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Accelerated Communication

Role of Ectodomain Lysines in the Subunits of the Heteromeric P2X_2/3 Receptor

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom (W.J.W., R.A.N.); and Department of Biomedical Science, University of Sheffield, Sheffield United Kingdom (L.-H.J., A.S.)

Received May 15, 2006; accepted July 13, 2006

	Abstract

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Lysine residues near each end of the receptor ectodomain (in rat P2X₂ Lys⁶⁹ and Lys³⁰⁸) have been implicated in ATP binding to P2X receptors. We recorded membrane currents from human embryonic kidney cells expressing P2X subunits and found that lysine-to-alanine substitutions at equivalent positions in the P2X₃ receptor (Lys⁶³ and Lys²⁹⁹) also prevented channel function. Heteromeric P2X_2/3 receptors are formed when P2X₂ and P2X₃ subunits are expressed together; they can be distinguished by their relatively sustained response to β-methylene-ATP. By coexpression of wild-type P2X₃ and mutated P2X₂ subunit, we found that the heteromeric P2X_2/3 channel functioned normally when either lysine in the P2X₂ subunit was mutated to alanine (i.e., [K69A] or [K308A]) but not when both lysines were mutated to alanine (i.e., [K69A, K308A]). However, coexpression of wild-type P2X₂ with a mutated P2X₃ subunit ([K68A] or [K299A]) produced no functional heteromers. The rescue of the single lysine mutant P2X₂ subunit by wild-type P2X₃ (but not the converse) suggests that the heteromeric channel contains one P2X₂ and two P2X₃ subunits and that the receptor functions essentially normally as long as two subunits are not mutated. The failure to rescue function in the P2X₂ subunit with both lysines mutated by wild-type P2X₃ suggests that these residues from two different subunits interact in agonist binding or channel opening.

There is good evidence that P2X subunits form channels as trimers (Nicke et al., 1998; North, 2002; Jiang et al., 2003; Barrera et al., 2005). Like other ligand-gated channels, P2X receptors can form by homo- or hetero-oligomeric assembly of subunits (for review, see North, 2002). Although both P2X₂ and P2X₃ subunits are able to form homomeric channels, the heteromeric P2X_2/3 receptor that is also formed can be distinguished by its unique functional properties (Lewis et al., 1995). P2X₂ receptors are activated by ATP but not by the analog β-methylene-ATP (βmeATP), and the current is largely sustained through a 2-s agonist application. P2X₃ receptors are activated by both ATP and βmeATP, and the current desensitizes very rapidly (<100 ms). The current through heteromeric P2X_2/3 channels can be identified as the sustained component when βmeATP is the agonist (Lewis et al., 1995).

The molecular operation of P2X subunits is not well understood. Each receptor subunit has intracellular amino and carboxyl termini and two transmembrane domains (TM1: approximately residues 30-50; TM2: approximately residues 330-354) that are joined by an ectodomain of approximately 280 residues. The ectodomain is glycosylated and disulfide-bonded. There are no obvious "canonical" ATP-binding amino acid sequences in the ectodomain. In the case of the P2X₂ homomeric receptor, two conserved ectodomain lysine residues have been suggested to play a role in binding the negatively charged phosphate moiety of ATP (Lys⁶⁹ and Lys³⁰⁸ in rat P2X receptor), but the evidence is most complete for Lys⁶⁹ (Jiang et al., 2000). A similar role for the corresponding lysines has also been identified in the case of the P2X₁ receptor (Lys⁶⁸ and Lys³⁰⁹; Ennion et al., 2000). These lysines are situated approximately 20 amino acids from the outer end of each TM. Experiments on the P2X₁ receptor using partial agonists also suggest a role for aromatic residues Phe¹⁸⁵ and Phe²⁹¹ to coordinate the binding of the adenine ring of ATP (Roberts and Evans, 2004; Vial et al., 2004). An alternative model of ATP binding has been proposed by Yan et al. (2005) in the case of the P2X₄ receptor. This is based on secondary structure homology between part of the receptor (Lys¹⁸⁰-Lys³²⁶) and class II aminoacyl-tRNA synthetases (Freist et al., 1998).

View larger version (22K): [in this window] [in a new window]   Fig. 1. A, mutation of conserved ectodomain lysines causes loss of function at rat P2X receptors. Pairs of traces show currents at wild-type and lysine mutant receptors. Agonists were: for P2X1, βmeATP (300 µM); for P2X4, ATP (1 mM); and for P2X7, BzATP (300 µM). Each was applied for 2 s. B, mutation of Lys69 or Lys308 at P2X2 causes a loss of function (ATP, 1 mM), and equivalent mutations to Lys63 and Lys299 in P2X3 have a similar effect (βmeATP, 300 µM).

