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Originally published In Press as doi:10.1074/jbc.M600442200 on March 13, 2006

J. Biol. Chem., Vol. 281, Issue 22, 15044-15049, June 2, 2006
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A C-terminal Lysine That Controls Human P2X4 Receptor Desensitization*

Samuel J. Fountain1 and R. Alan North

From the Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester, M13 9PT, United Kingdom

Received for publication, January 17, 2006 , and in revised form, March 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor desensitization can determine the time course of transmitter action and profoundly alter sensitivity to drugs. Among P2X receptors, ion currents through homomeric P2X4 receptors exhibit intermediate desensitization when compared with P2X1 and P2X3 (much faster) and P2X2 and P2X7 (slower). We recorded membrane currents in HEK293 cells transfected to express the human P2X4 receptor. The decline in current during a 4-s application of ATP (100 µM) was about 30%; this was not different during whole-cell or perforated patch recording. Alanine-scanning mutagenesis of the intracellular C terminus identified two positions with much accelerated desensitization kinetics (Lys373: 92% and Tyr374: 74%). At position 373, substitution of Arg or Cys also strongly accelerated desensitization: however, in the case of K373C the wild-type phenotype was fully restored by adding ethylammonium methanethiosulfonate. At position 374, phenylalanine could replace tyrosine. These results indicate that wild-type desensitization properties requires an aromatic moiety at position 374 and an amino rather than a guanidino group at position 373. These residues lie between previously identified motifs involved in membrane trafficking (YXXXK and YXXGL) and implicates the C-terminal also in rearrangements leading to channel closing during the presence of agonist.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
P2X receptors belong to a family of ATP-gated ion channels, seven subunits of which have been currently identified and cloned (P2X1–7) (1). Functional P2X receptors probably form as trimers, incorporating the same or different subunits (2, 3). The P2X subunits are considered to have two hydrophobic transmembrane domains with intracellular N and C termini and a large extracellular ectodomain containing conserved cysteines (1); the ectodomain also contains residues that appear to contribute to the binding site for ATP (4, 5).

In the central nervous system, P2X receptors have been implicated in mediating ATP-dependent fast excitatory neurotransmission (6–9). The P2X4 subunit is highly abundant in the central nervous system, with expression in both spinal cord and brain, including dentate gyrus, CA1/CA3 pyramidal, and cerebellar Purkinje cells (10–12); they are particularly localized to glutamatergic synapses (12). Upon activation, P2X4 can regulate cellular Ca2+ levels via direct permeation and by the activation of voltage-dependent Ca2+ channels. P2X receptor activity has therefore been proposed to be important in synaptic plasticity (13, 14).

The time course of the effect of ATP at P2X receptors is strongly influenced by receptor desensitization, a feature common to most other ligand-gated ion channels. Desensitization manifests itself as a decline in current amplitude during agonist occupancy of the receptor, and in the case of P2X4, it can be modified pharmacologically (15). Recombinant P2X receptors display varying degrees of desensitization; membrane current recordings show that P2X1 and P2X3 undergo fast desensitization (tens of milliseconds), P2X4 exhibits moderate desensitization (several seconds), and P2X2, P2X5, and P2X7 show less desensitization (1, 16, 17). For various P2X receptors, desensitization can be regulated by cellular signaling events, which further suggests its importance in P2X receptor physiology. Several studies have demonstrated regulation of P2X desensitization by protein kinase activity, either via direct N-terminal phosphorylation (at a highly conserved protein kinase C site) or via phosphorylation of an associated protein (18, 19). P2X3 desensitization can be decreased by the calcineurin-mediated dephosphorylation of the N terminus (20).

Experiments using chimeric constructs of P2X1 and P2X2 subunits identified desensitization critical domains as the transmembrane segments and the intracellular juxtamembrane regions (up to 15 amino acids in length). Other domains within the C terminus of P2X2 have been identified by functional analysis of splice variants (21, 22). P2X receptors have very different C-terminals, and the regions important for P2X4 subunits are not known. Given the likely importance of the P2X4 receptor in central synaptic physiology, it seemed useful to understand further the molecular mechanisms that may contribute to P2X4 desensitization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Human P2X4 subunit with a C-terminal EMYPME epitope tag subcloned into pcDNA3.1 (+) was used as the template for all mutagenesis reactions. Mutagenesis was achieved using Pfu turbo polymerase and QuikChange methodology (Stratagene). Truncated receptors were generated by the introduction of a premature stop codon by mutagenesis. HEK293 cells were grown in 10% fetal calf serum in Dulbecco's minimal essential medium at 37 °C with 5% CO2 in a humidified incubator. Cells were transiently transfected with plasmids encoding wild-type or mutant P2X receptor (1 µg) and enhanced green fluorescent protein (0.1 µg) using Lipofectamine 2000 (Invitrogen).

