From the Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Plan-les-Ouates, 1228 Geneva, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The extracellular signaling properties of nucleotides are mediated through two distinct families of membrane proteins. These are the P2Y receptors, coupled to G proteins and second messenger pathways, and the P2X receptors, which are ligand-gated ion channels (1). Seven subunits of the P2X receptor family (P2X1-7) have been characterized at the molecular level (reviewed in Refs. 2 and 3). These show a broad expression pattern compared with other ligand-gated channels, with the various forms being found in central and peripheral nervous system, different types of immune cells, glands, and smooth and skeletal muscles (4). The P2X receptor subunits can form channels as homomultimers or, in some cases, as heteromultimers (5, 6). The number of subunits in each channel molecule is not known.
P2X receptor subunits are 36-48% identical to one another at the amino acid level. All seven proteins have similar hydrophobicity profiles, with only two hydrophobic regions sufficiently long to span the plasma membrane. These regions display the features often seen in transmembrane segments such as aromatic residues at interfacial regions, and they have an excess of positively charged amino acids at their presumed cytoplasmic ends (7). Together with the absence of signal peptide sequence after the initiating methionine, this suggests that P2X receptors may have intracellular N and C termini, and two transmembrane domains separated by a large extracellular loop (8, 9). This proposed topology differs from that of the nicotinic and glutamate superfamilies of ligand-gated ion channels (reviewed in Ref. 10), but resembles that shown for the pore-forming subunits of epithelial sodium channels (11, 12). There is, however, no detectable similarity of amino acid sequence between P2X receptors and epithelial sodium channels (3).
There are other experimental results that are consistent with this
topological model of P2X receptor subunit. First, P2X1 receptors are activated by the ATP analog
meATP,1 whereas
P2X2 receptors are not; transferring the putative
extracellular loop (approximately residues 50-320) from the
P2X1 receptor into the P2X2 receptor confers
meATP sensitivity (13). Second, changing one amino acid within
the loop of the P2X4 receptor (E249K) causes a large
increase in the blocking action of the slowly reversible antagonist
pyridoxal-5-phosphate-6-azophenyl-2'-4'-disulfonic acid (14). Third,
the difference in sensitivity to
pyridoxal-5-phosphate-6-azophenyl-2'-4'-disulfonic acid between human
and rat P2X4 receptors can be transferred by exchange of a
segment within the first half of this loop (15). Fourth, evidence for
N-glycosylation of Asn184 of the
P2X1 receptor has been obtained, indicating that this must
be located extracellularly (16).
The recent identification of residues contributing to the pore of the P2X2 receptor has also provided evidence for an intracellular location for the C-terminal part of the protein (17). Amino acids in and around the second hydrophobic domain of the P2X2 receptor (residues 316-354) were mutated individually to cysteine, the proteins were expressed, and ATP-activated currents were measured. Inhibition of the current by polar methanethiosulfonate derivatives was then used to determine whether the residue was likely exposed to the aqueous solution. One substitution was identified (D349C) at which extracellularly applied methanethiosulfonate ethylamine inhibited the ATP-evoked current. However, the inhibition required channel opening. Because methanethiosulfonate ethylamine can permeate the open channel, these results indicate that Asp349 normally lies internal to the channel "gate." The residue is close to the C-terminal end of the second hydrophobic region; this therefore implies that the C terminus of the protein is within the cytoplasm, because there is no further hydrophobic domain long enough to span the plasma membrane.
These results are consistent with the topology for P2X receptors initially proposed (8, 9), but alternative models are also possible. For example, the data generated so far on cloned P2X receptors might as well be explained with a model in which only the second hydrophobic domain spans the plasma membrane, placing the entire N-terminal part of the protein extracellularly. Therefore, we have used the N-glycosylation site tagging approach to determine the precise topology of P2X2 receptors. During protein biosynthesis, extracellular domains of the protein are facing the lumen of the endoplasmic reticulum where they are able to be glycosylated. Due to the strict compartmentalization of N-glycosylating enzymes at the luminal face of the endoplasmic reticulum, it is possible to assess the extracellular location of different region of a given protein by N-glycosylation site tagging. This approach has been used to determine the topology of other membrane proteins including channels and transporters (18, 19).
We first identified the endogenous N-glycosylation of P2X2 receptor, and then by mutagenesis obtained a form in which no natural N-glycosylation of the protein remained. The cDNA for this modified form of the receptor was then used as a background plasmid to engineer artificial N-glycosylation sites at different locations in the protein, with respect to the two hydrophobic domains. The extent of N-glycosylation was assayed by gel shift assay after functional expression of epitope-tagged forms of the P2X2 receptor in Xenopus oocytes. Finally, we concatenated cDNAs and expressed the tandem constructs to show that the N and C termini reside on the same side of the plasma membrane.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutagenesis--
In all experiments, a P2X2
receptor cDNA was used that carried a C-terminal epitope
(DPGLNEYMPME), cloned into the expression vector pcDNA3
(Invitrogen) (17, 20). Point mutations in P2X2 receptor
cDNA were introduced by full-length polymerase chain reaction
amplification of plasmid DNA with sense and antisense mutated primers.
