-PAGE (7.5 % acrylamide) and transferred to a

PVDF membrane. Non-specific binding sites were blocked for 2 h with 5 % nonfat dry milk

in Tris-buffered saline/Tween (TBST: 10 mM Tris HCl, pH 7.4; 140 mM NaCl; 0.3 % Tween

20). The δ-ENaC subunit was detected with a rabbit anti δ-ENaC antibody (Santa Cruz,

California, USA) with a concentration of 1:200 diluted in 5 % nonfat dry milk/TBST

overnight at 4 °C. The ENaC α-subunit was detected with a rabbit anti-α-ENaC antibody

(PA1-920A, Dianova, Hamburg, Germany) with a concentration of 1:250 diluted in 5 % non-

fat dry milk/TBST at 4 °C overnight. After washing in TBST the membrane was incubated for

1 h at room temperature with the goat anti-rabbit IgGs conjugated with alkaline phosphatase

(Santa Cruz, California, USA) diluted 1:10,000 in 5 % non-fat dry milk/TBST. The

membrane was washed again in TBST and detection was carried out with NBT (nitroblue

tetrazolium) and BCIP (5-bromo-4-chloro-3- indolyl phosphate).

As positive control we used MIA Paca-2 cell lysate (Santa Cruz) to verify the specificity of

the anti δ-ENaC antibody and a neutralizing peptide (PEP-088, Dianova) to confirm the α-

ENaC antibody specificity.

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Antibody staining

Antibody staining was performed as described previously (27). Briefly, human nasal cells

were gently washed 5 times in phosphate buffered saline (PBS, in mM: 140 NaCl, 2 KCl, 4

Na2HPO4, 1 KH2PO4, pH 7.4) at room temperature after fixation (0.05 % glutaraldehyde) and

incubated afterwards for 30 min in 100 mM glycine/PBS solution. The cells were washed

again 5 times by gentle shaking in PBS and then blocked with 10 % normal goat serum (NGS)

at room temperature for 1 h. For the immunofluorescence experiments we used antibodies that

bind to the extracellular loop of the ENaC subunits, respectively. The primary polyclonal

rabbit anti α-ENaC (Santa Cruz) and anti δ-ENaC (Santa Cruz) antibodies were diluted

1:1000 and 1:200, respectively in 10 % NGS and applied to the cells.

After 1 hour of incubation with the primary antibody the cells were washed 5 times in PBS.

For Quantum Dot (QD)-labelling we incubated the cells with QD655 goat F(ab´)2 anti rabbit

IgG conjugates (Invitrogen) (1:100) at room temperature in the dark for 1 h. Cells were then

washed again five times in PBS, two times in HEPES (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1

CaCl2, 5 glucose, 10 HEPES) and fixed with 0.05 % glutaraldehyde in HEPES buffer for 15

min.

As a negative control, we incubated human nasal epithelial cells only with the secondary

antibody (figure S1 A, supplemental information). The background staining of the cells is due

to fixation with glutaraldehyde, which allows the identification of cell borders.

For further control experiments we incubated human nasal cells with a primary anti-Tubulin

antibody (1:200) (Tubulin, alpha, DLN-09993, Dianova, Hamburg, Germany) and Cy3-

labelled anti mouse secondary antibody (1:500) (Dianova) to ensure that the cells are not

permeabilized during preparation (figure S1 B, supplemental information). For comparison

only, we detected α-Tubulin in the cells by permeabilization with paraformaldehyd (0.37 %)

(figure S1 C, supplemental information). The staining with the anti-Tubulin antibody of non-

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permeabilized HNE cells was checked in every experiment with an inverted fluorescence

microscope.

Image analysis

Immunofluorescence images were acquired with an inverted fluorescence microscope

(Axiovert 200, Zeiss, Jena, Germany) equipped with a 100 x 1.45 oil immersion objective.

We used the following filter: QD655, 420 nm excitation, 655 nm emission (XF302-1 filter,

Omega Optical, Brattleboro, USA). Cell surface areas of HNE were selected manually and

QD-labelled ENaC molecules in the plasma membrane of the cells were detected by focusing

on the upper surface of the cells. Data acquisition and analysis were performed with the

Metavue Software (Visitron, Puchheim, Germany). Therefore, numbers of QD-labelled α- and

δ-ENaC molecules at the cell surface were counted manually. Subsequently, means were

corrected with the negative control values (HNE cells incubated only with the secondary

antibody).