Whole-cell recordings were made from HEK293 cells at room temperature, 24–48 h after transfection. The extracellular solution contained (mM): 145 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, 13 D-glucose, and 10 HEPES. The intracellular pipette solution contained (mM): 145 NaCl, 10 HEPES, and 10 EGTA. Solutions had pH 7.3 after titration with 5 M NaOH. Chemicals were purchased from Sigma (Poole, UK). Pipettes had resistances of 3–5 M{Omega}. For perforated patch-clamp, perforation was achieved by the addition of 120 µg/ml amphotericin in the patch pipette solution. Perforation typically occurred 5–8 min after gigaseal formation. ATP was applied using an RSC 200 system (Biological Science Instruments, Grenoble, France). Ethyl ammonium methane thiosulfonate (MTSEA2: Toronto Research Chemicals) was prepared as a concentrated stock, stored at –20 °C, and diluted in extracellular solution immediately prior to application. MTSEA was applied by superfusion at a rate of ~2 ml/min.


Figure 1
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  		FIGURE 1.
Differences between the decline in current with repeated ATP applications and decline in current during a single application. a, representative current trace for human P2X4 currents in whole-cell (top trace) and amphotericin perforated patch (bottom trace) configurations. ATP (100 µM) was applied (bars) for 2 s at 1 min intervals. Cells were voltage clamped at –60 mV. b, peak amplitudes as a function of time for recordings made in whole-cell (open circles) or perforated patch (closed circles) configurations (n = 12–25 cells). Currents are normalized to the amplitude of first response. c, decline of current during ATP application is not different in whole-cell versus perforated patch configurations but is slowed in the presence of ivermectin (IVM, 3 µM). Representative scaled traces showing currents during ATP application (100 µM, 5 s) were recorded in whole-cell (with or without IVM) and amphotericin-perforated patch configuration. Cells were voltage clamped at –60 mV.

 
For immunohistochemistry, transfected cells were fixed with 4% paraformaldehyde for 5 min followed by permeabilization with 0.1% Triton X-100. Cells were blocked for 30 min (phosphate-buffered saline, 0.5% bovine serum albumin) and incubated with primary antibody (rabbit anti-EE, 1:500; Bethyl Laboratories) in blocking solution for 1–2 h at room temperature. After washing, cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit, 1:200; Jackson ImmunoResearch) for 1–2 h at room temperature.

Numerical data are expressed as mean ± S.E., and differences were tested by Student's t test and analysis of variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Decline in Response with Repeated ATP Application—We distinguish two forms of desensitization at the human P2X4 receptor. The first is the decline in the peak current with repeated brief applications of ATP. Thus, in whole-cell recording, the peak current amplitude progressively declined when ATP (100 µM, 2 s) was applied at 1-min intervals (Fig. 1a). This decline was well fit with a single exponential ({tau} = 93 ± 6.2 s, n = 25 cells), reaching a plateau level at ~20% of the initial peak amplitude. We observed no recovery from desensitization with repeated application when the application interval was increased to 5, 10, or 20 min (data not shown).

P2X4 currents recorded using the perforated patch configuration (amphotericin 120 µg/ml) did not show decline with repeated applications (Fig. 1, a and b). However, we were unable to influence the decline in response by a range of other perturbations. Ivermectin (3 µM, 3 min) did not alter the kinetics of current decline ({tau} = 98 ± 5.4 s; n = 8, p > 0.05) (Fig. 1a), although it increased peak current amplitudes ~3-fold. The rate of current decline was unchanged in external solutions containing 0 mM calcium and also not different when the recording pipette contained ATP (1 mM), GTP (1 mM), staurosporine (100 nM), genistein (100 µM), phosphatidylinositol 4,5-bisphosphate (50 µM), or U73122 [GenBank] (10 µM). P2X4{Delta}Y378, which is truncated so as to lack the endocytic motif YEQGL (Fig. 2), showed properties not different from wild type receptors ({tau} = 91 ± 5.2 s, n = 12 cells, p > 0.05).

