Desensitization masks nanomolar potency of ATP for the P2X1 receptor.

ATP-gated P2X1 receptors feature fast activation and fast desensitization combined with slow recovery from desensitized states. Here, we exploited a non-desensitizing P2X2/P2X1 chimera that includes the entire P2X1 ectodomain (Werner, P., Seward, E. P., Buell, G. N., and North, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15485-15490) to obtain a macroscopic representation of intrinsic receptor kinetics without bias arising from the overlap of channel activation and desensitization. From the stationary currents made amenable to analysis by this chimera, an EC50 for ATP of 3.3 nM was derived, representing a >200- and >7000-fold higher ATP potency than observed for the parental P2X1 and P2X2A receptors, respectively. Also, other agonists activated the P2X2/P2X1 chimera with nanomolar EC50 values ranging from 3.5 to 73 nM in the following rank order: 2-methylthio-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, adenosine 5'-O-(3-thiotriphosphate). Upon washout, the P2X2/P2X1 chimera deactivated slowly with a time constant (ranging from 63 to 2.5 s) that is inversely related to the EC50 value for the corresponding agonist. This suggests that deactivation time courses reflect unbinding rates, which by themselves define agonist potencies. The P2X2/P2X1 chimera and the P2X1 receptor possess virtually identical sensitivity to inhibition by the P2X1 receptor-selective antagonist NF279, a suramin analog. These results suggest that the P2X1 ectodomain confers nanomolar ATP sensitivity, which, within the wild-type P2X1 receptor, is obscured by desensitization such that only a micromolar ATP potency can be deduced from peak current measurements, representing an amalgam of activation and desensitization.

Extracellular ATP is a ubiquitous signaling molecule that exerts fast effects by directly gating cation-conducting channels designated P2X receptors, which exist on a large variety of cells, including many excitable cells and different leukocytes (1). Seven genes encoding P2X subunits (P2X 1-7 ) are known in mammals. All P2X subunits share a common membrane topology, with two hydrophobic membrane-spanning segments (M1 and M2) separated by a large extracellular loop of ϳ300 amino acid residues, which comprise the ATP-binding domain. At least part of the pore of the P2X receptor is lined by M2 (2), which includes a conserved glycine residue that is likely to constitute the channel gate (3), but also residues of the Cterminal cytoplasmic tail appear to play a role in controlling channel permeability (4). The cytoplasmic N-terminal tails of the various subunit isoforms are approximately the same length (ϳ30 amino acid residues), whereas the cytoplasmic C-terminal tails are highly variable in length. Both chemical cross-linking studies and blue native polyacrylamide gel electrophoresis analysis of wild-type and concatenated P2X 1 and P2X 3 receptors indicate that P2X receptors feature a trimeric architecture, which is unique among ligand-gated ion channels (5)(6)(7). Further details of the structure of P2X receptors are unknown because neither high resolution electron micrographs nor x-ray or NMR analyses have been published.
Various homo-and heteromeric P2X receptors have been characterized in heterologous expression systems. Based on their sensitivity to the ATP analog ␣,␤-MeATP 1 and the rate of current desensitization, homomeric P2X receptors are generally divided into at least two categories: rapidly desensitizing (P2X 1 and P2X 3 ) and slowly or non-desensitizing (P2X 2A , P2X 4 , and P2X 7 ) receptors. The term P2X 2A distinguishes the nondesensitizing full-length P2X 2A subunit from the desensitizing C-terminal splice variant termed P2X 2B , which lacks a stretch of 69 amino acids C-terminal to M2 (8). The different rates of desensitization have been attributed by mutational analysis to various structural motifs, including N-and C-terminal domains (9, 10) and a highly conserved protein kinase C site (11).