Pfu polymerase (Stratagene) was used to reduce the rate of
contaminating mutations. Parental (wild type) DNA was digested with 10 units of DpnI (New England Biolabs) for 1 h at
37 °C; 2.5 µl of the reaction was then directly used for transformation of Escherichia coli DH5 strain. DNA
fragments carrying the mutation were digested with appropriate
restriction enzymes and subcloned in a background vector that had not
been amplified. All mutants were sequenced on both strands.
Construction of Dimeric cDNAs-- A concatenated tandem P2X2 receptor cDNA was constructed in pBluescript vector. Briefly, two P2X2 cDNAs were modified by in-frame addition of an EcoRI site either at the 5' or 3' end of the coding sequence, respectively. These two cDNAs were ligated through the EcoRI site and a unique site from the backbone of the vector. The resulting construct contains two P2X2 subunits linked from the C terminus of one (-KGLAQL) to the N terminus of another (MVRRLA-); the deduced amino acid sequence at the junction site is -KGLGIRLA-. The mutant subunit P2X2-T336C (17) was also constructed in pcDNA3; it also had the C terminus tag. To create the wild type-mutant dimer, a fragment from the Bluescript dimer was obtained by BstEII digestion and subcloned into the BstEII site of P2X2-T336C. The resulting plasmid encodes a wild type subunit followed by a mutant subunit and has the C-terminal epitope tag.
Gel Shift Assay--
Stage V-VI oocytes were removed from
ovaries of anesthetized Xenopus laevis as described (21).
Ovarian lobules were treated with 1 mg/ml collagenase (Sigma) for
2 h at room temperature in calcium-free solution (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 5 mM sodium pyruvate, 5 mM
Hepes, 10 units/ml penicillin, and 10 units/ml streptomycin). Healthy
oocytes were selected and allowed to recover overnight prior to
injection. Plasmid DNA was directly injected into oocyte nuclei. All
plasmids were co-injected with an enhanced green fluorescent protein
(EGFP)-containing plasmid (pEGFP, Life Technologies, Inc.). From 24 to
48 h after injection, EGFP-positive oocytes were sorted using a
fluorescent microscope. Crude membrane fractions were prepared from
batches of 12 oocytes; oocytes were homogenized in 240 µl of buffer H
(100 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine hydrochloride, 200 µg/ml pepstatin A, 250 µg/ml
aprotinin, 260 µg/ml 1-antitrypsin, 100 ng/ml soybean
trypsin inhibitor, 200 µg/ml bestatin). Homogenates were shaken for
15 min at 4 °C and centrifuged at 14,000 × g for 2 min. After denaturation, membrane-containing supernatants were
separated by SDS-polyacrylamide gel electrophoresis on a 8% Tris
glycine gel (Novex). Separated proteins were electro-transferred onto
nitrocellulose membranes (2 h, 25 V, 4 °C). Membranes were blocked
with 5% milk, 0.2% Tween 20 in phosphate-buffered saline for 1 h
at 37 °C. Monoclonal antibody against EYMPME (22) was added at
1:1000 dilution and incubated for 16 h at 4 °C, in 2.5% milk,
phosphate-buffered saline solution. A secondary antibody (goat
anti-mouse IgG) conjugated to horseradish peroxidase was added at a
1:1000 dilution in 2.5% milk and incubated for 1 h at 37 °C.
Proteins were detected by a chemiluminescent assay (ECL detection kit,
Amersham Pharmacia Biotech).
Endoglycosidase H Treatment--
Membrane extracts (15 µl)
were incubated with 10 µl of endoglycosidase H for 1 h at
37 °C in the presence of 0.2% SDS and 0.4%
2--mercaptoethanol.
HEK Cell Transfection, Electrophysiological Recording, and Immunocytochemistry-- Transfection of human embryonic kidney 293 (HEK) cells, and electrophysiological recording from oocytes and HEK cells has been described previously (17, 21). HEK cells were co-transfected with EGFP cDNA (0.5 µg/ml; CLONTECH) and the relevant P2X2 receptor cDNA (1 µg/ml), and recordings were made 18-48 h later. All EGFPpositive (i.e. fluorescent) cells exhibited ATP-evoked current.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of Natural N-Glycosylation-- We determined if it was possible to detect tagged P2X2 protein by Western blot from a crude oocyte membrane preparation. As shown in Fig. 1, when oocytes were injected with the wild type P2X2 cDNA, the anti-EE antibody recognized a protein of apparent molecular mass around 65 kDa. The calculated size of the P2X2 receptor protein is 52.6 kDa; the observed difference is due to N-glycosylation because a mutated subunit lacking all three asparagines found at consensus glycosylation sites (N-X-S/T) migrated at approximately 50 kDa. The broad smear of the wild type Western blot may result from the fact that crude membrane preparations were used; these would include not only plasma membrane but also the membrane of intracellular organelles (endoplasmic reticulum and Golgi) in which the identity and length of sugars added to proteins might be variable. Uninjected or EGFP-injected oocytes showed no staining or very weak bands (Fig. 1B).