Decline in Response during ATP Application—The second form of desensitization is the decline in the membrane current during the continued application of ATP, and this is the subject of the remainder of this paper. Currents evoked by ATP (100 µM) rose to their peak amplitude in 410 ± 8.2 ms, and the average peak amplitude was 151 ± 30 pA (n = 25 cells). During a 5-s application of ATP (100 µM), wild-type currents declined to a value that was 32 ± 7.9% (n = 25 cells) of their initial peak amplitude. These values were obtained in the whole-cell configuration, but the decline in current during the application was not different when recorded using the perforated patch configuration (27 ± 5.2%, n = 12 cells, p > 0.05) (Fig. 1c). However, the decline during the application was substantially slowed by ivermectin (3 µM, 3 min; 2.4 ± 0.8%, n = 12 cells, p < 0.01) (Fig. 1c) (15). The decline in response was independent of ATP concentration (1 µM, 30 ± 1.8%, 3 µM, 31 ± 1.4%, 30 µM, 31 ± 4.1%, 300 µM, 34 ± 2.0%; n = 6 cells). Further experiments were carried out with 100 µM ATP. The rate of current decline was not different when the extracellular calcium concentration was 0 mM as compared with 2 mM; it was also independent of holding potential (–60 mV versus +60 mV).


Figure 2
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  		FIGURE 2.
P2X4 receptor C-terminal truncations. a, left, schematic representation of human P2X4 receptor subunit showing topology and sites of C-terminal truncations used. Right, alignment of relevant parts of C terminus tail of P2X receptors. Boxed, YXXXK motif common to all receptors. Underlined, P2X4 motif that binds to AP2 protein. Bold, Lys373 and Tyr374. b, representative evoked currents (ATP; 100 µM, 5 s) for wild-type and three C-terminally truncated mutants ({Delta}Y378, {Delta}K373, and {Delta}K362). Cells were voltage clamped at –60 mV. {Delta}K373 and {Delta}K362 were non-functional (n = 8 cells each). Currents shown were obtained during the first ATP application.

 
Truncations and Alanine Scanning in the C Terminus—The C terminus of the human P2X4 receptor extends from residue Leu358, at the end of the second transmembrane domain, to Gln388. A truncation that deleted the final eleven amino acids ({Delta}Y378) had properties not different from wild type P2X4; it showed a similar desensitization during the application (Fig. 2) and a similar decline with repeated applications. Removal of the residues from and including Lys373 or Lys362 resulted in receptors that were non-functional (Fig. 2b).

Receptors containing a singly substituted alanine at the 27 positions from Tyr359 to Gln388 had normal currents in response to ATP, with several exceptions (Fig. 3). Substitution of alanine for Tyr367, Lys371, or Tyr372 produced non-functional receptors that have been described previously (23, 24). Non-functional receptors reached the plasma membrane when wild-type and mutant receptor immunocytochemistry was compared (Fig. 3c). All mutant receptors gave current densities not significantly different from wild-type when challenged with 100 µM ATP, with the exception of P2X4[D377A] and P2X4[E379A] (wild-type, 121 ± 10 pA/pF; P2X4[D377A], 20 ± 4 pA/pF, 25 ± 5 pA/pF, p < 0.01, n = 5 cells). Substitution of alanine at Lys373 and Tyr 374 resulted in currents with much accelerated current decline in the presence of ATP (K373A: 92 ± 3.2%, n = 20; Y374A: 74 ± 4.2%, n = 15, p < 0.01). The double mutant receptor P2X4[K373A,Y374A] had properties not different from the single mutant P2X4[K373A](decline in current 93 ± 2.8%; n = 8 cells; Fig. 3). Both P2X4[K373A] and P2X4[Y374A] had accelerated rundown kinetics compared with wild-type in whole-cell patches ({tau} = 41 ± 2.2 s and 61 ± 3.8 s, respectively; n = 15–20, p < 0.01). All other alanine mutants had wild-type run-down kinetics (data not shown). Sequence alignment of the C-terminal regions of the seven P2X subunits shows that P2X4 (and P2X1) subunits differ from the others at positions 373 and 374 (Fig. 2a). P2X4 and P2X1 have a positively charged residue at position 373, whereas all the others are negative. P2X4 and P2X1 subunits have an aromatic tyrosine residue at position 374, whereas in the others it is charged or polar (P2X7). We investigated the effect of substituting equivalent residues from P2X2 (a non-desensitizing receptor) for residues at positions 373 and/or 374 in P2X4. However, currents at P2X4[K373D], P2X4[Y374K], or P2X4[K373D,Y374K] receptors were not significantly different from those seen with the alanine-substituted equivalents (92 ± 5.4%, 93 ± 3.2%, and 92 ± 4.8%, respectively, n = 8–12 cells, p > 0.05). Phenylalanine substituted fully for tyrosine at position 374: P2X4[Y374F] showed currents and desensitization not different from wild type (28 ± 6.2%, n = 8 cells, p > 0.05) (Fig. 3a).