View larger version (21K): [in this window] [in a new window]   Fig. 2. ATP elicits significant currents when lysine-mutant P2X2 subunits are coexpressed (n = 9). However, receptors P2X2[K69A] and P2X2[K308A] expressed separately do not respond to ATP at concentrations up to 3 mM (n = 12). Human embryonic kidney, nontransfected cells (n = 3). Inset shows a representative trace elicited by 1 mM ATP when lysine mutants are coexpressed; scale bars, 50 pA and 1 s.

	Materials and Methods

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Molecular and Cell Biology. Rat P2X receptor cDNAs were used as described previously (Jiang et al., 2000). Mutations were introduced using QuikChange site-directed mutagenesis (Stratagene, La Jolla CA) and confirmed by sequencing. Wild-type and mutated receptors were transiently expressed in human embryonic kidney 293 cells using Lipofectamine 2000 (Invitrogen, San Diego CA) according to the manufacturer's instructions. Transfected cells were harvested and replated on glass coverslips 24 to 48 h after transfection and kept at 37°C for 18 to 48 h before electrophysiological recording. P2X₂ plasmid (0.2 µg) was cotransfected with P2X₃ plasmid (1 µg), these ratios having been established to produce equal amounts of protein when detected by Western blot with a common C-terminal epitope tag (Jiang et al., 2003). Enhanced green fluorescent protein cDNA (0.1 µg) was also included. Immunocytochemistry of transfected cells was as described, using a C-terminal EYMPME epitope (Jiang et al., 2000, 2003).

Electrophysiology. Whole-cell patch-clamp recordings were made at 19 to 22°C using a HEKA EPC9 amplifier (Heka, Lambrecht, Germany). The holding potential was -60 mV. Patch pipettes (3-6 M) contained 145 mM NaCl, 10 mM HEPES, and 10 mM EGTA, pH adjusted to 7.3 with NaOH. The external solution contained 147 mM NaC1, 2 mM KC1, 2 mM CaC1₂, 2 mM MgCl₂,10 mM HEPES, and 13 mM glucose, pH adjusted to 7.3 with NaOH. Both internal and external solutions were 290 to 310 mOsM. Agonists were applied using the RSC 200 rapid solution changer (Biologic Science Instruments, Grenoble, France), with flow-pipe tips approximately 150 µm from cell. All chemicals were purchased from Sigma (Poole, UK) and were made up daily from aliquots stored at -20°C.

	Results

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Homomeric Channels. Previous work on homomeric human P2X₁ (Ennion et al., 2000) and rat P2X₂ (Jiang et al., 2000) receptors has indicated that Lys⁶⁹ and Lys³⁰⁸ (rat P2X₂ numbering) are essential for channel function. We found that alanine substitution at the equivalent positions in the P2X₃ subunit also led to a nonfunctional homomeric channel. We observed no current with applications of βmeATP (up to 1 mM; n = 5) (Fig. 1) or ATP (up to 1 mM; n = 7). Immunocytochemistry of transfected cells showed no difference in the pattern or distribution between wild-type and mutated subunits. Figure 1 also shows that equivalent mutations in other rat P2X receptors also prevented any response to agonist [P2X₁[K68A]: ATP (300 µM) 23 ± 9 pA, n = 8; βmeATP (300 mM) no current, n = 3; P2X₄[K67A]: ATP (1 mM) no current, n = 8; P2X₇[K64A]: ATP (3 mM) no current, n = 3].

Neither P2X₂[K69A] nor P2X₂[K308A] gave any detectable current in response to 1 or even 3 mM ATP (Fig. 1). However, coexpression of these two mutated receptors clearly did (Fig. 2). The currents were small compared with wild-type P2X₂ currents (>600 pA/pF) but not obviously different in other properties (Fig. 2). It was striking that coexpression of two "dead" subunits resulted in formation of a channel that was responsive to 100 or 300 µM ATP. If we assume that neither mutation affects expression or assembly, then one would expect three eighths of all trimeric channels to be [K69A]₂·[K308A] and three-eighths to be [K69A]·[K308A]₂. This result therefore indicates that the effects of these two point mutations are not independent.

