From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
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ABSTRACT |
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The Helix aspersa Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channel is formed by homomultimerization of several FMRFamide-activated Na+ channel (FaNaCh) proteins. FaNaCh is homologous to the subunits that compose the amiloride-sensitive epithelial sodium channel, to Caenorhabditis elegans degenerins, and to acid-sensing ionic channels. FaNaCh properties were analyzed in stably transfected human embryonic kidney cells (HEK-293). The channel was functional with an EC50 for FMRFamide of 1 µM and an IC50 (25 °C) for amiloride of 6.5 µM as assessed by 22Na+ uptake measurements. The channel activity was associated with the presence of a protein at the cell surface with an apparent molecular mass of 82 kDa. The 82-kDa form was derived from an incompletely glycosylated form of 74 kDa found in the endoplasmic reticulum. Formation of covalent bonds between subunits of the same complex were observed either after formation of intersubunit disulfide bonds following cell homogenization and solubilization with Triton X-100 or after use of bifunctional cross-linkers. This resulted in the formation of covalent multimers that contained up to four subunits. Hydrodynamic properties of the solubilized FaNaCh complex also indicated a maximal stoichiometry of four subunits per complex. It is likely that epithelial Na+ channels, acid-sensing ionic channels, degenerins, and the other proteins belonging to the same ion channel superfamily also associate within tetrameric complexes.
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INTRODUCTION |
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The protein FaNaCh1 is expressed in the Helix aspersa nervous system (1). It is the first identified member of a new class of ionotropic receptors that can be directly activated by peptides. FaNaCh expression in Xenopus oocytes generates an amiloride-sensitive Na+ conductance activated by the molluscan cardioexcitatory peptide FMRFamide (1). FaNaCh is related to the three homologous proteins that constitute the pore-forming subunits of the epithelial Na+ channel (ENaC) (2-5), the membrane proteins DEG-1, MEC-4, MEC-10, UNC-105, UNC-8, and DEL-1 from Caenorhabditis elegans, involved mainly in mechanosensation (6-10), and the mammalian acid-sensing ionic channels ASIC (11) (also called BNC2) (12), mammalian degenerin homologue (13) (also called BNC1) (14), and dorsal root ganglia ASIC (15). This new gene superfamily encodes ionic channels expressed in epithelial (i.e. ENaC) as well as excitable tissues (i.e. FaNaCh, ASIC, or degenerins) (16).
The proteins belonging to this family are characterized by a large extracellular domain located between two large hydrophobic zones forming the transmembrane regions. The NH2- and COOH-terminal domains are cytoplasmic (17). Despite increasing information on the structure and function of these channels, the stoichiometry of the functional complexes remains unknown.
Biochemical handling of FaNaCh is facilitated by several properties:
(i) unlike ENaC which comprises ,
, and
subunits, active
FMRFamide Na+ channels are formed by a homomultimerization
of the FaNaCh channels; (ii) unlike ENaC, the FaNaCh channel is totally
silent in the absence of FMRFamide, and therefore not toxic for cells
that express it. The determination of the oligomeric arrangement of the
FaNaCh channel might serve as a model for other channels within this family (ENaC, degenerins, and ASIC). After extensive functional and
biochemical characterization of the channel complex, we show that four
FaNaCh subunits participate in the formation of the FMRFamide-activated
sodium channel.
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EXPERIMENTAL PROCEDURES |
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Constructs and Transfection--
FaNaCh cDNA structure has
been previously described by Lingueglia et al. (1). The
cDNA was inserted either into the plasmid pBSSK-SP6-globin (18) for efficient expression in
Xenopus oocytes or in pRC/CMV plasmid (Invitrogen) for
transfection of eucaryotic cells. A tagged FaNaCh was constructed by
polymerase chain reaction, leading to the addition of the FLAG sequence
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the COOH terminus of the protein.
This epitope, which does not alter the channel activity (not shown),
can be detected by a commercial monoclonal anti-FLAG antibody (Eastman
Kodak Co.) with a high specificity and a low background. Stable
transfection was carried out on HEK-293 cells with the FaNaCh or
FaNaCh-FLAG pRC/CMV plasmid (Invitrogen) by the
Ca2+-phosphate technique (19).