|
|
Introduction of Artificial N-Linked Glycosylation Sites--
We
introduced three point mutations resulting in consensus sites for
N-glycosylation into the 3N-P2X2 receptor
between the initiating methionine and the beginning of the first
hydrophobic regions (C9N, Y16N, and R28T) (Fig.
3A). Fig. 3B shows
that the protein had the same apparent molecular weight as
3N-P2X2, indicating that this N-terminal region is not
accessible to the glycosylating enzymes. Thus, this experiment failed
to provide any evidence for an extracellular location of the N
terminus.
|
|
|
Expression of Dimeric cDNAs-- The failure to observe glycosylation when consensus sequences were introduced into the region N terminus to the first hydrophobic domain is unhelpful with respect to assignment of this domain to one or other side of the membrane, because not all consensus sites are used (24). We therefore sought to determine whether the N and C termini were located on the same side of the membrane by expressing a concatenated cDNA (25). Fig. 6B shows a Western blot from oocytes injected with the dimeric construct. A single band was observed at a molecular mass of about 120 kDa, indicating that the intact dimeric protein is expressed. Recordings made from the same oocytes, prior to membrane preparation for Western blotting, showed inward currents evoked by ATP (30 µM) which were not obviously different from those seen in oocytes expressing the monomeric wild type protein (data not shown). We have previously shown that substitution of Cys for Thr at position 336 in the P2X2 receptor produces a channel in which methanethiosulfonate ethyltrimethylammonium (MTSET) almost completely blocks the current evoked by ATP (17). MTSET inhibited the currents by about 50% when the Cys to Thr substitution was made in either the first or second (Fig. 6C) domain of the dimeric construct, and by close to 100% when the mutation was in both domains (25).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These experiments show that the P2X2 receptor is a glycoprotein, in which all three consensus sequences for N-linked glycosylation (N-X-S/T) are used. Among the seven P2X receptor subunits, the number of such consensus sequences ranges from three (P2X2, P2X5, P2X6) to seven (P2X4), and in no case is the consensus sequence found at a corresponding position in all seven sequences. However, the sequence around Asn182 of the P2X2 receptor (N-F-T) is present in all except the P2X5 receptor, and it is also found within a region of the protein showing relatively high homology among all seven subunits. This suggests that it may be glycosylated in them all (except P2X5); indeed, the corresponding residue in the P2X1 receptor provides the only previously known glycosylation site in the P2X receptor family (16). Our finding that fully functional ATP-gated channels are found with the point mutation N182S is consistent with the inference from the P2X5 receptor sequence that glycosylation at this position is not essential for channel assembly and function.
The second of the two sites identified in the present study (Asn239 of N-F-T) also occurs in a relatively highly conserved region of the protein, but in this case the consensus sequence is found only in the P2X2 and P2X7 receptors. The third of the three sites found to be glycosylated is the Asn298 of N-G-T; the consensus sequence is found at this position also in the P2X1 and P2X3 receptors but not in the other receptors. A further conserved consensus site that is missing in the P2X2 receptor is found in receptors P2X4-7 (corresponding to Asn255 in the P2X4 receptor), and it will be interesting to determine whether this is also glycosylated.
We found that the currents induced by ATP in HEK293 cells expressing P2X2 receptors lacking any one of the three asparagines were essentially normal (Fig. 2), suggesting that none of the attached sugars play a significant role in agonist recognition and binding. On the other hand, receptors in which two or three asparagines were substituted by serine responded to ATP either very poorly or not at all (Fig. 2). We have visualized the cells immunohistochemically using standard fluorescent or confocal laser microscopy: all showed a pattern of plasma membrane localization similar to that seen in the wild type, but the fluorescence intensity was significantly reduced in the receptors lacking two or three (but not one) glycosylation sites.2 This finding implies that full glycosylation is not necessary for correct protein folding and transport to the plasma membrane. However, further immunohistochemical studies of these receptors tagged in the extracellular domain would be useful to examine whether the unglycosylated protein is correctly inserted in the plasma membrane.