Heteromeric Channels. For the P2X_2/3 receptors, there was a striking difference in the effect of a single point mutation of either of the lysine residues, depending whether it was present in the P2X₂ or P2X₃ subunit. The currents in cells expressing mutant P2X₂ receptors with wild-type P2X₃ subunits were essentially the same as those observed in wild-type heteromeric receptors (Fig. 3). The βmeATP concentrations giving half-maximal currents (EC₅₀) were: for P2X_2/3 wild-type, 27 ± 3.3 µM (n = 5); for P2X₂[K69A] + P2X₃, 22 ± 2.6 µM (n = 5), and for P2X₂[K308A] + P2X₃, 44 ± 2.6 µM (n = 8) (the last two values are significantly different, p < 0.0001). In contrast, cells expressing wild-type P2X₂ with P2X₃[K63A] subunits showed no currents in response to βmeATP up to 100 µM. At such high concentrations, P2X₂ homomeric receptors are sensitive to βmeATP (n = 5) (Fig. 3) (see also Jiang et al., 2003), and the small current observed was presumably passing through such receptors. Likewise, coexpression of wild-type P2X₂ subunits with P2X₃ [K299A] gave no currents in response to βmeATP (up to 100 µM, n = 6) (Fig. 3). These results indicate that a wild-type P2X₃ subunit can rescue function when it enters a heteromer with a mutated P2X₂ subunit, but not vice versa.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Coexpression of P2X2 and P2X3 subunits. A, currents elicited by a 2-s application of 30 µM βmeATP. P2X2[K69A] and P2X2[K308A] can be rescued to form functional channels by expression with wild-type P2X3. The corresponding mutations in the P2X3 subunits are not rescued by wild-type P2X2 subunits. The double-mutant P2X2[K69A,K308A] subunit does not function when coexpressed with wild-type P2X3. However, the rapidly desnsitizing homomeric P2X3 responses were typically observed on the first application of βmeATP. B, concentration-response curves for some of the combinations shown in A. Responses are expressed as a percentage of the maximal response (mean ± S.E.M., five to eight cells at each point).

View larger version (38K): [in this window] [in a new window]   Fig. 4. A, schematic illustration of the P2X2 and P2X3 subunits used, and summary results. Small solid circles indicate positions of lysine-to-alanine substitution near outer end of TM1 or TM2. B, schematic interpretation. Channels with two P2X3 subunits and one P2X2 subunit might provide two subunit contacts with two lysine residues (arrows, putative binding site) if only one of the P2X2 lysines were substituted by alanine (left, middle) but would have only one such contact when P2X2 subunit has both lysines changed to alanine. 1, tested with ATP; 2, tested with βmeATP; 3, normal wild-type currents were 522 ± 74 pA/pF (n = 9; measured 200 ms after beginning superfusion); 4, dead indicates no detectable current (<2 pA/pF); 5, ATP also evoked a sustained current, suggesting that the involvement of these residues is not specific to βmeATP.

	Discussion

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The two lysines that are the focus of the present study are very highly conserved among P2X subunits. Lys⁶⁹ is present in 41 of 43 vertebrate sequences (including seven human, seven rat, seven mouse, one guinea pig, three chick, two Xenopus laevis, one bullfrog, nine zebrafish, and six pufferfish); the zebrafish P2X₂ receptor has Asp at this position, and it cannot be activated by ATP when heterologously expressed (Kucenas et al., 2003). This lysine is also present in the one invertebrate (schistosome) sequence that is known to operate as an ATP-gated channel (Agboh et al., 2004). Lys³⁰⁸ is even more highly conserved; it is found in all the above 44 sequences and in two of the five related Dictyostelium discoideum sequences. Of the eight positively charged residues in the P2X₂ receptor ectodomain, Lys⁶⁹ and Lys³⁰⁸ are the two where replacement by alanine causes the greatest loss of channel function; even replacement by arginine results in more than 100-fold reduction in sensitivity to ATP (Jiang et al., 2000). The present experiments confirm that the lysine in the first of these positions is critical also for function of other P2X receptors, including P2X₃.

The heterotrimeric P2X_2/3 receptor must contain either one or two copies of the P2X₃ receptor subunit. The simplest interpretation of the one-way rescue of P2X₂ mutants by P2X₃ (but not vice versa; Fig. 3), is that the P2X_2/3 receptors activated by βmeATP have two copies of P2X₃ and one copy of P2X₂. In other words, a functional channel can be formed and activated by βmeATP even though it contains one dead P2X₂ subunit (e.g., P2X₂[K69A]) but no opening can be elicited from a receptor comprising two dead P2X₃ subunits with a wild-type P2X₂ subunit. The rescue of wild-type P2X₃ was complete in the case of P2X₂[K69A] and slightly less so for P2X₂[K308A] (Fig. 3), indicating that the two P2X₂ lysines do not have precisely equivalent roles.

