GB2396414A - Modulators of human epithelial sodium channels(hENaC) - Google Patents

Abstract

An assay method for the isolation of compounds that alter the human ENaC epithelial ion channel activity comprising: (i) preparing an expression system comprising human a -, b -, and q -subunits; (ii) adding a test compound to the expression system and: (iii) determining if there is a change in hENaC ion channel activity resulting from the addition of the test compound. Also claimed is a gene cassette under the control of a promoter and a transfer vector both comprising the three subunits above

Description

fat T?- 1 ASSAY FIELD OF THE INVENTION

The present invention relates to a method and a kit for a salt enhancer assay.

Furthermore, the present invention relates to gene cassettes, transfer vectors, 5 cells and an expression system for use in the assay and kit.

BACKGROUND OF THE INVENTION

Salt (sodium chloride) is a ubiquitous food additive which has been used for centuries as a flavour enhancer and a preservative. However, it is now 10 recognised that excess salt intake is deleterious to health, for example it can cause or exacerbate hypertension.

Salt receptors in the mouth are responsive to the sodium ion, although they are also responsive to other highly water soluble cations such as lithium and 15 potassium. Clearly, if the receptors could be made more responsive to salt, its flavour enhancing effects could be achieved at lower levels of intake, potentially leading to health benefits.

Salt receptors are known to function by virtue of epithelial sodium channels 20 (ENaC's) in the neuronal cell membrane. ENaC's are multimeric integral membrane proteins formed by the association of highly homologous, a-,,8-, y-ENaC subunits. Sodium ions entering the cell by virtue of the concentration gradient across the membrane cause neutralization of the electrical charge difference between the inner and outer membrane surfaces. This sudden 25 membrane depolarization cause the neuron to signal the perception of saltiness. The sodium channels are in effect, pores which constantly open and close. If they could be caused to be open more often, the perception of salt flavour would be enhanced.

To screen many potential compounds as salt enhancers, a high throughput screening (HTS) method would be needed. Also regulatory constraints for new or unapproved molecules indicate the need for an in vitro assay. In theory, it would be possible to culture the requisite neuronal cells, wash them 5 with a solution of a candidate compound and detect its uptake and/or the cellular response. However, in practice, to expose cultured neuronal cells to agents which maintain the sodium channel in the open configuration effectively results in a "leaky" cell, leading to rapid cell death. This means that the cells have to be continuously cultured which makes such an assay 1 0 impractical.

Rat ENaC's have been expressed in insect cells as disclosed in Rao et a/., Anal. Biochem. (2000) 286:206-213, using a known Sf9 cell-baculovirus expression system. It was found that the conventional strategy of infection of 15 cells with a multiple recombinant baculovirus vectors using a triple promoter cassette derived from pAcBA3 (Belyaev and Roy, 1993, Nucl. Acids Res.) was inefficient when dealing with the multimeric rat ENaC protein. This reference discloses a procedure that utilises a baculovirus vector to coexpress all three cDNAs of rat ENaC in Sf9 cells in high amounts. It was 20 found that the expression was at a maximum when the transcription units were present tandemly in the baculovirus genome. The presence of two transcription units in opposite directions was found to prevent efficient transcription possibly due to unwinding of the DNA during transcription from both ends and probable supercoiling in the middle region. In this reference a 25 crude non-purified membrane fraction of the rat ENaC was isolated. The functional test applied to the membrane fraction, involved the reconstitution of single ion channels in a planar lipid bilayer membrane. This is a very sensitive technique as only one channel complex is needed. However, this technique is very unreliable with respect to artefacts.