The main aim of the present work was to delineate the boundaries of the putative transmembrane domains. We interpret the finding of glycosylation at residues P62N, I66N, and K324N to indicate that these positions are exposed at the extracellular aspect of the membrane; Pro62 is the 11th amino acid residue following the final hydrophobic residue of the first hydrophobic domain (Val51). The failure to observe glycosylation at sites closer to the hydrophobic domain (Q52N, Q56N, and S58N) might be because they are situated within the interfacial region and inaccessible to glycosylating enzymes (23), or because these mutations prevent normal folding and membrane expression. The latter interpretation seems less likely in view of the finding of normal functional responses in the wild type P2X2 receptor carrying mutations Q52N, S58N, or P62N. The residues corresponding in position to Gln52, Ser58, and Pro62 are highly variable among the seven P2X receptor subunits, and may be presumed to tolerate asparagine. The lack of functional responses in the case of mutations Q56N and I66N indicates that these substitutions are not tolerated; the residues in these positions are identical (all glutamine) or highly conserved (six valine, one isoleucine) among the seven subunits. Altogether, the results are consistent with the interpretation that Q52N and S58N are not glycosylated because they are located too close to the plasma membrane, although other structural reasons can not be excluded.
The second hydrophobic domain probably begins with Ile331, although residues at positions 327 and 328 are conserved hydrophobic amino acids in all seven P2X receptor subunits. The present finding that K324N was glycosylated shows clearly that this position is located on the extracellular surface of the cell. In the P2X2 receptor in which the three native glycosylation sites were left intact, K324C functions normally and appears not to contribute to the ion permeation pathway (17). This Lys residue occurs within a completely conserved motif (G-X-X-G-K-F), which might be thought to resemble that observed in some ATP-binding proteins (26); the finding that Lys324 can be replaced by Cys without obvious change in function argues against any such critical role. In the case of Ile328, substitution by Cys results in a channel which is sensitive to block by MTSET (17). One interpretation of that result is that Ile328 is situated within the outer vestibule of the channel; such close proximity to the membrane could be the reason why no glycosylation was observed when asparagine was introduced at this position.
The failure to observe any glycosylation in the case of three engineered Asn residues in the region of the molecule preceding the first hydrophobic domain N terminus is consistent with an intracellular location for this segment. However, direct evidence that both the N and the C termini are located on the same aspect of the membrane was obtained by the construction and expression of a concatenated dimeric cDNA (25). Taken together with previous work, which suggested that the C terminus should be intracellular (17), these findings indicate that both N and C termini are located within the cell. The observation that the substitution T336C confers responsiveness to MTSET when introduced into either the first or second domain of concatenated subunits is consistent with both subunits of the dimer contributing to the pore (25). Further quantitative experiments on dimers, trimers, and larger multimers might be interpretable with respect to the channel stoichiometry and are presently in progress.
The main conclusion of the present work is that each subunit of the
P2X2 receptor, and presumably the other homologous family members, has intracellular N and C termini, two hydrophobic domains (approximately residues 31-51 and 331-353), and a large extracellular loop (52-330). We cannot exclude the possibility that regions of this
long loop fold back into the plasma membrane, although we note that any
such regions that are predominately hydrophobic are only six or seven
amino acids in length. The identification of the natural glycosylation
sites indicates that residues 184, 239, and 298 are extracellular. This
overall topology is thus similar to that described for the subunit
of the epithelial sodium channels (11). The sequence homology between
the epithelial sodium channels and the FMRFamide-activated (27) and
proton-activated (28) channels makes it likely that these other
ligand-gated channels share the same overall pattern of membrane
insertion, with two transmembrane domains and a large, cysteine-rich
extracellular loop. This contrasts clearly with the other large
families of ligand-gated ion channels which have either three
(glutamate family) or four (nicotinic acetylcholine family)
membrane-spanning segments per subunit (see Ref. 10).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kathryn Radford for advice on Western blotting, Danielle Estoppey for tissue culture, and Denis Fahmi for oocyte preparation and injection.
![]() |
FOOTNOTES |
---|
* 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.
Present address: School of Biological Sciences, University of
Manchester, Manchester M13 9PT, United Kingdom.
§ Present address: Institute Biologie Cellulaire et Morphologie, University of Lausanne, CH1005 Lausanne, Switzerland.
¶ Present address: Dept. of Pharmacology, Glaxo Wellcome Research and Development, 37135 Verona, Italy.
Present address: Inst. of Molecular Physiology, University of
Sheffield, Sheffield 510 2TN, United Kingdom.
** To whom correspondence should be addressed. Tel.: 41-22-706-9739; Fax: 41-22-794-6965; E-mail: rassendren{at}serono.com.
1
The abbreviations used are: meATP,
adenosine 5'-(
,
-methylene)triphosphate; Endo H, endoglycosidase
H; MTSET, methanethiosulfonate trimethylammonium; EGFP, enhanced green
fluorescent protein.
2 A. Newbolt, R. Stoop, C. Virginio, A. Surprenant, R. A. North, G. Buell, and F. Rassendren, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|