This result contrasts with earlier findings in which a highly conserved intracellular C-terminal lysine was mutated (Chaumont et al., 2004). This lysine is required in homomeric P2X₂ and P2X₃ receptors for retention in the plasma membrane. In that case, wild-type P2X₃ subunits could restore the appearance and retention in the membrane of mutant P2X₂ subunits (P2X₂[K366A]), and this rescue was fully reciprocal because wild-type P2X₂ also restored the appearance of P2X₃[K357A]) (Chaumont et al., 2004). Taken together, these results imply that only a single wild-type subunit is needed for successful retention in the membrane, but two wild-type (P2X₃) subunits are needed for channel function. The conclusion that the functional heteromer contains two P2X₃ subunits and one P2X₂ subunit agrees well with the results of a completely independent approach in which subunits were joined by disulfide bonds between engineered cysteine substitutions at the outer ends of the TMs (Jiang et al., 2003). The finding is also consistent with the observation that the limiting slope of the Hill plot for activation of the heteromeric P2X_2/3 receptor is close to two, whereas for the homomeric P2X₃ and homomeric P2X₂ receptors, it is close to three (Jiang et al., 2003). However, the present analysis can not distinguish between different numbers of ligand binding sites and different degrees of intersubunit cooperativity in the heteromeric versus the homomeric channels.

For the homomeric P2X₁ and P2X₂ receptors, there is evidence that Lys⁶⁹ and Lys³⁰⁸ contribute to an ATP binding site (Ennion et al., 2000; Jiang et al., 2000; Roberts and Evans, 2004). Therefore, our present finding that the heteromeric channel operates normally even though it incorporates a mutated P2X₂ subunit may be interpreted in this context. This would imply that a heterotrimeric P2X receptor could open from a less than fully liganded state. For example, the tetrameric -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (Rosenmund et al., 1998) and the pentameric glycine receptor (Beato et al., 2002) can open from less than fully liganded states. But other interpretations are possible. For example, the P2X₃ (though not P2X₂) subunit lysines may be critical for multimeric channel assembly. Or the mutation might impede the conformational change of gating: a lysine-to-alanine substitution in a single P2X₂ subunit of the trimer might provide insufficient energetic impediment, whereas two such independent substitutions in the P2X₃ subunits might prevent it completely.

We were surprised to find that cells cotransfected with P2X₂[K69A,K308A] subunit and wild-type P2X₃ subunit produced no detectable heteromeric current (Fig. 3). In other words, the wild-type P2X₃ subunits can rescue function in a channel containing a P2X subunit with a single mutation ([K69A] or [K308A]) (a dead P2X₂) but not in a channel containing both those mutations (a "double-dead" P2X₂). This could have a simple energetic explanation of the type introduced above; namely, both P2X₃ subunits become liganded by βmeATP, but the mutations provide too great an energy barrier for opening to occur. Or it could indicate that the effects of the lysines are not independent and that an interaction occurs between Lys⁶⁹ and Lys³⁰⁸. This would be consistent with the finding that in homomeric P2X₂ receptors, ATP can elicit currents when P2X₂[K69A] and P2X₂[K308A] are coexpressed, but not when either is expressed alone.

If one assumes that at least two agonist binding sites are required for channel activation (see above, and Ding and Sachs, 1999; Jiang et al., 2003), then these observations can be explained if both Lys⁶⁹ of one subunit and Lys³⁰⁸ of another (or equivalent for the P2X₃ subunits) are needed at one ATP binding site. The presence of a dead P2X₂ subunit would still provide for two βmeATP binding sites (Fig. 4B, left and middle), but a double-dead P2X₂ subunit would prevent βmeATP binding at two sites and therefore prevent channel function (Fig. 4B, right). The interpretation that the agonist site is contributed from two different subunits in a heteromeric channel has parallels with nicotinic acetylcholine receptors (Sine, 2002). Moreover, P2X₂ subunits carrying mutations of an allosteric zinc binding site have been covalently joined in tandem from concatenated cDNAs; these experiments indicated that histidines from different subunits contribute to the zinc binding site (Nagaya et al., 2005).

The conclusion that the βmeATP binding sites forms at the P2X₃/P2X₃ and P2X₂/P2X₃ interfaces is consistent with the observations that both nucleotide (Virginio et al., 1998; Burgard et al., 2000) and non-nucleotide antagonists (Jarvis et al., 2002, 2004) mostly fail to discriminate between P2X₃ homomeric receptors and P2X_2/3 heteromeric receptors. A more complete understanding of the agonist binding site will inform the further development of small molecules that act as competitive antagonists at the P2X_2/3 heteromeric receptors; such molecules are likely to have value as pain-relieving drugs.

	Acknowledgements

 
We thank Helen Broomhead, Louise Almond, and Sandy Dossi for their technical assistance.

	Footnotes

 
The work was supported by the Wellcome Trust

ABBREVIATIONS: βmeATP, β-methylene-ATP

¹ Current affiliation: Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

Address correspondence to: R. Alan North, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. E-mail: r.a.north{at}manchester.ac.uk

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