We have found that human ENaC (hENaC) was more difficult to express than rat ENaC. To date, construction of a transformed mammalian cell line expressing hENaC encountered many difficulties, such as the toxicity problems mentioned above in relation to "leaky" cells. As described in 5 Lingueglia et a/., FEDS Lett. (1993) 318:95-99, a- rat ENaC produced about 100-150 nA channel current. Whereas, a-human ENaC produced about 50 100 nA channel current, as described in Voilley et al., PNAS (1994) 91:247 251 We have carried out experiments on transient expression of human ENaC in oocytes from Xenopus laevis and transient short-term expression in 10 different mammalian cell lines (MOCK-, HEK-, A549-, HeLa- and CHO-cells).

However, it turned out to be virtually impossible to obtain a mammalian cell line with stable and functional expression of the human ENaC multimeric protein in significant amounts over time periods of several months. Problems encountered included compromised vitality and cell growth in cells expressing 15 even low levels of human ENaC.

Thus, to date the generation of a stable cell line with functional expression of human ENaC has not been reported. However, we have now found that this problem can be overcome by using an expression system comprising the a-, 20,8-, y-subunits of human EnaC. This expression system may be used as the basis for a salt enhancer assay.

DEFINITION OF THE INVENTION

25 Thus, a first aspect of the present invention provides an assay method for the isolation of compounds that alter human ENaC ion channel activity comprising: (i) preparing an expression system comprising human ENaC a-,, 3-, y subunits; 30 (ii) adding a test compound to the expression system; and

(iii) determining if there is a change in hENaC ion channel activity resulting from the addition of the test compound.

According to a second aspect of the invention, there is provided a gene 5 cassette comprising a-, if-, y-subunits of human ENaC under the control of a promoter, preferably early or late viral promoters or inducible promoters.

According to a third aspect of the invention there is provided a vector comprising the o-,,8-, and y-subunits of human ENaC.

According to a fourth aspect, there is provided an expression system comprising cells, preferably insect cells, transfected with the vector as described above or infected with recombinant baculovirus vectors resulting from a previous transfection of the vector.

According to a fifth aspect, there is provided a salt enhancer kit comprising the expression system as described above.

According to a sixth aspect, there is provided a multimeric hENaC protein 20 obtainable from the expression system or cell as described above.

DETAILED DESCRIPTION OF THE INVENTION

In the detailed description of embodiments of the invention, reference is made

to the following figures.

Figure 1 (a), (b) and (c) are the sequence data for the o-,,3-,y -hENaC subunits used in this study, respectively.

Figure 2A is a construction of a quadruple transfer plasmid for the bacmid 10 system and Figure 2B Schematic illustration of the baculovirus expression vectors used in the experimental protocol.

Figure 3 shows the result of a western blot derived from SDSelectrophoresed proteins of HEK293 mammalian cells transfected with the o,'-,y-hENaC 15 subunit genes in comparison to the expression levels achieved in T. ni High Five insect cells infected with the recombinant baculoviruses carrying the same genes. Detection of hENaC subunit expression is probed with antibodies specifically detecting the three hENaC subunits and reveals several-fold higher expression levels in insect cells compared to expression 20 levels detected in mammalian cells.

Figure 4 shows experimental results obtained by measuring the the functionality of the hENaC in insect cell membranes by fluorometric determination of the membrane potentials using bis-(1,3-dibutylbarbituric 25 acid)trimethine oxonol (DiBac4). The cells carrying the hENaCexpression construct in addition to the marker gene display an increased depolarization if challenged with Na±containing buffer and compared to the depolarization induced in control cells, being either uninfected or infected with a vector carrying the marker gene (FP) only. The increased fluorescence in the cells 30 containing the Na±channel genes demonstrates increased depolarization.

As shown in Figure 4, the high level expression of hENaC in insect cells allows the application of a different assay methodology, including (i) Detection of Na±channel activity in membrane vesicles loaded with dyes, such as Na-

5 indicator dyes, and following the increase in intravesicular Na±levels.

Figure 5 shows the results of assay methodology (i) where the fluorescence ratio was determined for a membrane vesicle preparation derived from control cells are compared to signals from hENaC-expressing cells.