Transepithelial measurements

After reaching confluence, the nasal epithelial cells were mounted in modified Ussing

chambers designed by Prof. Willy Van Driessche (KU Leuven, Belgium). The electrical

measurements were performed as described in more detail elsewhere (28). Briefly, the two

compartments of the Ussing chamber were continuously perfused with cell culture Ringer at

37 °C. For voltage current-measurements we used KCl electrodes, which were connected to

Ringer solution through agar bridges. For the measurements the transepithelial potential (Vt)

was clamped to 0 mV with a low noise voltage clamp. The short-circuit current (Isc) was

continuously monitored using the computer program ImpDsp1.4 (Prof. Willy Van Driessche,

KU Leuven, Belgium). The measured area had a size of 0.5 cm2, while the electrical

parameters were normalized to an area of 1 cm2.

siRNA transfection procedures

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To downregulate δ-ENaC, HNE cells were transfected with 10 nM siRNA using HiPerfect

Transfection reagent (Qiagen). 7 days after seeding the cells on the membrane we normally

obtained confluent monolayers and carried out the transfection procedure following the

manufactures protocol. The HiPerfect/siRNA complex remained on the apical side of the

support for 24 h and the functional properties were measured in Ussing chamber experiments.

We determined transfection efficiency by semi-quantitative RT-PCR. We used commercially

available siRNA complementary to the δ-ENaC subunit (Qiagen, Hs_SCNN1D_2 HP

siRNA).

Semi-quantitative RT-PCR

2 µl of cDNA first strand products were amplified with 1.25 units TopTaq polymerase

(Qiagen) in the presence of the specific primers, respectively (δ-ENaC sense: 5` CAG CAT

CCG AGA GGA CGA GGT G 3`, δ-ENaC antisense: 5` CGG TAG TTG AGC TCC TGG

TAG ACG 3`). The oligonucleotide primers for RT-PCR were selected to bind specifically to

human cDNA and created spanning Exon/Exon boundaries to prevent amplification of

genomic DNA. As external control we used human Glycerin-Aldehyd-3-Phosphat-

Dehydrogenase (GAP-DH) specific primers (GAP-DH sense: 5` GAC ATC AAG AAG GTG

GTG AAG CAG 3`, GAP-DH antisense: 5` GCC ATG AGG TCC ACC ACC CTG 3`),

which produce a PCR fragment of 218 bp. The conditions were chosen that none of the

cDNAs analyzed reached a plateau but are situated in the exponential phase of the

amplification. A first cycle of 3 min at 94 °C, 45 seconds at 59 °C and 1 min at 72 °C was

followed by 45 seconds at 94 °C for 32 cycles.

siRNA control experiments

For control experiments we used the AllStars Negative Control siRNA (Qiagen), which is a

scrambled, nonsilencing siRNA with no homology to any known mammalian gene.

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Furthermore, the gene expression analyses was carried out on cells that have not been treated

(non-transfected control).

Semi-quantitative analyses

Images of the RT-PCR loaded on ethidium bromide-stained agarose gels were recorded with a

digital camera (Canon, Krefeld, Germany) combined with the BioDocAnalyze system

(Biometra, Göttingen, Germany) and quantification was performed by densitometry using

ImageJ analysis software 1.36 (Sun Microsystems, Inc., California, USA). The ratio between

the sample RNA and GAP-DH was calculated to normalize the initial variations in sample

concentration and as control for reaction efficiency.

Solutions

Cell culture Ringer was composed as follows (in mM): 130 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 5

glucose, 10 HEPES. In the Na+-free solution used, NaCl was replaced by an equivalent

amount of TMACl (tetramethylammonium chloride). For investigation of the amiloride-

sensitive α- and δ-ENaC currents the apical compartment of the Ussing chamber was perfused

with amiloride (100 µM) and Evans Blue (300 µM) in cell culture Ringer, respectively.

Statistics

Where applicable, data are expressed as arithmetic means ± SEM; n is the number of

experiments. Statistical analysis was made by the t-test where appropriate, and a significant

difference was assumed at p < 0.05 (*) or p < 0.01 (**).