Further Studies at Lys373—When arginine replaced lysine at position 373 (P2X4[K373R]) the receptors exhibited a current not significantly different from P2X4[K373A] (89 ± 3.4%, n = 8 cells, p > 0.05) (Fig. 3a). This indicates that the arginine side chain (–CH2)3–NH–C(NH2) = NH+2) cannot substitute for that of lysine Formula at position 373. We examined further the difference between the [cepsilon]-amino group and the guanidino moiety by expressing the P2X4[K373C] receptor. Current evoked by ATP at this receptor declined similarly to that seen with P2X4[K373A] (87 ± 3.4%, n = 8 cells, p > 0.05). However, MTSEA (1 mM; 10 min) restored the wild-type phenotype (38 ± 6.3%, n = 6 cells, p > 0.05) (Fig. 4a). This effect of MTSEA was prevented by intracellular cysteine (20 mM: in the recording pipette; Fig. 4b). MTSEA had no effect on wild type (33 ± 4.2%, n = 6–10) or P2X4[K373A] receptors (90 ± 4.2%, n = 8, p > 0.05), indicating that the effect of MTSEA was a specific modification of the cysteine at position 373 (Fig. 4).

These results indicate that maintained channel opening in the presence of ATP requires Lys373 (or the equivalent side chain Formula. We asked therefore in how many subunits of a homotrimeric receptor was the lysine required? We co-expressed wild-type and P2X4[K373A] receptors in nominally equal amounts. Whole-cell recordings showed typical ATP-activated currents, but the decline during the application was intermediate between that seen for cells expressing either wild-type or P2X4[K373A] receptors alone (Fig. 4c). In fact, the current declined by 58 ± 8.4% over 4 s (n = 12 cells) during a 4-s application. This decline in the current was not different from that which would have been observed from an equal mixture of wild type and [K373A] substituted receptors. If one assumes that heterotrimer formation occurs readily between subunits containing alanine and those containing lysine and that only two (rather than four) phenotypes can occur, then this result indicates that a single alanine-containing subunit does not exert a dominant effect with respect to the desensitization kinetics (Fig. 4c).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results distinguish two modes of P2X4 receptor desensitization. In our whole-cell experimental conditions, P2X4 responses declined both during ATP applications lasting a few seconds and with applications repeated at 1-min intervals (Figs. 1a and 2a). With perforated patch recording to minimize dialysis of intracellular contents, the decline with repeated application ("run-downP") was completely prevented, whereas the decline during ATP applications was the same as in whole-cell recordings (Fig. 1). The prevention of P2X4 rundown by perforated patch recording is comparable with the work by Lewis and Evans (25), who showed that the run-down of a P2X1-like current in smooth muscle cells did not occur with amphotericin-permeabilized recordings. The phenomena of run-down has been observed for P2X1, P2X3, P2X4, and P2X7; other P2X receptors give robust steady-state currents with repeated ATP applications. A likely explanation for P2X4 run-down in whole-cell recording is that a diffusible cytosolic factor that tonically regulates the receptor is lost during intracellular dialysis. The identity of such a factor is not obvious, but from our experiments it seems not to be ATP, GTP, or phosphatidylinositol 4,5-bisphosphate. The mechanism underlying the decline in P2X4 response with repeated ATP application is also not likely to involve AP2-mediated receptor internalization. This is because the truncation P2X4[{Delta} Y378], and the point mutations Y378A and L382A, each of which lack the AP2-dependent endocytic motif YXXG{Phi} responsible for receptor internalization in neurons (27), exhibited a decline in response with repeated ATP application that was not different from wild type receptors. These truncated receptors would presumably use the YXXV motif (beginning at Tyr372) for their normal trafficking (24).