Only a small response in both vesicle suspensions can be seen after the addition of KCI-buffer (Ctl-0 and hENaC-0), only a moderate increase in the SBFI-signal is seen in the control vesicle suspension (Ctl-50) after increasing extracellular Na±levels to 50 mM, whereas a pronounced signal can be 15 observed in hENaC-containing membrane vesicles (hENaC-50) after increasing extracellular Na±levels to 50 mM.

The invention will now be described, by way of example, as a series of embodiments. The present invention relates in general to a salt enhancer assay method using an expression system comprising the a-,,8-, y-subunits of human ENaC.

The assay is used to identify compounds which alter the human ENaC ion channel activity, in order to determine if a compound may be used as a salt 25 enhancer.

A preferred embodiment of the present invention involves inserting a (sodium) salt sensor gene, more particularly human ENaC, into cells which normally live in a low sodium environment to cause expression of sodium channels in 30 their membranes and, for example, harvesting the membranes so that they

1 7 form vesicles and introducing a sodium marker (e.g. indicator dye) into the vesicles. This method can be used to form the basis of a salt enhancer assay.

The sequences of the human ENaC a-,,8-, y-subunits are shown in Figures 1 5 (a), (b) and (c). Human EnaC a-, '3-, y-subunits which have 80%, preferably 90%, more preferably 95%, even more preferably 99% homology with the sequences of Figure 1 (a), (b) and (c) may be used in accordance with the present invention.

10 The expression system used is preferably an insect cell - Baculovirus expression system. Suitable cells grow under low sodium conditions and can be infected easily with baculovirus vectors. The cells are preferably Tn-5B1-4 cells (Wickham et al. 1992, Biotechnol Progr 8:391-396), also known as H5 or High Five' cells (Invitrogen), but may also be Sf21 or Sf9 cells or any other 15 cells that can be infected by Autographa califomica multiple-capsid nucleopolyhedrovirus (AcM N PV).

More preferably, the cell is cotransfected with baculovirus. However, other insect cell expression systems such as transgenic lines using insect or 20 baculovirus immediate early promoters to drive the expression may also be used. Drosphila cells may also be used.

The expression system used provides transient expression of the ENaC ion channel proteins and, thus, the toxicity problem associated with mammalian 25 cells is overcome. In addition, the amount of protein present in the insect cells can be controlled by modulating the harvest time thereby effecting the duration of the infection before the assay is started. The ideal harvest time depends on the cell line used. Infected cells can be easily harvested and plated in assay format. The membrane fraction may also be isolated and used

1 8 for assaying salt enhancers. This assay method is highly suitable for use in HTS. In preferred embodiments, there is provided a gene cassette and/or transfer 5 vector comprising the o-, '3-, y-subunits of human ENaC each driven by a promoter, preferably an inducible promoter.

A specific embodiment exemplified herein shows a transfer vector including a quadruple transcription unit (see Fig. 2) based on baculovirus very late 10 promoters. In this construct pairs of promoters point outwards from the centre.

The centre two promoters have an opposite orientation (see pUR8135 in Figure 2). According to Rao et a/, such an arrangement of the expression vector would not work, since the centre two promoters were thought to interfere with each other.

The quadruple vector of Figure 5 has a less functional promoter (ph next to the p-subunit of pUR8135), which is used to drive marker gene expression, than used in Rao et al. This promoter has the same position as the p10 promoter used by Rao at al for their ENaC,B-subunit.

Alternatively, baculovirus early promoters or inducible promoters may be used. Additionally, expression vectors with all the transcription units in tandem might be used.

25 The multimeric human ENaC protein may be obtained from the expression system by harvesting the membrane fraction of the cell and forming membrane vesicles from the membrane fraction. Suitable techniques are well known and described in, for example, Aubel et al., Mol.Pharm. (1998) 53: 1 062-7.