We have demonstrated recently that nanomolar ATP concentrations drive significant fractions of the rapidly desensitizing P2X 1 receptor pool into a long lasting refractory closed state, from which it recovers only slowly with a time constant of ϳ12 min (12). A 50% steady-state P2X 1 receptor desensitization was achieved by the sustained exposure of rP2X 1 receptor-expressing oocytes to as little as 3 nM ATP. Several lines of experimental evidence suggest that the P2X 1 receptor enters the desensitized state both at low and high ATP concentrations exclusively through the open conformation. Such behavior can be adequately described by a minimal three-state model, closed-open-desensitized, according to which P2X 1 receptor activation and desensitization follow the same reaction pathway, i.e. without significant closed-to-desensitized transition. Recovery from desensitization occurs without channel opening, i.e. exclusively from desensitized to closed.
One conclusion of our previous desensitization study (12) is that the nanomolar potency of ATP for receptor desensitization can be attributed to the large ratio between the fast activation rate and the slow recovery rate from desensitization. The affinity for binding of ATP to the resting state of the P2X 1 receptor could not be deduced from our experiments, but model calculations based on simulated data allowed us to estimate an upper limit of 100 nM for the ATP affinity of this step. Up to this concentration, the ATP binding affinity seems to have no influence on the EC 50 values for receptor activation and desensitization. This suggests that the resting P2X 1 receptor state possesses a higher affinity for ATP than revealed by peak current measurements, which provide only an EC 50 value determined under non-steady-state conditions that is significantly biased by the rapid rate of P2X 1 receptor desensitization.
Desensitization can be fully eliminated in the P2X 1 receptor by simply replacing the first 47 amino acids of the P2X 1 subunit with a complementary portion of the P2X 2 subunit corresponding to the cytoplasmic N-terminal tail and almost the entire first transmembrane domain (9). Because these regions comprise only intracellular portions of the subunit and ATP interacts exclusively with the ectodomain (13,14), the ATP binding properties of such a P2X 2 /P2X 1 chimera can be expected to be identical to that of the genuine P2X 1 receptor. In this study, we exploited this non-desensitizing P2X 2 /P2X 1 chimera for a more rigorous evaluation of agonist and antagonist potencies under steady-state conditions without the bias of desensitization. We found that elimination of desensitization unmasks nanomolar ATP potency for the P2X 1 receptor.
Electrophysiology-Two-electrode voltage clamp recordings were performed 1-3 days after cRNA injection. Microelectrodes were pulled from borosilicate glass, filled with 3 M KCl, and broken at the tips to achieve electrical resistances below 1.0 and 1.5 megaohms for the current and potential electrode, respectively. Currents were recorded with a Turbo TEC-05 amplifier (npi electronic, Tamm, Germany), low pass-filtered at 200 Hz, and sampled at 500 Hz (INT-10 AD/DA converter, npi electronic) using commercially available software (Cell-Works, npi electronic). To avoid activation of endogenous Cl Ϫ channels, a nominally Ca 2ϩ -free oocyte Ringer's solution (MgCl 2 substituted for CaCl 2 and therefore designated MgCl 2 /oocyte Ringer's solution) was used for superfusion of oocytes. Measurements were performed at ambient temperature (20 -22°C). The holding potential was Ϫ60 mV. Recordings were made in a small chamber (bath volume of Ϸ10 l), which was superfused by gravity flow at a constant rate of Ϸ200 l/s to allow for rapid solution exchange. Current signals were elicited by switching automatically via magnetic valves between agonist-free and agonist-containing MgCl 2 /oocyte Ringer's solutions in an order controlled by the CellWorks software as described in detail previously (12).
Data Analysis-rP2X 1 receptor responses were quantified by measuring the peak current amplitude relative to the base-line holding current recorded immediately preceding agonist application. Analysis of the non-desensitizing rP2X 2 receptor and the rP2X 2 /P2X 1 chimera was performed using steady-state currents. Concentration-response curve parameters were determined by nonlinear curve fitting of the Hill equation to the data using Origin software (Version 5.0, Microcal Corp., Northampton, MA). The concentration of agonist giving half-maximal activation (EC 50 ) was obtained using Equation 1.