Results

Molecular cloning of δ-ENaC human nasal epithelium

The δ-ENaC subunit was cloned and sequenced and the amino acid sequence was compared

in a multi-sequence alignment with the previously published sequences of δ-ENaC

(http://www.ncbi.nlm.nih.gov). Using reverse transcription-polymerase chain reaction (RT-

PCR) we showed that the transcript and protein encoding δ-ENaC (isoform 1) is expressed in

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HNE. The coding region of the δ-ENaC subunit comprises 1917 bp translated into 638

aminoacids. The nucleotide sequence of δ-ENaC cDNA was submitted to GenBank, accession

number: EU489064.

Semi-quantitative RT-PCR of δ-ENaC expression in HNE

For subsequent investigations it was required to downregulate δ-ENaC expression. Therefore,

we used specific siRNA directed against δ-ENaC and carried out semi-quantitative RT-PCR

to confirm the knockdown. The δ-ENaC specific primer produced a fragment of 474 bp. For

normalization we used human GAP-DH specific primers (239 bp fragment). We observed that

the expression of the δ-subunit is reduced (47.2 ± 3.19 %) by about 52.8 % by specific siRNA

compared to the non-transfected cell control (n = 7) (figure 1A). The non-silencing,

scrambled siRNA that we used as negative control (NC) caused no considerable changes in δ-

ENaC expression (98.14 ± 13.5 %) (figure 1B). Representative images of the semi-

quantitative RT-PCR are shown in figure 1C.

Detection of the δ-ENaC protein

For the determination of the presence and abundance of δ-ENaC in HNE on the protein level

we performed Western blot analyses. These experiments were repeated three times and a

representative experiment is shown (figure 2). According to the manufactures` specifications

the anti δ-ENaC antibody detected one distinct and specific band in the range of 100 kDa,

representing the δ-ENaC subunit in HNE. Furthermore, we demonstrated the knockdown of

the δ-subunit with siRNA. We detected a specific band of δ-ENaC in non-transfected HNE

cells (lane 1) and a decreased band in the siRNA transfected sample (lane 2). As positive

control we used MIA PaCa-2 cell lysate (Santa Cruz) (lane 3).

In order to demonstrate that the expression of α-ENaC is unaffected by the transfection of the

δ-ENaC siRNA, we identified the α-subunit in the range of 65 kDa in non-transfected (figure

S2, lane 1, supplemental information) and in δ-ENaC siRNA transfected HNE cells (figure

S2, lane 2, supplemental information). As reviewed by Rossier and Stutts, this 65 kDa band

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represents an endogenous proteolytic cleaved form of the α-ENaC protein (29). Furthermore,

a neutralizing peptide was used to confirm α-ENaC antibody specificity (figure S2, lane 3,

supplemental information). The signal of the 65 kDa band detected in lane 1 and 2 was

abolished by the neutralizing peptide (lane 3).

Additionally, we carried out immunofluorescence experiments to detect δ-ENaC exclusively

at the cell surface with the specific anti-ENaC antibodies, which bind to the extracellular loop.

For this approach we used QD-labelled anti-rabbit F(ab´)2 conjugates as secondary

antibodies. Experiments were performed with material from five different patients and at least

30 cells were analyzed. Using this technique we were able to show that the δ-subunit is

expressed at the cell surface and/or in nearby regions. Using this method, we compared the

expression of δ-ENaC with the classically expressed α-subunit and quantified ENaC

molecules at the cell surface. Representative images for both subunits are shown in figure 3

(A, B). We counted the abundance of QD-labeled ENaC molecules/µm2 apical cell surface of

HNE for each cell, thereby demonstrating that the amount of α-ENaC molecules is only

slightly larger (0.164 ± 0.008 ENaC molecules/µm2) than of δ-ENaC molecules (0.146 ±

0.025 ENaC molecules/µm2) (figure 3C), suggesting that in HNE both subunits are expressed

in a similar manner.

Transepithelial measurements

The electrophysiological parameters in HNE were determined in modified Ussing chambers.

We used amiloride (100 µM) to identify total ENaC current. Subsequent application of the δ-

ENaC subunit specific blocker Evans Blue (300 µM) revealed the portion of transepithelial

current mediated by the δ-subunit. Figure 4A shows the short-circuit current (Isc) of cultured

monolayers from HNE. Isc is markedly inhibited by amiloride, demonstrating the Na+

absorption via ENaC, which represents 39.1 ± 2.3 % of total Na+ absorption, determined by

removing Na+ from the apical Ringer. In a second step the effects of Evans Blue in the

absence and presence of amiloride are shown. When Evans Blue was applied to the apical

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side the Na+ absorption was inhibited by 19.8 ± 1.9 % in average. The simultaneous

application of amiloride and Evans Blue led to a decrease of the transepithelial Na+ absorption

by 40.1 ± 2.2 % (figure 4B). Thus, the portion of Evans Blue-sensitive current dependent on

the amiloride-sensitive Na+ current is ~ 50 %. To verify the specificity of Evans Blue and the

contribution of δ-ENaC to native Na+ transport across epithelia we downregulated δ-ENaC

expression with siRNA complementary to this subunit.