22Na+ Uptake Experiments-- HEK-293 cells were seeded in collagen-coated 24-well clusters (Falcon). Confluent cells were rinsed twice with the flux buffer (140 mM choline chloride or N-methyl-D-glucamine chloride, 20 mM HEPES/Tris, pH 7.4, 1.8 mM CaCl2, 0.8 mM MgSO4) and then incubated with 200 µl of flux buffer supplemented with 2 mM 22NaCl (0.5-1 µCi/ml Amersham Pharmacia Biotech, 12 Ci/mmol) with or without amiloride or FMRFamide. External 22Na+ was washed out by four 1-ml washings with cold flux buffer, and trapped radioactivity was measured using a Packard 1600 CA liquid scintillation counter. Dose-dependent activations and/or inhibitions were measured after a 150-s incubation.
Antibodies-- The cytoplasmic COOH-terminal domain of FaNaCh (amino acids 570-625) was produced in the glutathione sulfhydryl transferase fusion protein expression vector pGEX-3X according to the manufacturer's instructions (Amersham Pharmacia Biotech). The peptide was injected intradermally into a rabbit as described previously (17). Antibodies were first characterized by enzyme-linked immunosorbent assay on pure antigen, then used for immunoprecipitation experiments. Some experiments were carried out using the FaNaCh-FLAG construct. The FLAG was detected by the commercial monoclonal M2 anti-FLAG antibody (Kodak).
Immunoblot Experiments--
Cells were rinsed with Hank's
balanced salt solution, scraped, and recovered in a lysis buffer
containing 50 mM Tris/H2SO4, pH
7.4, 100 mM K2SO4, 5 mM
EDTA, and protease inhibitors (100 µM leupeptin, 1 mM aprotinin, 1 µM pepstatin, 1 mM phenylmethylsulfonyl fluoride). After lysis in a Potter
and slow centrifugation, the supernatant was centrifuged 1 h at
30,000 × g. Pellets were resuspended at about 2 mg/ml
in lysis buffer and stored at 70 °C. 10-20 µg of membrane
proteins were heated at 65 °C in SDS-PAGE sample buffer (2.5% SDS,
2 M urea, bromphenol blue, with or without 4%
-mercaptoethanol) and applied to SDS-PAGE. After migration, proteins
were transferred to a nitrocellulose membrane (Hybond C extra, Amersham
Pharmacia Biotech). Nonspecific binding was blocked by a 1-2-h
incubation in 140 mM NaCl, 20 mM Tris, 5%
milk. The filter was incubated overnight in the same buffer with the
monoclonal anti-FLAG antibody (dilution 1/1000, Kodak), rinsed 3 times
for 10 min with 140 mM NaCl, 20 mM Tris, 0.05%
Tween 20, incubated 45 min with a goat anti-rabbit antibody coupled to
peroxidase (Jackson Immunotech), rinsed, and revealed by
chemiluminescence (SuperSignal, Pierce).
Metabolic Labeling, Cell Surface Iodination, and Immunoprecipitation-- Confluent cells were incubated in a methionine- and cysteine-deficient medium for 30 min prior to the addition of 50 µCi/ml [35S]methionine (Trans35S-LABEL, ICN, 1077 Ci/mmol). After a pulse of 2-3 h (or 30 min for pulse-chase experiments), the cells were rinsed and incubated in complete medium (plus 10% fetal calf serum) for various lengths of time. Cells were subsequently pelleted and lysed with 50 µl of chilled radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% C12E8 (Nikko Chemicals), protease inhibitors)/35-mm dish. After preclearing with pansorbin (Calbiochem), the lysate was incubated overnight with the polyclonal antibody (1/100), and pelleted using protein A-Sepharose (Sigma). After washing, samples were heated in SDS-PAGE sample buffer, and applied to 6.5 or 8% SDS-PAGE. Gels were treated with 1 M salicylic acid for 30 min, dried, and exposed to a phosphorimager imaging plate.