The concentration-inhibition curves and the resulting IC 50 values were derived from nonlinear least-squares fits of the Hill equation to the pooled data points (Equation 2), where I max is the current response in the absence of NF279, I is the current response at the respective NF279 concentration, and n H is the Hill coefficient. Agonist concentration-response curves and EC 50 values in the presence of different concentrations of NF279 were obtained using Equation 3.
Results are presented as means Ϯ S.E. from n experiments. Further details of data analysis are given under ''Results'' and in the figure legends.

RESULTS
Unlike the rP2X 2A Receptor, the rP2X 2 /P2X 1 Chimera Closes Slowly upon ATP Washout- Fig. 1 shows typical current traces recorded by the two-electrode voltage clamp technique in oocytes expressing the rP2X 1 or rP2X 2A receptor or an rP2X 2 / P2X 1 chimeric subunit containing the N-terminal part (amino acids 1-47) of the rP2X 2A subunit and the complementary C-terminal part (amino acids 48 -399) of the rP2X 1 subunit (9). The two portions were joined using Val 48 , which is a conserved residue in both subunits, as a junction point.
At a near-saturating ATP concentration of 10 M, the rP2X 1 receptor-mediated inward current rose fast to its peak and then declined in ϳ1 s, leading to a complete loss of the response in the sustained presence of ATP as a result of receptor desensitization (Fig. 1A). The term desensitization as used here refers to a reversible functional inactivation by a conformational transition of the receptor itself rather than to down-regulation by receptor internalization and related cellular processes, which lead to a reduction in the number of receptor molecules in a given plasma membrane (18). In contrast to the rP2X 1 receptor, the rP2X 2A receptor generated a stationary current as long as superfusion with ATP was maintained. Removal of ATP from the bath was accompanied by a virtually immediate channel closure (Fig. 1B), the time course of which could not be resolved because it occurred with a time constant below the solution exchange time of our system (t 10 -90% ϭ 150 ms). On the other hand, the rP2X 2 /P2X 1 chimera showed little or no desensitization and thus exhibited a P2X 2A receptor-like phenotype, confirming that the first 47 amino acid residues fully eliminate desensitization in the P2X 1 receptor (9). However, in striking contrast to the behavior of the rP2X 2A receptor, ATP washout was followed by a rather slow deactivation of the rP2X 2 /P2X 1 chimera, as inferred from the slow monoexponential decay ( ϭ 63 Ϯ 2 s, n ϭ 15) of the current to the base-line level (Fig. 1C).
One feature that characterizes some (although not all) P2X receptor channels is that they conduct inward current more easily than outward current (16), a characteristic referred to as inward rectification. To examine whether the rP2X 2 /P2X 1 chimera exhibits rectification, current-voltage (I-V) relationships were determined. The I-V relationships of the rP2X 2 /P2X 1 chimera and the rP2X 1 receptor were virtually indistinguishable, but differed markedly from that of the rP2X 2A receptor, which showed stronger rectification (Fig. 1D).
ATP Activates the rP2X 2 /P2X 1 Chimera with Nanomolar Potency-To examine the possibility that the slow channel closure of the rP2X 2 /P2X 1 chimera is indicative of a high ATP potency conferred by the rP2X 1 ectodomain, we established ATP concentration-response curves. In the rP2X 1 and rP2X 2A receptors, half-maximal inward currents were produced by 0.7 Ϯ 0.1 M ATP (n H ϭ 1.1 Ϯ 0.1) and 24 Ϯ 2 M ATP (n H ϭ 1.6 Ϯ 0.1), respectively (Fig. 2B). In contrast to the micromolar EC 50 values of ATP for the parental P2X receptors, the rP2X 2 /P2X 1 chimera was half-maximally activated by 3.3 Ϯ 0.1 nM ATP (n H ϭ 1.6 Ϯ 0.1) (Fig. 2, A and B), corresponding to almost 4 orders of magnitude higher potency than for the rP2X 2A receptor and still Ͼ200-fold higher potency than for the rP2X 1 receptor. Strikingly, virtually the same concentration of 3.2 nM ATP was previously found to half-maximally desensitize the wild-type rP2X 1 receptor by sustained exposure to low levels of ATP (12).