-l. 9 Using the expression system, it is possible to determine if there is a change in ENaC ion channel activity and, hence, determine if a compound is a salt enhancer. Such techniques may include (a) measuring the change in membrane potential induced by human ENaC ion channel activity and/or (b) 5 measuring the activity of the human ENaC ion channel with sodium indicator dyes. The techniques used in the assay method measure the overall activity in the test sample and are, thus, much better controllable, reliable and suited for 10 HTS than electrophysiological approaches. Preferably, the techniques (a) and (b) involve: (a) Detection of channel activity with potentiometric dyes The cell membrane potential may be measured with potentiometric dyes. This 15 dye is used to monitor changes in the membrane potential due to activity of human EnaC. This can be achieved, for example, by increasing the extracellular /extravesicular sodium concentration, causing a depolarization and a measurable fluorescence signal of the indicator dye. Compounds to be tested can be added to test whether they result in faster or slower 20 depolarization in order to determine whether these compounds are salt enhancer. The membrane potential may be measured using a potentiometric dye, preferably oxonol dyes such as bis-(1,3- dibutylbarbituric acid)trimethine oxonol (DiBac4,) or alternatively with carbocyanine (Indo-, this- or oxa-

carbocyanins) or styryl dyes (Di-4-ANEPPS or Di-8-ANEPPS).

(b) Functional analysis with membrane vesicles The vesicles may be loaded with Na±ionselective indicator dyes for detection of changes in intravesicular Na±levels after addition of Na±ions to the extravehicular medium. The activity of the ENaC channel can be measured 30 (kinetic measurement). Thus, when test compounds are added it can be

assessed whether it takes a shorter or longer time than the control, and hence whether a compound will act as a salt enhancer. Suitable dyes include SBFI (1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13diazacyclopenta-

decane-7, 13- dlylbis(5-methoxy-6, 1 2-benzofurandlyl)]bis-, tetrakis [(acetyl 5 oxy) methyl] ester), Sodium Green, Corona-Red or other sodium indicator compounds. EXAMPLES

10 The present invention is now further illustrated by way of the following examples

hENaC-EXPRESSION EXPERIMENTS 1 5 cDNAs a-,,3-, & y-hENaC were supplied by M. Lazdunski, CNRS Valbonne.

Sequence information for the a-,,3-, y-subunits as used in this study are provided in Figures 1 (a), (b) and (c).

20 Outline of the Bacmid-Method Recombinant baculovirus vectors were constructed via the bacmid-method described by Luckow et al., (1993) J Virol 67:4566-79. The bacmid strategy allows the generation of recombinant baculoviruses in Escherichia cold and also allows the testing of multiple constructs in a short period of time. The 25 AcMNPV bacmid is a bacterial plasmid carrying the AcMNPV genome with T7 transposon acceptor sites at the polyhedrin locus. The transfer plasmids used to insert foreign genes into the baculovirus genome carry T7 transposon donor sites, flanking the cassette with the genes of interest. DH10Bac cells also contain a helper plasmid supplying the T7 transposase.

1 1 1 Construction of baculovirus transfer vectors carrying hENaC cDNAs As shown in Figure 2A, the transfer plasmid used in this example, 5 pFastBacTetra (pUR8132), was derived from pFastBac1 (Invitrogen) and pAcAB4 (Belyaev and Roy, (1993) Nucleic Acids Res 21, 1219-1223). The full sequence of pAcAB4 can be found on http://www.bdbiosciences.com/cgi-

bin/sequences?vect=pAcAB4. A 4525 bp Bst1 1071-Kpol fragment of pFastBac1 was combined with a 1098 bp EcoRV-Kpnl PCR product carrying 10 two dual promoter cassettes consisting of an AcMNPV p10 and a polyhedrin promoter. The dual promoter cassettes point outwards and are flanked by a polyhedrin and an SV40 terminator, respectively. This PCR product was generated using pAcAB4 as template and 5'CGCGCGCCGGATATCCGTCGAGTCMTTGTACACTMCG3' and 15 5'GCGCGCGGGTACCCAGCTGGMT3' as primers, generating EcoRV and Kpnl sites, respectively.