Cell surface labeling was performed according to Dalemans et al. (20). Confluent cells were rinsed with PBS+ (phosphate-buffered saline + 1 mM CaCl2 + 1 mM MgCl2). 250 µl PBS+, 10 units of lactoperoxidase (Sigma), and 500 µCi of 125I (NEN Life Science Products, 2200 Ci/mmol) were added to each 35-mm dish at 4 °C. Reactions were initiated by adding 10 µl of H2O2 0.01%; this addition was repeated twice (every 30 s). Cells were rinsed several times in PBS+, and resuspended in the lysis buffer containing 1% Triton X-100, 0.5% deoxycholate. Proteins were immunoprecipitated as described above and analyzed by SDS-PAGE. Gels were dried and exposed to phosphorimager imaging plate.Cross-linking Experiments-- Cells were solubilized after membrane preparation, metabolic labeling, or cell surface iodination in the cross-linking assay buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM EDTA). Samples were incubated for 15 min at room temperature with DSS (suberic acid bis(N-hydroxysuccinimide ester, Sigma) at pH 9.4 with SADP (N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate, Sigma) or with NHS-ASA (N-hydroxysuccinimidyl-4-azidosalicylic acid, Pierce) at pH 7.4. Reactions were stopped with 50 mM Tris/HCl, pH 7.4. SADP- or NHS-ASA-treated samples were then exposed for 10 min to an UV source (252 nm). Following cross-linking, samples were applied to a 5% Laemmli or 3.5% Weber-Osborn polyacrylamide gel (21).
Sucrose Gradient Centrifugation and Gel Filtration
Analysis--
Membrane proteins were solubilized and centrifuged at
170,000 × g for 15 min. In some experiments, the
solubilized material was also cross-linked using 500 µM
DSS, or the formation of intersubunit disulfide bonds was prevented by
the addition of 10 mM iodoacetamide. The solubilized
material was recovered and applied to the top of a linear 5-15%
sucrose gradient formed in water or in D2O (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 0.1% Triton X-100 or CHAPS, protease inhibitors).
-galactosidase (100 µg), catalase (100 µg), alkaline phosphatase
(67 units), and cytochrome c (100 µg), used as markers,
were also applied on top of the same gradient. After a 17 h run at
39,000 rpm (190,000 × g) on a SW41-TI rotor (Beckman),
300-µl fractions were recovered. They were assayed for the presence
of the standards and either immunoprecipitated overnight with the
anti-FaNaCh antibodies (for metabolic and cell surface labelings) or
assayed by Dot-blot with the anti-FLAG antibody. Gel filtration was
performed on a Superdex 200 column (Amersham Pharmacia Biotech)
equilibrated in a buffer containing 150 mM NaCl, 10 mM EDTA, 0.1% Triton X-100, at a flow rate of 0.4 ml/min.
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(Eq. 1) |
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RESULTS |
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Functional Expression of FMRFamide-activated Sodium Channels in FaNaCh-transfected HEK-293 Cells-- FMRFamide-activated sodium channel activity was measured after stable transfection of the FaNaCh cDNA into HEK-293 cells. Fig. 1 shows that the properties of the channel observed in FaNaCh-transfected HEK-293 cells were identical to those of the native channel recorded in snail neurons (25) and to those of the channel recorded in Xenopus oocytes injected with the corresponding cRNA (1). 22Na+ uptake was largely increased after stimulation with 30 µM FMRFamide in transfected cells (Fig. 1A). The time course of the uptake was biphasic. After a relatively fast rise to a plateau, the 22Na+ uptake declined to a lower intermediate value. The initial event is consistent with the rapid development of a Na+ permeability. The decline of the signal observed after 2.5 min is likely due to the inactivation of the channel (25) combined with a sustained Na+/K+-ATPase activity. A Michaelis-Menten relationship was observed between the external concentration of sodium and the initial ratio of 22Na+ uptake (Fig. 1B) with a half-maximal activation of 1.4 mM. A similar value (K0.5 = 5 mM) has been reported for the external sodium dependence of the ENaC activity after expression in Xenopus oocytes (4).
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FaNaCh Is Expressed at the Cell Surface as a 82-kDa Glycoprotein-- The protein expressed in transfected cells was first characterized using a polyclonal antibody directed against the COOH-terminal domain. In pulse-chase experiments, the polyclonal antibody immunoprecipitated a 74-kDa protein, which matured after 2-3 h into a 82-kDa protein (Fig. 2A). After deglycosylation of the immunoprecipitated material with N-glycosidase F (data not shown), or pretreatment of the cells with tunicamycin (a blocker of N-glycosylation), a 67-kDa protein was detected in good agreement with a predicted molecular mass equal to 71 kDa for the polypeptide (Fig. 2B).