The nanomolar potency of ATP for the rP2X 2 /P2X 1 chimera could be observed only with completely defolliculated oocytes, which are virtually devoid of ecto-ATPase activity (19). Expression of the chimera in incompletely defolliculated oocytes resulted in a profound reduction of ATP potency (Fig. 2C) in a manner attributable to rapid ATP breakdown by ecto-ATPases located on residual follicle cells (20).
Deactivation Rates Define Agonist Potencies for the rP2X 2 / P2X 1 Chimera-The stable ATP analog ␣,␤-MeATP is a high potency agonist for the rapidly desensitizing P2X 1 and P2X 3 receptors, but a low potency agonist for the P2X 2A receptor (EC 50 Ͼ 300 M) (21). Accordingly, ␣,␤-MeATP can be used to distinguish between P2X 1 -and P2X 2 -like agonist properties. Consistent with P2X 1 receptor-like agonist properties, the rP2X 2 /P2X 1 chimera was efficiently activated at low concentrations of ␣,␤-MeATP (Fig. 3G). Virtually equal currents could be elicited by maximally effective ATP and ␣,␤-MeATP concentrations of 1 M each (Fig. 3A). Upon ␣,␤-MeATP washout, the current declined also monoexponentially ( ϭ 2.5 Ϯ 0.3 s, n ϭ 8), but at a 25-fold higher rate than after ATP washout (Fig. 3F), although still slow enough to be resolved unbiased by the rate of solution exchange in our system. Strikingly, a similar 20-fold ratio difference existed also between the EC 50 values for ␣,␤-MeATP and ATP, 67 Ϯ 3 nM (n H ϭ 1.7 Ϯ 0.1) and 3.3 Ϯ 0.1 nM (n H ϭ 1.6 Ϯ 0.1), respectively (Figs. 2B and 3G). This led us to hypothesize that deactivation rates are inversely related to EC 50 values. Individual I-V curves were normalized to the response at Ϫ100 mV and averaged. The P2X 2A receptor showed stronger rectification than the rP2X 1 receptor and the rP2X 2 /P2X 1 chimera, which behaved identically. In this and all other figures, the error bars were omitted when they were smaller than the symbols used.
Antagonist Binding Properties of the rP2X 2 /P2X 1 Chimera-In a further attempt to characterize the ligand binding properties of the rP2X 2 /P2X 1 chimera, we took advantage of NF279, a derivative of the classical P2X antagonist suramin. The P2X receptor selectivity profile of NF279 is different from that of suramin. In particular, NF279 allows one to distinguish between recombinant rP2X 1 and rP2X 2A receptors, which are blocked at 1 and 10 M ATP with IC 50 values for NF279 of 19 nM and 0.76 M, respectively (22) (see also Fig. 4B). To examine the inhibitory potency of NF279 for the rP2X 2 /P2X 1 chimera, we co-applied 1-100 nM NF279 and 5 nM ATP (Fig. 4A), a concentration that elicited an approximately half-maximal response in the absence of NF279 (Fig. 2). The extent of NF279induced inhibition of the rP2X 2 /P2X 1 chimera is apparent from the current amplitudes recorded before and after NF279 washout in the sustained presence of ATP (Fig. 4A). Fig. 4B shows the corresponding concentration-inhibition curves. A fit of the Hill equation to the data yielded an IC 50 value of 16.4 Ϯ 0.8 nM (n H ϭ 2.0 Ϯ 0.2) compared with an IC 50 value of 19 Ϯ 0.8 nM for the wild-type rP2X 1 receptor (22). The almost identical IC 50 values and the virtually overlapping concentrationinhibition curves indicate that NF279 produces a equipotent inhibition of the ATP response for both the rP2X 1 receptor and the rP2X 2 /P2X 1 chimera. For half-maximal inhibition of the rP2X 2A receptor, an ϳ50 times higher NF279 concentration is required.