Figure 3
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  		FIGURE 3.
Effect of amino acid substitutions in the P2X4 C-terminal tail on the decline in response during ATP application. a, representative traces showing currents evoked by ATP (100 µM, 5 s) recorded from cells expressing wild-type, K373A, K373R, Y374A, Y374F, and K373A/Y374A receptors. Currents shown were obtained during the first ATP application. b, summary of C-terminal alanine scanning mutagenesis on the decline in response during ATP application (values are percent amplitude after 4-s application, n = 6–25 cells; **, p < 0.01 Student's t test). nf = non-functional). c, representative images showing comparable localization of wild-type and non-functional mutant EE-tagged receptors expressed in HEK293 cells."control"indicates experiments performed in the absence of anti-EE tag primary antibody. Images were taken at x60 magnification.

 
Ivermectin increases the currents evoked by ATP at P2X4 receptors (26). A recent careful study by Priel and Silberberg (15) distinguished two effects: the first is an increase in the maximum current that is seen at concentration around 0.3 µM, and the second is a slowing of deactivation that occurs at 10-fold higher concentrations. Both these effects were observed in our experiments, using 3 µM ivermectin. However, in ivermectin there was no change in the decline of the peak currents observed with repeated application of ATP. Parenthetically, we also noted that the increase in current by ivermectin was observed in the truncated P2X4 receptor P2X4[{Delta}Y378].

The C terminus of P2X4 is short (30 residues) relative to that of other P2X receptors: 62% of these residues are either charged or aromatic (tyrosine) (Fig. 2a). By alanine scanning we identified two residues (Lys373 and Tyr374) as being important for the time course of desensitization during the ATP application. Both K373A and Y374A mutations resulted in receptors with much accelerated decline in response during ATP application as compared with wild-type receptor currents (Fig. 3a). These residues lie between two other functionally important regions for P2X receptors. These are the YXXXK motif that is found in all P2X family members (Tyr367 to Lys371, human P2X4) and the YEQGL sequence (Tyr378 to Leu382) found uniquely in P2X4 subunits. The first of these contributes to receptor stabilization at the plasma membrane (23). The second has been shown to bind directly to the µ2 subunit of the adaptor protein AP-2, where it occupies a site comparable to that seen for the more common endocytic motif YXX{Phi} (24). This binding is key to clathrin-mediated receptor internalization undergone by P2X4 receptors (27). These two residues that we have identified form the heart of the YKYV motif that is not normally used for clathrin-mediated endocytosis of P2X4 receptors (24, 27).


Figure 4
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  		FIGURE 4.
Reconstitution of wild-type properties by MTSEA-induced cysteine modification of P2X4[K373C]. a, representative superimposed normalized traces showing currents at P2X4[K373C] and effect of extracellular perfusion with MTSEA (1 mM). The right panel illustrates the absence of effect of MTSEA on wild-type receptors. b, summary data showing the time-dependent effect of MTSEA on current decline during ATP application (100 µM, 4 s). There is no effect of MTSEA on wild-type receptor (open squares). P2X4[K373C] receptor currents show almost 100% decline, in the absence of MTSEA (filled circles). P2X4[K373C] receptor currents show wild-type decline after 10 min in MTSEA (closed triangles). MTSEA effect on P2X4[K373C] current was prevented by intracellular cysteine (20 mM, open diamond). n = 6–10 cells each; p < 0.01 Student's t test, compared with K373C in the absence of MTSEA. c, averaged and superimposed macroscopic currents recorded from cells expressing either wild-type or P2X4[K373A] receptor alone or co-expressing wild-type and P2X4[K373A] receptors; standard error is shown at 200-ms intervals; n = 8–15 cells each. The gray line is the macroscopic current predicted to be observed if two independent populations of wild-type and P2X4[K373A] receptors exist and assuming equal contribution. Currents shown were obtained during the first ATP application. d, schematic showing predicted receptor stoichiometry and ratios assuming equal mixing between wild-type (white) and P2X4[K373A] (black) receptor subunits following co-expression.