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Solubilization by Triton X-100 Favors the Formation of Intersubunit
Disulfide Bonds and Leads to the Formation of Covalent
Tetramers--
When SDS-PAGE migration was performed under nonreducing
conditions, oligomeric forms of FaNaCh were observed (Fig.
3A). These forms were
sensitive to -mercaptoethanol, suggesting the existence of disulfide
bonds between subunits. When membranes were prepared in the presence of
10 mM iodoacetamide, the monomer was the only form detected
(Fig. 3A). This result shows that formation of intersubunit disulfide bonds is not occurring in the native channel, but rather that
it results from a redox potential alteration created by the experimental conditions during cell lysis or the following steps.
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Reticulation of the Complex by Bifunctional Cross-linkers Generates Up to Tetramers-- After a lactoperoxidase labeling of the cell surface proteins with 125I and covalent reticulation with DSS, the material was immunoprecipitated and analyzed by SDS-PAGE. Cross-linked proteins were detected on a 3.5% Weber-Osborn gel (nonreducing conditions) at apparent molecular masses of 180 (dimer), 260 (trimer), and 345 kDa (tetramer) (Fig. 3C). Most of the cross-linked material appeared as trimers, but the presence of a tetramer was detected in three out of eleven experiments as a diffuse band around 345 kDa.
When membranes were solubilized with Triton X-100 then cross-linked with the heterobifunctional reagent SADP (Fig. 3B), the tetramer was the main form detected by Western blot. Fig. 3B also shows an increase of the tetrameric form when SADP concentration was increased from 100 to 500 µM. The formation of covalent tetramers was not restricted to Triton X-100-solubilized material. They were also detected on CHAPS-solubilized material after a reticulation with the heterobifunctional cross-linker NHS-ASA (Fig. 3D).Hydrodynamic Analysis of Solubilized FaNaCh Demonstrates the Tetrameric Organization of the Complex-- The pool of FaNaCh proteins expressed at the cell surface was analyzed by sedimentation experiments through sucrose gradients after a solubilization with 1% Triton X-100 + 0.5% deoxycholate. A unique peak characterized by an apparent sedimentation coefficient of 9.5 S was observed. This peak was not modified after cross-linking of the membrane proteins with DSS (not shown). This proved that a unique FaNaCh complex (formed by a given number of subunits) was expressed at the cell surface and was resistant to solubilization by 1% Triton X-100 + 0.5% deoxycholate.
Hydrodynamic characterization of the complex solubilized in 1% Triton X-100 was then performed. The apparent sedimentation coefficients in a linear 5-15% sucrose gradient formed in water and in D2O were determined (Figs. 4, A and B). The sedimentation coefficient at 20 °C in water (s20,w) of the Triton X-100-protein complex was equal to 9.12 ± 0.02 S, and the partial specific volume
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DISCUSSION |
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The properties of the FMRFamide-gated sodium channel expressed in HEK-293 cells are quite consistent with the properties of the channel recorded in native tissues or after expression of the cloned cDNA into Xenopus oocytes. Since FaNaCh expression in HEK-293 cells and in Xenopus oocytes is able to generate a large FMRFamide-induced inward current, it is likely that the functional channel corresponds to a homomultimeric structure, and does not need association with endogenous subunits. The transfected cells therefore represent a useful model to study the pharmacological and biophysical properties of the FMRFamide-gated sodium channel. These results also demonstrate that correct assembly of the channel complex is possible in these transfected cells, which can be used to characterize the quaternary structure of the channel.
The main purpose of this study was to analyze the maturation pattern and the quaternary structure of the FMRFamide-gated sodium channel. In transfected HEK-293 cells, FaNaCh glycosylation occurs in two steps. An immature precursor core-glycosylated form is produced into the endoplasmic reticulum and is then converted into a fully glycosylated form abundantly expressed at the cell surface, which generates the expected channel activity. Figs. 3 and 5 show that the immature form is already assembled into an oligomeric complex that sediments at the level of a unique peak on the sucrose density gradient. This proves that tetrameric assembly occurs at an early step of maturation in the endoplasmic reticulum. A similar type of assembly has also been reported for the tetrameric potassium channel Shaker (29).