In these functional transfection analyses we could demonstrate that the application of

amiloride only slightly inhibited the transepithelial Na+ absorption compared to the

untransfected monolayers, thereby representing the remaining α-ENaC current (figure 5A). As

expected, the addition of Evans Blue revealed only a marginal change of the Isc, dependent on

the downregulation of δ-ENaC. Thereby, we could show that after 24 h the Evans Blue-

sensitive current was reduced from 19.8 % to 0.7 % (figure 5B) and therefore almost

completely abolished (reduction: 96.7 %). Thus, we can assume that Evans Blue is also in

native tissue δ-ENaC specific and it is possible to determine its contribution to the native Na+

transport of human primary cultured cells.

Discussion

The detection of the δ-ENaC subunit in human nasal epithelium (HNE) alters the point of

view on ENaC function. In previous studies we demonstrated that α-, β-, and γ-ENaC are

expressed in this tissue (21). Here we show that also the δ-ENaC subunit is constitutively

expressed in respiratory epithelia. Co-expression of these subunits was reported previously for

cell cultures of human lung origin (H441 and A549) (14), human epidermis and keratinocytes

(15). But no functional proof for δ-ENaC expression was found in native tissue yet. In a first

approach we cloned and sequenced the cDNA of the δ-subunit and could show that at least

isoform 1 is expressed in HNE. This verification was followed by the detection of the δ-ENaC

protein using Western blot analyses. Thus, we could show that the δ-subunit is present in the

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total membrane protein extract of human nasal cells. By using immunofluorescence

experiments we compared the expression of the δ-subunit with the classically expressed α-

subunit at the apical cell surface and showed that the quantity of both subunits is almost

similar. These results revealed that the amount of α-ENaC molecules is only slightly higher

than of δ-ENaC molecules, suggesting that in HNE both subunits are expressed in a similar

manner. This observation indicates that the δ-ENaC subunit plays an important role in Na+

transport in respiratory epithelia.

To obtain functional data of the δ-subunit in the apical membrane of HNE we carried

out electrophysiological tests using Ussing chamber measurements. Previously, it was shown

that amiloride is a well known blocker of both channel complexes (αβγ, δβγ), while Evans

Blue inhibits the δ-subunit current specifically, but does not affect the α-subunit current (24).

With the transepithelial measurements we observed an Evans Blue-sensitive current that was

clearly distinguishable from the amiloride-sensitive current by using Evans Blue alone in

comparison to an amiloride/Evans Blue mixture. The portion of the δ-ENaC mediated Na+

current was 19.8 ± 1.9 % in average of total ENaC current. When both blockers were applied

40.1 ± 2.2 % of ENaC current was inhibited. Thus, the Evans Blue-sensitive Na+ current

contributes about 50 % to the amiloride-sensitive current. To proof that the Evans Blue-

sensitive Na+ current is indeed mediated by the δ-subunit we downregulated the expression of

δ-ENaC in primary cultures of HNE with δ-ENaC specific siRNA. We showed by semi-

quantitative RT-PCR and Western blot analyses an effective knockdown of δ-ENaC

expression. Furthermore, the Western blot reveals that the expression of the α-subunit remains

unaffected (see figure S2, supplemental information). Subsequently, we demonstrated this

downregulation in Ussing chamber experiments thereby demonstrating that the Evans Blue-

sensitive current is nearly completely reduced after 24 h (96.7 %). Although several studies

revealed that Evans Blue is able to modulate receptors and ion channels (30-33), we could not

observe any effect on other anion channels in our cell preparations, verified by the addition of

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Na+-free Ringer solution in combination with Evans Blue in Ussing chamber experiments

(data not shown). The high specificity of such siRNA molecules prompted us to assume that

Evans Blue is δ-ENaC specific in native tissue and it is possible to determine its contribution

to the native Na+ transport of human primary cultured cells.