The polyhedrin terminator corresponds to position 5681 to 5271 of the AcMNPV C6 strain genome sequence (GeneBank L22858). The SV40 20 terminator was originally derived from Simian Virus 40. A control vector (pUR8133) was derived from the pFastBacTetra-plasmid (pUR8132) by inserting a marker protein, such as fluorescent protein (FP), under control of the ph-promoter on the right end of the gene cassette (Fig. 2B) .

25 The final construct (pUR 8135) carries the o-,,8-, y-subunits hENaCsubunits Construction of recombinant bacmids DH10Bac cells carrying the Autographa califomica (Ac) MNPV bacmid were made heat-shock competent and transformed with 50 ng plasmid DNA of the 30 various transfer vectors (pUR8122, pUR8124, pUR8128, pUR8130,

1 12 pUR8133, pUR8135 and pUR8131). Transformed bacterial cells were incubated at 37 C in SOC-medium by shaking at 220 rpm for 4 h and plated onto LB-agar plates containing 7,ug/ml gentamycine, 50,ug/ml kanamycin and 10,ug/ml tetracylcin, 40,ug/ml IPTG and 100,ug/ml Bluo-gal (Invitrogen).

5 White colonies were streaked on fresh plates and incubated overnight. Liquid cultures were prepared in LB containing gentamycine and kanamycin, and bacmid DNA was isolated from 1.5 ml cultures as described in the Invitrogen BAC-TO-BACO Baculovirus Expression Systems Instruction manual (CAT.

NO. 10359-016 and 10608-016). The transposition event was checked by 10 PCR on purified bacmid DNA using an M13 forward primer (5'GCCAGGGTTTTCCCAGTCACGA3') (Invitrogen) annealing to the pFastBacTETRA vector sequence and a gentamycin resistance gene-specific primer (5'AGCCACCTACTCCCMCATC 3'). In this way PCR products were generated only if transposition had occurred. A control with the M13 forward 15 and the M13 reverse primer was performed to check for the absence of wild type bacmid. This resulted in bacmids pUR8133b (FP-control), pUR8135b (aphENaC), and pUR8131b (ac,0-hENaC only) (fig. 2).

Transfection of insect cells and the neneration of recombinant baculovirus 20 stocks Sf21 insect cells (Vaughn et a/., (1978) In Vitro 13, 213-217) were seeded at a density of 1.5 x 106 per 35 mm culture dish in 2 ml of Grace's supplemented insect medium (Grace's Insect Medium, Supplemented (1X), liquid with L glutarnine, lactalbumin hydrolysate, yeastolate) 25 httP://www. Iifetech.com/content. cfm?paneid=3387&cfid=553759&cftoken=63118675 with 10% Fetal Bovine Serum (FBS) (Invitrogen). After two hours the medium was replaced with Grace's minimal insect medium without any supplements and cells were incubated overnight at 27 C.

Cells were transfected with 10 al of the isolated bacmid DNA for the constructs mentioned above by mixing DNA with 5,ul of sterile water and 10,ul CellfectinO reagent (Invitrogen CAT NO. 10367-010), and incubated for 15 min at room temperature. The medium was replaced with 0.3 ml Grace's 5 insect medium without FBS. The Cellfectin/DNA mixture was supplemented with 0.3 ml Grace's insect medium without FBS and transferred drop by drop to the culture dish. After 1 hour incubation at 27 C another 0.5 ml of Grace's insect medium was added. After 3 hour incubation at 27 C, 1 ml of Grace's supplemented insect medium with 20% FBS was added and the cells were 10 further incubated for 4 days. Infection was detected by cytopathic effects and detection of the marker protein FP.