Covalent tetramers were observed after use of three distinct chemical cross-linkers, i.e. DSS, SADP, and NHS-ASA. They were also detected after oxidation of the protein through the formation of interchain disulfide bonds. Triton X-100 increased the latter covalent bonding, but tetramers were also observed after solubilization in the presence of other detergents such as CHAPS, followed by chemical cross-linking (Fig. 3). It is therefore unlikely that tetramers only represent artifactual denaturation due to Triton X-100. Thus, cross-linking experiments provided a minimal stoichiometry of the complex equal to 4, and hydrodynamic properties of the solubilized FaNaCh complex provided a maximal stoichiometry strictly below 5. Taken together, these results led us to the conclusion that the FaNaCh complex is composed of four subunits. Since the FMRFamide-gated sodium channel is a member of the same structural family as ENaC, degenerins, and ASIC, a stoichiometry of 4 can also be expected for these channels.
McNicholas and Canessa (30) analyzed the properties of epithelial
Na+ channels generated after coexpression of ENaC and
ENaC, or
ENaC and
ENaC. Using different ratios of injected
cRNAs, they showed an optimal signal after injection of equal amounts
of
ENaC +
ENaC and
ENaC +
ENaC. This would also be
consistent with a tetrameric organization in which two
subunits
would be associated with one
and one
subunit. The same type of
conclusion is also suggested by the results of Gründer et
al. (31). These authors have analyzed the residual ENaC current
generated after mutation of a highly conserved region located before
the first transmembrane region of
,
, or
ENaCs. As assessed by
the residual current recorded after expression of a mutant subunit with
two other wild-type chains, the role of the three subunits was not
strictly equivalent. A stronger effect was observed after mutation of
ENaC than after mutation of
or
ENaCs. A tetrameric
organization of the ENaC complex with two copies of
ENaC, one copy
of
ENaC, and one copy of
ENaC might suggest that the dominant
negative effect would directly depend upon the number of mutated
subunits participating in the complex.
Gain-of-function mutations affecting MEC-4 and MEC-10 degenerins, which are likely subunits of a common complex expressed in the six touch-receptor neurons of C. elegans, cause neurodegeneration of these cells. Modulation of MEC-4 and/or MEC-10-induced degenerations by a supplementary loss-of-function mutation in trans was analyzed for MEC-10 (8) and MEC-4 (32). It was concluded that there are at least two copies of MEC-10 and two copies of MEC-4 in the complex. A tetrameric organization of the MEC-4/MEC-10 complex formed by two copies of MEC-10 and two copies of MEC-4 would then imply that the other mec gene products that build up the complex such as MEC-6 (8) are not degenerins.
This work is the first direct demonstration that one member of the ENaC/FaNaCh/ASIC/degenerins superfamily can form tetramers. It is interesting to note that the family of inward rectifying K+ channels, in which each of the subunits is also characterized by the presence of only two transmembrane domains, but which displays no homology with proteins of the ENaC/FaNaCh/ASIC/degenerins family, also has a tetrameric organization (33). It might be tempting to speculate that the P2x subtype of purinergic receptors, which also displays a structural organization with two transmembrane domains per subunit, (34) also forms tetramers.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. Guy Champigny for initial electrophysiological characterization of FaNaCh transfected HEK-293 clones, to Dr. Joëlle Bigay for help with FPLC experiments, to Dr. Amanda Patel for careful reading of the manuscript, and to Valérie Friend, Danièle Moinier, Franck Aguila, and Dahvya Doume for expert technical assistance.
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FOOTNOTES |
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* This work was supported by CNRS, INSERM, and the Association Française de Lutte contre la Mucoviscidose (AFLM). Thanks are due to Bristol Myers Squibb for an unrestricted award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of Grant 91815-48/A000 from the Délégation
Générale pour l'Armement (DGA).
§ To whom correspondence should be addressed. Tel.: 33-04-93-95-77-20 or 02; Fax: 33-04-93-95-77-04; E-mail: ipmc{at}ipmc.cnrs.fr or pbarbry{at}itsa.ucsf.edu.
1 FaNaCh, Phe-Met-Arg-Phe-amide-activated Na+ channel; FMRFamide, Phe-Met-Arg-Phe-amide; ENaC, epithelial Na+ channel; ASIC, acid-sensing ionic channel; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DSS, suberic acid bis(N-hydroxysuccinimide ester); SADP, N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate; NHS-ASA, N-hydroxysuccinimidyl-4-azidosalicylic acid; PCR, polymerase chain reaction.
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REFERENCES |
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