Up to now the physiological and pathological role of δ-ENaC in non-neuronal tissues

is still unclear, but our findings raise the question if the δ-ENaC subunit possesses important

regulatory functions and if it interacts with other ENaC subunits or members of the

DEG/ENaC family in the human respiratory epithelium. Former in vitro studies of other

groups showed that the δ-subunit leads to differentiated biophysical and pharmacological

ENaC properties, that could modify functional properties of the channel (14). They

demonstrated that the selectivity for monovalent and divalent cations, single channel

conductance, amiloride affinity, gating kinetics, and proton activation of αβγδ-ENaC were

different from those of αβγ- and δβγ-ENaC. Furthermore, Yamamura et al. demonstrated that

homomeric δ-ENaC is sensitive for protons (13) and Ji et al. showed the same for the δβγ

complex (34). Additionally, it is known that ENaC subunit expression as well as regulation is

tissue specific. It was shown that several cells in the lung express only the α- and γ-ENaC

subunits (35) while in colon and kidney ENaC expression is extremely dependent on

aldosterone stimulation (12, 36). Thus, a tissue specific subunit composition with modified

ENaC properties of the channel is likely. The modulated subunit formation of ENaC could

enable epithelia cells to respond to different extracellular parameters by an altered subunit

expression and stoichiometry. Thus, it could be possible that these combinations lead to

different biophysical and also pharmacological features of the channel. These are certainly

speculative presumptions and there is no evidence of this modulation in vivo yet. A variety of

studies showed that also culture conditions, cellular protein polarization, subunit composition,

and splice variants can be responsible for the diversity in channel properties (37-39).

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In conclusion, we found that the δ-ENaC mRNA and protein are expressed in HNE

and functional measurements support the presumption that this subunit plays a prominent part

in the whole Na+ transport in this particular tissue. The observance of α-, β-, γ- and δ-ENaC

co-expression in HNE provides an insight in variability of ENaC expression and distribution

that enlarges the possibilities of regulation and channel properties in respiratory epithelia. Our

data confirm and extend the existing knowledge with regard to ENaC distribution and

function. This offers new perceptions to the complex and tissue specific appearance of the

large variety of epithelial sodium channels.

Acknowledgement

We thank Ann-Kathrin Tilly and Ruth Kläver for the kind help in some experiments.

Furthermore, we thank Prof. Dr. Claudia Rudack (HNO-Klinik, University of Muenster) and

Dr. Hartmut Winzer (Maria-Josef-Hospital, Greven) for kindly providing us with human nasal

specimen. Parts of the work were supported by Mukoviszidose e.V. Giessen and Deutsche

Foerderungsgesellschaft zur Mukoviszidoseforschung e.V. Rhede.

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Figure legends

Figure 1: Semi-quantitative RT-PCR of δ-ENaC expression in HNE.

Data were acquired by semi-quantitative RT-PCR and are shown as mRNA expression (%) ±

SEM. Relative mRNA expression of non-transfected (NT) HNE cells was normalized to

100 %. The expression of the δ-subunit (47.2 ± 3.19 %) is reduced by about 52.8 % by

specific siRNA compared to the NT cell control (n = 7) (A). The non-silencing, scrambled

siRNA that we used as negative control (NC) caused no considerable changes in δ-ENaC

expression (98.14 ± 13.5 %) (B). Representative images of the semi-quantitative RT-PCR (C):

lane 1: 1kb ladder plus (Fermentas), lane 2: non-transfected cell control; lane 3: δ-ENaC

siRNA transfected cells; lane 4 non-transfected control; lane 5: negative control siRNA

transfected cells. The δ-ENaC specific primers produce a fragment of 474 bp. For

normalization we used human GAP-DH specific primers (239 bp fragment).

Figure 2: Western blot analyses of δ-ENaC in human nasal epithelium.

Total membrane proteins (30 µg) from primary HNE cells were isolated by using 2 % Triton

X-100 and separated on a 7.5 % SDS-PAGE. We detected a specific band of δ-ENaC in the

range of 100 kDa in non-transfected HNE cells (lane 1) and a decreased band in the siRNA

transfected sample (lane 2). As positive control we used MIA PaCa-2 cell lysate (lane 3)

(Santa Cruz). A representative Western blot is shown (n = 3).