Four days after transfection the supernatant of the transfected cells was harvested. Cell debris was removed by centrifugation at 1600 rpm for 10 min 15 and followed by filtration through a 0.45,um non-pyrogenic filter (Millipore product # SLHA 02505). To grow high titre virus stocks, two million Sf21 cells were seeded in 25 cm2 flasks in Grace's insect medium with 10% FBS and inoculated with 300 Saul of the transfection supernatant for each recombinant virus, respectively. The culture medium was harvested 3 days after infection, 20 cleared from cell debris as mentioned above and 100,ul was used to infect 10 million Sf21 cells seeded in 75 cm2 flasks. The culture medium containing budded viruses was harvested 3-4 days after infection. The titer of the virus stocks was determined by end point dilution assays (King and Possee, 1992) to determine the tissue culture infective dose using the marker protein as a 25 marker for infection.

Baculovirus infection of Insect cells H5-cells are grown in Grace's insect cell medium containing 10 % fetal bovine serum (FBS), counted and infected with 5-fold excess of infectious particles 30 (recombinant virus particles) for 2 hours in a overhead shaker and

al 14 subsequently washed with fresh medium before cultivation for additional 40 hours. Expression of hENaC subunit proteins in Mammalian Cells vs insect cells 5 24 and 48 h after infection the cells were washed from the flasks and harvested by centrifugation for 2 minutes at 1500 g. The cells were then suspended in 375 Ill PBS (Phosphate-buffered saline, ex Invitrogen) + E64 (protease inhibitor, 30,ug/ml) and ultrasonified for 30 sec.

10 125 pi 4 x sample buffer (8%. SDS, 40% glycerol, 0.01% Bromophenol blue, 400 mM Dithiothreitol, 200 mM TEIS pH B.8) was added and 20 Ill of this mixture was loaded on a 10% SDS-PAGE gel.

After blotting and blocking with PBS containing 5% milk powder (Marvel), the 15 blots were treated with the first antibody in 10 ml PBS with 0.1% marvel. The primary rabbit antibodies Anti-a (Troll2, from M. Lazdunski, CNRS-lnstitute Valbonne, France) was used in a dilution of 1: 2000, Anti-, 3 (Sophie 2, from M. Lazdunski, CNRS-lnstitute Valbonne, France) was used in a 1: 500 dilution, whereas the V5-tagged y-hENaC subunit was detected with a mouse anti V5 20 antibody (Invitrogen) at a 1: 2000-dilution.

As second antibody for the and,3 anti-rabbit-lgG antiserum coupled to Alkaline Peroxidase (AP, Promega) was used in a 1: 5000-dilution, whereas the -specific labelling was obtained with the anti-mouse IgG-antiserum 25 coupled to AP (Promega) in a 1: 5000-dilution.

Results from Western Blots derived from membrane preparation of HEK293-

cells (mammalian cells) vs. H5-cells (insect cells), probed with the a-,, 8- or y-

specific primary antibodies are shown in Figure 3.

Al 15 It can be seen that hENaC expression is detectable in mammalian cells, although being very low (at least 50-times less) if compared to expression as detected in homogenates from insect cells expressing the hENaC genes.

5 Assav with insect cells 24 or 48 hours after infection the cells were harvested by centrifugation for 2 minutes at 1500 g, suspended in Graces's supplemented insect medium with 10% FBS and seeded into polylysine-coated microtiter plates 42 h after infection the the plate was centrifuged for 2 minutes at 1500 g, the medium 10 was removed by pipetting and 200 al KCI buffer (100 mM KCI, 6.75 mM CaCI2, 11.21 mM MgCI2, 11.28 mM MgSO4, 4.17 mM NaHCO3, 178 mM sucrose, 7.34 mM maleic acid pH 6.1) was added, centrifuged for 2 minutes at 1500 g. The cells were washed two times.

