Allosteric nature of P2X receptor activation probed by photoaffinity labelling.

BACKGROUND AND PURPOSE: In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate. EXPERIMENTAL APPROACH: We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors. KEY RESULTS: Irradiation of the P2X2/1 receptor chimera - BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists. CONCLUSIONS AND IMPLICATIONS: Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding.

NF449: a subnanomolar potency antagonist at recombinant rat P2X1 receptors.

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 >> P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.

Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor.

P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).

The suramin analogue NF279 is a novel and potent antagonist selective for the P2X(1) receptor.

The suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)) bis(1,3,5-naphthalenetrisul fonic acid) (NF279) was analysed with respect to its potency and P2X receptor subtype selectivity. Two-electrode voltage-clamp measurements were performed with Xenopus laevis oocytes expressing homomultimeric rat P2X(1), P2X(2), P2X(3) and human P2X(4) receptors. For the fast desensitising P2X(1) and P2X(3) receptors, IC(50) values strongly depended on whether oocytes were pre-incubated with NF279 prior to ATP superfusion or exposed to NF279 simultaneously with ATP. With a 10 s pre-incubation period of NF279, IC(50) values of 19 nM and 1.62 microM were obtained for rat P2X(1) and P2X(3), respectively. Without pre-incubation, IC(50) values amounted to 2 microM and 85.5 microM for P2X(1) and P2X(3), respectively. For the non-desensitising rat P2X(2) receptor NF279 appeared to act as a competitive antagonist with an IC(50) value of 0.76 microM and a K(B) value of 0.36 microM, as derived from Schild analysis. P2X(4) receptors were the least sensitive subtypes for NF279 (IC(50)>300 microM). The antagonism was fully reversible at all P2X subtypes analysed. Our results indicate that NF279 is a potent P2X(1) receptor-selective and reversible antagonist.

The novel pyridoxal-5'-phosphate derivative PPNDS potently antagonizes activation of P2X(1) receptors.

Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.

Novel ligands for P2 receptor subtypes in innervated tissues.

Among suramin analogues, the properties of P2 receptor subtype blockade and ecto-nucleotidase inhibition appear to be controlled by different structural parameters (Fig. 1 and 2, Table 1; Van Rhee et al., 1994; Beukers et al., 1995; Bültmann et al., 1996; Damer et al., 1998a, 1998b; and this study): the molecular size of the compounds, the position of the sulfonic acid residues in the naphthalene rings, the substitution pattern of the benzoyl moieties and the 3'- or 4'-aminobenzoyl-linkages of the phenyl rings "1" and "2". As a result, compounds with different receptor selectivity profiles were obtained. A maximum in potency at and selectivity for P2X1 receptors is reached in NF279, which is a specific P2 receptor antagonist and the compound with the highest P2X1 vs. P2Y receptor and ecto-nucleotidase selectivity presently available.

Novel properties of the depolarization-induced endogenous sodium conductance in the Xenopus laevis oocyte.

It has been shown by means of the two-microelectrode voltage-clamp technique that in membranes of Xenopus laevis oocytes a Na+-selective permeability can be activated by long-lasting or repetitive depolarization (R.T. Kado and C. Baud, Journal of Physiology, Paris, 77:1113-1117, 1981). In this study the permeability in inside-out giant membrane patches with diameters of 20-30 microm was analysed. Once induced, the Na+ permeability has a voltage-dependent open probability that increases with positive potentials and half-maximally activates at about 0 mV. Sudden changes of membrane potential elicit transient currents with strongly voltage-dependent time constants of from less than 1 ms at -150 mV to several hundreds of milliseconds at positive potentials. In contrast to the on-cell configuration, the permeability ceases completely within a few minutes in the cell-free inside-out configuration. This rundown can be prevented by including MgATP, but not Mg2+ or ATP alone, in the intracellular solution. Intracellular Mg2+ ions, in addition to being a co-factor for ATP in the activation process, decrease the permeability in a dose-dependent manner. Steady-state fluctuation analysis gave no evidence that an increased noise level is caused by open-close kinetics of an ion channel, suggesting that the single-channel conductance is below 1 pS if a channel-like structure is the origin of the endogenous Na+ permeability.

Blue native PAGE as a useful method for the analysis of the assembly of distinct combinations of nicotinic acetylcholine receptor subunits.

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma delta >> alpha 1 beta 1 gamma > alpha 1 beta 1 delta > alpha 1 gamma delta >> alpha 1 delta >> alpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.

Voltage-dependent inhibition of the Na+,K+ pump by tetraethylammonium.

Tetraethylammonium (TEA+) is an effective inhibitor of a variety of K+ channels, and has been widely used to reduce K+-sensitive background conductances in electrophysiological investigations of the Na+,K+-ATPase. Here we demonstrate by combination of two-electrode voltage clamp (TEVC) and giant patch clamp of Xenopus oocytes, and measurements of the activity of purified ATPase of pig kidney that TEA+ directly inhibits the Na+,K+-ATPase from the outside. The KI value in TEVC experiments at 0 mV is about 10 mM increasing with more negative potentials. A similar voltage-dependent inhibition by TEA+ was observed in the excised membrane patches except that the apparent KI value at 0 mV is about 100 mM, a value nearly identical to that found for inhibition of purified kidney ATPase. The voltage-dependent inhibition can be described by an effective valency of 0.39 and is attributed to an interference with the voltage-dependent binding of K+ at an external access channel. The apparent dielectric length of the access channel for K+ is not affected by TEA+.