Figure 3: Detection and quantification of α- and δ-ENaC at the apical surface of human

nasal epithelia cells. Single ENaC molecules were detected with the combination of a

specific polyclonal anti α-ENaC antibody (A) or anti δ-ENaC antibody (B) and a QD-labelled

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secondary antibody (representative images). The background staining of the cells is due to

fixation with glutaraldehyde, which allows the identification of cell borders.

The abundance of QD-labeled ENaC molecules/µm2 at the apical surface of HNE was

counted for each cell (N = 5, n = 30). In the bar diagram (C) mean values are given (0.164 ±

0.008 α-ENaC molecules/µm²; 0.146 ± 0.025 δ-ENaC molecules /µm²).

Figure 4: Inhibition of ENaC current by Evans Blue and amiloride.

Time course of a typical experiment (A). Shown is the short-circuit current (Isc) of cultured

monolayers from human nasal epithelia. Isc is markedly inhibited by amiloride, demonstrating

the Na+ absorption via ENaC. Then, we removed Na+ from the apical Ringer to determine

overall Na+ absorption of the cells. In a second step the effects of Evans Blue in the absence

and presence of amiloride are shown. Statistical evaluation of the short-circuit current (Isc)

(B). Shown is the inhibition of the amiloride-sensitive (39.1 ± 2.3 %) and the Evans Blue-

sensitive current (19.8 ± 1.9 %), as well as the blockage of both in primary cultured HNE

cells (40.1 ± 2.2 %) (n = 9) (amiloride = ami; Evans Blue = EB).

Figure 5: Inhibition of ENaC current by Evans Blue and amiloride after downregulation

with siRNA.

Time course of a typical experiment (A). Shown is the short-circuit current (Isc) of cultured

monolayers from HNE after siRNA transfection directed against the δ-ENaC subunit. Isc is

only slightly inhibited by amiloride, representing the remaining Na+ absorption via the α-

ENaC subunit. The removal of Na+ from the apical Ringer resulted in a strong decrease of the

current, demonstrating the overall Na+ absorption of the cells. In a second step the effects of

Evans Blue in the absence and presence of amiloride are shown. Evans Blue caused only a

weak change of the current, whereas the simultaneous application of Evans Blue and

amiloride decreased the current comparative to the exclusive addition of amiloride shown

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before. Statistical evaluation of the short-circuit current (Isc) in siRNA transfected HNE (B).

Shown is the inhibition of the amiloride-sensitive (20.8 ± 0.04 %) and the Evans Blue-

sensitive (0.7 ± 0.23 %) current, as well as the blockage of both in primary cultured nasal

epithelial cells after siRNA transfection directed against the δ-ENaC subunit (4.5 ± 1.67 %) (n

= 3) (amiloride = ami; Evans Blue = EB).

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Figure 1

47x87mm (600 x 600 DPI)

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Figure 2

44x38mm (600 x 600 DPI)

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53x78mm (600 x 600 DPI)

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Figure 4

63x75mm (600 x 600 DPI)

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Figure 5

63x75mm (600 x 600 DPI)

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Online Data Supplement

Characterization of the epithelial sodium channel delta subunit

in human nasal epithelium

Nadine Bangel-Ruland1*, Katja Sobczak1, Tina Christmann1, Dominik Kentrup2, Hanna

Langhorst1, Kristina Kusche-Vihrog3 & Wolf-Michael Weber1

Supplemental information

Figure S1: Detection of α-tubulin in permeabilized and non-permeabilized HNE

(representative images).

As a negative control, we incubated HNE cells only with the secondary antibody (QD655

goat F(ab´)2 anti rabbit IgG conjugates) (A). In non-permeabilized cells no α-tubulin

could be detected in the cytosol (B). In permeabilized HNE cells Cytosolic α-tubulin was

detected with the tubulin antibody and Cy3 as secondary antibody (C). This experiment

serves as a control for the staining of proteins at the cell surface.

Figure S2: Western blot analyses of α-ENaC in human nasal epithelium.

In order to demonstrate that the expression of α-ENaC is unaffected by transfection of the

δ-ENaC siRNA, we identified the α-subunit in the range of 65 kDa in non-transfected

(lane 1) and transfected (lane 2) HNE cells. A neutralizing peptide (PEP-088, Dianova)

was used to confirm α-ENaC antibody specificity (lane 3). A representative Western blot

is shown (n = 3).

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Figure S1

74x32mm (600 x 600 DPI)

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Figure S2

37x35mm (600 x 600 DPI)

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characterization of the epithelial sodium channel delta ... · enac and its isoform (δ2) can be...

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