15 The cells were loaded with DiBAC4 (5 I1M, Molecular Probes), 50 pI/well (5 pl 10 mM stock in DMSO in 10 ml KCI buffer) 100 p1 NaCI buffer (100 mM NaCI, 6.75 mM CaCI2, 11.21 mM MgCI2, 11.28 mM MgSO4, 4.17 mM NaHCO3, 178 mM sucrose, 7.34 mM maleic acid pH 20 6.1) or KCI buffer containing 5 M DiBAC4 was added and the fluorescence was measured at 538 nm with excitation at 485 nm wavelength on a microtiterplate reader.

As seen in Fig.4, it was found that only the hENaC-containing cells (infected 25 with the pUR8135-derived virus) display an increase in fluorescence signal (i.e. depolarisation) after addition of the Na+buffer, indicative of increased Na-

conductance in the hENaC-expressing cells, whereas the K±buffer leads in hENaC-expressing cells and cells infected with the control vector (pUR8133-

derived virus) to the expected reduction in fluorescence signals.

_--, 16

Assay with membrane vesicles derived from insect cell membranes The preparation of membrane vesicles from His-cells was carried out as described in Aubel et al., (1998) Mol.Pharm. 53, 1062-7. The infected cells 5 were washed from plates obtained above and the cells were harvested by centrifugation for 1 minute at 1500 9. The cells were then suspended in 10 ml hypotonic buffer (5 mM HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2 ethanesulphonic acid)) pH 7.0, 1 mM EDTA (Ethylenediaminetetraacetic acid), 3,ug/,ul Protease-lnhibitor E-64 (Ex Roche Diagnostics)) and stirred 10 gently for approximately 1.5 hours on ice. After centrifugation for 40 minutes at 100.000 g the pellet obtained was diluted with 3ml of isotonic buffer (250 mM Sucrose, 10 mM TRIS (Tris(hydroxymethyl) methylamine), 10 mM HEPES pH 7.4, 3,ug/,ul Proteaselnhibitor E-64) and homogenized with a homogenisator. After centrifugation for 10 minutes at 12.000 g the 15 supernatant was kept and the pellet resuspended in 1ml Isotonic buffer, and recentrifuged for 5 minutes. The supernatant was put onto a 38 % Sucrose solution cushion (38 % Sucrose, 5 mM HEPES-KOH pH 7.4, 3,ug/pl Protease-lnhibitor E-64) and centrifuged for 2 hours at 100.000 9. The interphase was collected and rehomogenized before centrifugation for 40 20 minutes at 100.000 9. The pellet obtained was diluted in 500 ul Transport buffer (100 mM KCI, 6.75 mM CaCI2, 11 mM MgCI2, 11 mM MgSO4, 4.2 mM NaHCO3, 178 mM Sucrose, 7.4 mM Maleic acid, 2 mM EGTA (Ethylene glycol-bis(-aminoethyl ether)-N,N,N'N'- tetraacetic acid), 10 mM HEPES pH 7.4, 100,uM SBFI, 3 p9/pl Protease- lnhibitor E-64), passed 30 times through 25 27-gauge needle and rapidly frozen in liquid nitrogen.

For the determination of the Na±Flux into the vesicles 50 pl of the membrane vesicle suspension was mixed with 501 of either KCI-buffer (100 mM KCI, 6.75 mM CaCI2, 11 mM MgCI2, 11 mM MgSO4, 4.2 mM NaHCO3, 178 mM 30 Sucrose, 7.4 mM Maleic acid, 2 mM EGTA, 10 mM HEPES pH 7.4) or NaCI

buffer (100 mM NaCI, 6.75 mM CaCI2, 11 mM MgCI2, 11 mM MgSO4, 4.2 mM NaHCO3, 178 mM Sucrose, 7.4 mM Maleic acid, 2 mM EGTA, 10 mM HEPES pH 7.4) , while the changes of the SBFI-Fluorescence were measured by a Fluorescence Reader at 340 and 380 nm Excitation wavelength. The SBFI 5 outside the vesicles reacts instantaneously with extracellular sodium, whereas the rise in intravesicular Na-levels can be followed from 200 ms after the mixing event up to 20 seconds by determination of SBFIFluorescence at 340 nm and 380 nm Excitation wavelength and calculation of the ratio between these two values.