To elucidate the mechanism of antagonism by NF279 for the rP2X 2 /P2X 1 chimera, ATP concentration-response curves were established in the absence and presence of increasing concentrations of NF279. Concentration-response curves were shifted to the right without altering the maximal ATP response (

DISCUSSION
This study shows that elimination of desensitization unmasks nanomolar ATP potency for the rP2X 1 receptor based on the analysis of a non-desensitizing rP2X 2 /P2X 1 chimera described first by others (9). Several lines of evidence strongly indicate that the rP2X 2 /P2X 1 chimera features the same agonist and antagonist binding properties as the wild-type rP2X 1 receptor. (i) Like the rP2X 1 receptor, the rP2X 2 /P2X 1 chimera was activated with high potency by ␣,␤-MeATP, which is a low potency agonist for P2X 2A receptors. (ii) The suramin analog NF279 blocked the rP2X 2 /P2X 1 chimera with virtually the same IC 50 value as for the wild-type rP2X 1 (23, 24). (iv) The rP2X 2 /P2X 1 chimera does not include extracellular portions of the P2X 2 receptor and hence no part of its ATP-binding site, which is located just extracellular to the transmembrane domains in P2X channels (13,14).
Interestingly, transplantation of the first transmembrane domain of the P2X 1 subunit to the P2X 2 subunit has been shown to confer ␣,␤-MeATP sensitivity to the corresponding receptor chimera, indicating that the first transmembrane domain participates in ␣,␤-MeATP-induced channel gating (21). However, the high ␣,␤-MeATP sensitivity of the rP2X 2 /P2X 1 chimera studied here appears to result from the rP2X 1 ectodomain because the first transmembrane segment originated entirely from the ␣,␤-MeATP-insensitive rP2X 2 subunit.
It must be mentioned that, in the original study, the EC 50 of ATP for an apparently identical rP2X 2 /P2X 1 chimera was found to be not different from the control value (9). Because it is known that the true potency of ATP to activate P2X receptors in native tissues can be profoundly reduced by ectonucleotidases, we consider degradation of ATP by the ecto-ATPase activity of folliculated oocytes as a potential explanation of the unchanged EC 50 value in the previous study. Completely defolliculated Xenopus oocytes, as used in this study, do not show significant extracellular hydrolysis of ATP (19). Indeed, we could artificially render the rP2X 2 /P2X 1 chimera non-responsive to low ATP concentrations using incompletely defolliculated oocytes for expression.
For theoretical reasons (25), a significantly lower EC 50 for The inset shows the region near the origin on an expanded scale to allow two overlapping data points to be seen separately. the non-desensitizing rP2X 2 /P2X 1 chimera than for the fast desensitizing rP2X 1 receptor is not surprising. Because virtually all activated P2X 1 receptors close rapidly by desensitization and not by direct transition to the reactivable closed state, peak current measurements provide no more than an EC 50 determination under non-steady-state conditions (12). By elimination of desensitization, stationary currents become amenable to analysis that allows for a determination of the EC 50 under steady-state conditions. The nanomolar ATP sensitivity of the rP2X 2 /P2X 1 chimera is compatible with our previous calculations based on a kinetic model of the rP2X 1 receptor showing that any intrinsic affinity value below 100 nM can account for the observed potency of 0.7 M and 3.2 nM ATP to activate and desensitize the wild-type rP2X 1 receptor, respectively (12). The reasoning that the observable EC 50 value for the wild-type rP2X 1 receptor is, to a large extent, independent of the intrinsic affinity is also supported by the observation that ␣,␤-MeATP and ATP␥S have an ϳ20-fold lower potency than ATP (or 2-MeSATP) for the rP2X 2 /P2X 1 chimera, but are almost equipotent with ATP (and 2-MeSATP) in activating the rP2X 1 receptor.