NF279: a novel potent and selective antagonist of P2X receptor-mediated responses.

8,8'-(Carbonylbis(imino-4, 1 -phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3, 5-naphthalenetrisulfonic acid) (NF279) antagonized P2X receptor-mediated contractions in rat vas deferens, evoked by alpha,beta-methylene ATP (10 microM; pIC50=5.71) without affecting responses mediated via alpha1A-adrenoceptors, adenosine A1 and A2B receptors, histamine H1, muscarinic M3 and nicotinic receptors. The low inhibitory potency of NF279 on P2Y receptors in guinea-pig taenia coli (pA2=4.10) and at ecto-nucleotidases in folliculated Xenopus laevis oocytes (IC50 > 100 microM) indicates that NF279 is a novel specific and selective P2X receptor antagonist.

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

UNLABELLED: P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. KEYWORDS: blue native PAGE/cross-linking/P2X receptor/quaternary structure.

Characteristics of Na+/K(+)-ATPase mediated proton current in Na(+)- and K(+)-free extracellular solutions. Indications for kinetic similarities between H+/K(+)-ATPase and Na+/K(+)-ATPase.

The Na+/K(+)-ATPase of an ouabain-resistant mutant of Torpedo californica and of rat was expressed in Xenopus oocytes by microinjection of mRNA coding for the alpha- and the beta-subunit of the protein. Electrophysiological measurements were performed by means of the extracellular giant-patch clamp technique. The pump currents were analyzed in nominal absence of extracellular Na+ and K+ ions. Under these conditions strongly inward rectifying, ouabain-sensitive current was detected with reversal potentials depending on extracellular pH. Presence or absence of intracellular Na+ or K+ ions had nearly no effect on the inward currents, removal of intracellular ATP caused their inhibition. The reversal potentials of the currents generated by the rat pump was shifted by 82.7 +/- 5.4 mV per 10-fold increase of extracellular proton concentration. This refers to an effective valency of 0.71 +/- 0.05. In the absence of a transmembrane proton gradient the pump current reversed at -64.2 +/- 4.4 mV. These findings are not compatible with a proton conducting channel formed by the pump molecule (Wang and Horisberger (1995) Am. J. Physiol. CP 37, C590-595). Therefore, a kinetic model based on the Post-Albers scheme with a modification derived from the reaction scheme of the gastric H+/K(+)-ATPase is proposed. Together with voltage-dependent binding of extracellular protons, this model is compatible with the observe pump currents.

Ion-selective channels in K562 cells: a patch-clamp analysis.

Four types of ion-selective channels were found by the patch-clamp technique in the human erythroleukemia K562 cells. I) in cell-attached configuration at potentials less negative than -40 mV an 8 ps channel was detected. The potential dependence of channel activity suggests that this is the TTX-sensitive Na+ channel. II) A cation-selective channel was observed with equal permeability for Na+ and K+ and a potential-independent single-channel conductance of 19 pS. The channel is activated by intracellular Ca2+ and inhibited by TEA, III) A predominantly anion-selective channel was identified with the selectivity sequence NO3- > J- > Cl- = Br- >> SO4(2-). The single-channel conductance shows outward rectification, and is in symmetrical NaCl solution 19 pS at -60 mV and 54pS at +50 mV. The open- and closed-time distributions suggest one open and at least four closed states. At submicromolar concentrations, the open state is blocked by H2DIDS leading to channel flicker between open and blocked channel; higher concentrations (apparent KI = 6.8 uM) lead to a longer-lasting blocked state. Both components of inhibition are reversible. IV) In addition, an 8 pS, Na(+)- and K(+)- selective channel could be induced by application of palytoxin. For channel activity, the presence of extracellular Na+ is essential. It is assumed that the Na+, K(+)-pump molecule is involved in the channel formation. Similarly, it is discussed whether the anion-selective channel represents a pore conformation of an electrically silent anion exchanger.

Inward-directed current generated by the Na+,K+ pump in Na(+)- and K(+)-free medium.

In Na(+)- and K(+)-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardiac glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na+,K+ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H+] in the external medium. This current is not observed when Mg2+ instead of Ba2+ is the only divalent cation present in the bath medium, and it does not depend on whether Na+ or K+ is present internally. At 5 to 10 mM Na+ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na+]. It is suggested that in low-Na+ and K(+)-free medium the Na+,K+ pump molecule can either form a conductive pathway that is permeable to Ba2+ or protons or operate in its conventional transport mode accepting Ba2+ as a K+ congener. A reversed pump mode or an electrogenic uncoupled Na(+)-efflux mode is excluded.