The result of such an experiment is shown in Fig. 5, where the fluorescence ratio was determined for a membrane vesicle preparation derived from control cells are compared to signals from hENaC-expressing cells. Only a small response in both vesicle suspensions can be seen after the addition of KCI 15 buffer, only a moderate increase in the SBFIsignal is seen in the control vesicle suspension after increasing extracellular Na±levels to 50 mM, whereas a pronounced signal can be observed in hENaC-containing membrane vesicles after increasing extracellular Na±levels to 50 mM.

Claims (22)

1. An assay method for the isolation of compounds that alter human ENaC ion channel activity comprising: 5 (i) preparing an expression system comprising human ENaC a-,,B-, y-subunits; (ii) adding a test compound to the expression system; and (iii) determining if there is a change in hENaC ion channel activity resulting from the addition of the test compound.

2. An assay method according to claim 1 wherein the human ENaC a-,,3-, y subunits have more than 80% homology with the sequences of fig 1 (a), (b) and (c).

15

3. An assay method as claimed in claim 1 or 2 wherein the expression system is an insect cell - baculovirus expression system.

4. An assay method as claimed in claim 3 wherein the insect cell is Sf9 or T. ni High Five.

5. An assay method as claimed in any of claims 1 to 4 wherein step (iii) comprises (a) measuring the membrane potential of the human ENaC ion

channel and/or (b) measuring the activity of the human ENaC ion channel using a sodium indicator dye.

6. As assay method as claimed in claim 5 wherein the sodium concentrion 5 changes are followed by indicator dyes selected from SBFI (1,3 Benzenedicarboxylic acid, 4,4'-[1,4,1 0-trioxa-7,1 3diazacyclopentadecane 7, 13- diylbis(5-methoxy-6,12-benzofurandlyl)]bis-, tetrakis [(acetyloxy) methyl] ester), Sodium Green or Corona-Red.

10

7. An assay method as claimed in claim 5 wherein the membrane potential is measured using a potentiometric dye, preferably using oxonolols, such as bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBac4), or carbocyanine, including Indo-, thia- or oxa-carbocyanins or styryl dyes, such as Di-4 ANEPPS or Di-8-ANEPPS.

8. A compound identified by the assay according to any of the preceding claims.

9. A compound according to claim 8, which is a salt enhancer compound.

10. A gene cassette comprising a-,,6-, y-subunits of human ENaC under the control of a promoter, preferably an inducible promoter or an early or late viral promoter.

_v 1 20

11. A gene cassette according to claim 10 wherein the human ENaC a-,,3-, y-

subunits have more than 80% homology with the sequences of fig 1 (a), (b) and (c), preferably 90% homology, more preferbably 95% homology.

12. A gene cassette as claimed in claim 10 or 11 wherein two of the human ENaC subunits are arranged in opposite orientation.

13. A transfer vector comprising the a-,,3-, y-subunits of human ENaC.

14. A transfer vector comprising the gene cassette of any of claims 10 to 12.

15. A transfer vector as claimed in claim 13 or 14 wherein the vector is a baculovirus transfer vector.

16. A cell transformed with the transfer vector of any of claims 13 to 15.

17. A cell as claimed in claim 16 wherein the cell is an insect cell, preferably H5 or Sf9.

18. An insect cell according to claim 17 wherein the cell is cotransfected with baculovirus.

l

19. An expression system comprising the cell as claimed in any of claims 16 to 18.

20. A kit for testing compounds that alter human ENaC ion channel activity 5 comprising the expression system of claim 19.

21. A multimeric hENaC protein obtainable from the cell of any of claims 16 to 18.

10

22. A process for obtaining the multimeric protein from the cell of any of claims 16 to 18 or from the expression system of claim 18 comprising harvesting the membrane fraction of the cell and forming membrane vesicles from the membrane fraction.