 
We asked how critical was the requirement for tyrosine at position 374 and found that P2X4[Y374F] functioned essentially as the wild-type receptor with respect to the decline in current during the ATP application (Fig. 3a). This indicates that the requirement for aromaticity at this position for the channel to remain open during agonist occupancy. It further indicates that Tyr374 is unlikely to be phosphorylated: for example, previous work (28) on P2X7 receptors has indicated that repeated activation leads to receptor dephosphorylation and that this underlies the decline in the responses in that case.

On the other hand, arginine could not substitute for lysine at position 373 (Fig. 3a). This suggests that the critical moiety is not simply the positive charge but other features of the lysine side chain (such as the hydrocarbon chain). We tested this by expressing the P2X4[K373C] receptor and using MTSEA to add a side chain of similar length Formula to that of lysine Formula. This completely restored the wild-type phenotype. Control experiments showing that MTSEA did not modify the wild-type channel or the fast desensitizing mutant P2X4[K373A], and that the effect of MTSEA was prevented by intracellular cysteine (29–31), strongly support the interpretation that MTSEA is acting by modification of the cysteine side chain at position 373. This indicates that lysine rather than arginine is required for structural reasons (e.g. the arginine side chain Formula is too long to be accommodated) or perhaps that the lysine is subject to posttranslational modification. For example, post-translational modification of lysine has been shown to be important for the function of other ion channels (32). However, there are no consensus motifs for lysine modification apparent at position 373 and the relatively fast time course (5–10 min: Fig. 4b) with which MTSEA restored the wild type phenotype effect also argues against such an interpretation. The simplest explanation of why K373A mutation accelerates the decline in response during ATP application is that the critical positioning of this positive charge is required for channel opening to be maintained when ATP is bound. This implies that gating transitions involve not only conformational changes in the ectodomain and transmembrane hydrophobic regions but also the first part of the C terminus (33–36). It is difficult to make further mechanistic interpretation, given that the residue at this position is lysine in one other P2X subunit (P2X1, which shows rapid desensitization) and negatively charged (Glu or Asp) in all others.

In a previous study by Koshimizu et al. (22), the region 376–381 (EDYEQG) was identified as being important in controlling P2X4 desensitization during sustained applications of ATP. They measured the decline in intracellular calcium concentration over periods of several hundred seconds, so it is difficult to interpret those results with respect to the present studies in which ionic current was measured over a period of 5 s. Under our experimental conditions, these residues do not participate in receptor desensitization, as determined by alanine substitution or truncation. Previous work by Royle et al. (27) has shown that this region contains the first four amino acids of the motif (YEQGL) that interacts with the µ2 subunit of the adaptor protein AP2 and is thus involved in receptor internalization. In general terms, this would be consistent with our interpretation that the decline in the current during the time frame of a few seconds that we have studied is unrelated to receptor internalization.

P2X receptors are generally considered to operate as trimers (1). Our final question was whether the rapid desensitization was observed when only one of the three subunits carried alanine at position 373 (i.e. P2X4[K373A]·P2X4·P2X4) (Fig. 4); in other words, did this mutation confer a dominant phenotype. The result of the co-expression of wild-type and mutant subunits in equal amounts (Fig. 4c) strongly indicates that accelerated desensitization is seen only in channels with two or more subunits carrying the alanine (i.e. P2X4[K373A]·P2X4[K373A]·P2X4). This would imply that normal channel function can occur even though one of the three subunits is mutated to alanine at this position. We cannot exclude the alternative explanation for the results of the co-expression, which is that the lysine to alanine mutation prevents the formation of heteromeric channels, and that the currents observed flow through approximately equal parts of the homomeric wild type P2X4 and mutant P2X4[K373A] channels. This seems unlikely given that the P2X4[K373A] has properties similar in many respects to wild type channels, which shows that alanines in this position (at least with three of them) do not themselves prevent formation of functional homomers.

In summary, we have identified Lys373 and Tyr374 in the C terminus of the P2X4 receptor as being key determinants of P2X4 receptor desensitization on the time scale of seconds. This implicates the juxtamembrane C terminus in playing an important role in determining the duration of the physiological action of ATP at the P2X4 receptors.


   FOOTNOTES
 
* This work was supported by The Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. E-mail: Samuel.fountain{at}manchester.ac.uk.

2 The abbreviation used is: MTSEA, ethyl ammonium methane thiosulfonate. Back


   ACKNOWLEDGMENTS
 
We thank H. Broomhead, L. Almond, and K. Dossi for technical assistance during this study.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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