Interestingly, there is experimental evidence that the rP2X 3 and rP2X 1 receptors share a high sensitivity to ATP. In GT1 cells, for instance, expressed rP2X 3 receptors can be detected only when apyrase is added to metabolize extracellular ATP (26), suggesting that that the low level of endogenous ATP secretion is sufficient to desensitize this receptor (10). Moreover, a chimera containing the extracellular domain of the rP2X 3 receptor flanked by the transmembrane and cytosolic domains of the rP2X 2A receptor exhibits a 30-fold lower EC 50 value compared with the rP2X 2A receptor (26). A leftward shift of the concentration-response curve is also observed when the native rP2X 2A ectodomain is substituted with the rP2X 4 ectodomain, suggesting that transmembrane domain-flanking sequences can also affect agonist potency (27).
Deactivation of the P2X 2 /P2X 1 Channel-mediated Stationary Current Reflects ATP Unbinding-The kinetic behavior of the rP2X 1 receptor can be adequately described by a minimal three-state reaction model: C ϩ ATP 7 O ATP 7 D ATP , where C is closed, O is open, and D is desensitized. Given that the N-terminal part and the ectodomain of the P2X 1 receptor determine desensitization and ligand binding, respectively, the rP2X 2 /P2X 1 chimera corresponds to a non-desensitizing rP2X 1 receptor represented by the following reaction diagram: C ϩ ATP 7 O ATP . The experimentally determined agonist potency is given by EC 50 ϭ K A /(1 ϩ E), where K A denotes the agonist equilibrium dissociation constant for the binding step, and E is the equilibrium constant for the closed- Note that the fast desensitization of the rP2X 1 receptor to ATP and the comparably slow binding of NF279 required a 10-s pre-equilibration of the rP2X 1 receptor with NF279 before stimulation with ATP. This period is sufficient to ensure that a binding equilibrium between NF279 and the rP2X 1 receptor is reached (22). In contrast, the steady-state inhibition of the non-desensitizing receptors can be directly deduced from the current plateaus attained in the simultaneous presence of ATP and NF279. C, the rightward shift of the ATP concentration-response curves at increasing concentrations of NF279 (0, 30, 100, and 300 nM) combined with the absence of depression of maximal current indicates that NF279 acts as a competitive antagonist with ATP for the rP2X 2 /P2X 1 chimera. A simultaneous fit of all the data to Equation 3 yielded a K B value for NF279 of 12.5 Ϯ 1.2 nM (n H ϭ 2.3 Ϯ 0.2 nM, n ϭ 5-6).
to-open conformation change (28). As apparent from this equation, EC 50 approximates K A solely under the restricted condition that E is Ͻ Ͻ1. To discuss our data in terms of K A and E, an expanded reaction diagram is necessary, given by Reaction 1.
The observed slow deactivation of the rP2X 2 /P2X 1 chimera after ATP washout could then be due to (i) a channel-inherent slow transition from O ATP to C ATP , followed by a faster dissociation of ATP from C ATP to C, or, alternatively, (ii) a slow ATP dissociation after fast channel closure. In both cases, the time course of deactivation reflects the time course of ATP dissociation, although not necessarily the microscopic dissociation rate.
A possible clue that allows one to distinguish between these two possibilities comes from the observation that the potency of several agonists to activate the rP2X 2 /P2X 1 chimera correlates well with the time constant of deactivation after washout of the corresponding agonist. Hence, the deactivation after agonist washout appears to reflect the unbinding properties of the agonist, C agonist 3 C ϩ agonist, and not the channel-inherent O agonist 3 C agonist transition. A similar relationship has been previously observed for the EC 50 values and agonist unbinding rates for nicotinic acetylcholine receptor and N-methyl-D-aspartate receptors and is thus in line with the widespread view that differences in agonist affinity are predominantly determined by the ligand unbinding rate (29,30). It should be noted, however, that a time constant of 63 s for ATP unbinding is still too short to account for slow recovery of the rP2X 1 receptor from the desensitized state, which occurs with a 10-fold longer time constant of ϳ12 min (12). In summary, current deactivation after removal of agonist directly reflects dissociation of ATP from the receptor. Circumstantial evidence favors the view that agonist dissociation represents the rate-limiting step for current deactivation. Accordingly, current deactivation may directly correspond to the microscopic dissociation rate of agonist.
Antagonism by the P2X 1 -selective Suramin Derivative NF279 -We have previously shown for the desensitizing rP2X 1 receptor that ATP concentration-response curves are shifted to the right and exhibit an increasing depression of the maximal response to ATP when oocytes are pre-equilibrated in the presence of increasing concentrations of the P2X 1 -selective antagonist NF279 (22). This behavior could be assigned to a so-called hemi-equilibrium resulting from the fast rise of the P2X 1 receptor-mediated current to its peak, leaving little time for the slowly dissociating NF279 to equilibrate with the fast binding ATP. An apparent K B value of 14 nM was estimated using a double-reciprocal plot (31), but this analysis did not allow us to confirm or reject a competitive type of antagonism by NF279 for the P2X 1 receptor. The present experiments with the nondesensitizing rP2X 2 /P2X 1 chimera clearly indicate that NF279 meets the criteria for competitive antagonism in that it causes parallel rightward shifts in the ATP concentrationresponse curve without depression of maximal current. The virtually identical IC 50 and K B values obtained with the rP2X 2 /P2X 1 chimera suggest that the simplifying assumptions we made previously were reasonable approximations. Because the NF279 binding properties of the rP2X 2 /P2X 1 chimera closely reflect those of the rP2X 1 receptor, the use of the chimera can be advantageous in unraveling the exact mechanism of antagonism, which cannot be determined reli-ably by non-steady-state measurements.
Physiological Implications-The present findings suggest that ATP sensitivity and unbinding are, in addition to ␣,␤-MeATP responsiveness and desensitization patterns, additional fundamental features that distinguish P2X receptors. Two principles seem to have evolved. (i) Highly ATP-sensitive P2X receptors such as the P2X 1 receptor (and potentially also the P2X 3 receptor) feature slow agonist unbinding combined with rapid and sustained desensitization, whereas (ii) moderately or poorly ATP-sensitive P2X receptors feature rapid agonist unbinding and slow desensitization. We postulate that the linkage of high ATP sensitivity and sustained desensitization is not by coincidence but is the result of a demand of high ATP sensitivity and related slow ATP unbinding: desensitization serves to ensure rapid termination of the P2X 1 receptor-mediated current despite the slow rate of ATP unbinding, thus shaping the response. Likewise, enduring elevated low levels of ATP, even if existing only occasionally, would lead without desensitization to cell toxicity problems arising from permanently open cation channels. Low ATP concentrations may, for instance, occur at the border of a synaptic cleft and, due to the high ATP sensitivity of the P2X 1 receptor, lead to a silencing of P2X 1 receptor responses when this synapse is repeatedly activated. On the other hand, moderately or poorly ATPsensitive receptors may serve to respond repeatedly to rapid rises to high ATP concentrations as they occur, for instance, under physiological conditions in a synapse, where the transmitter rises and falls rapidly. Due to their low ATP sensitivity, these P2X receptors shut immediately when ATP concentrations fall to the submicromolar level and are thus immediately prepared to respond to the next rise in the ATP level. Altogether, these two receptor principles may complement each other in subserving particular demands in neuronal excitability.