Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X
4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X
4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s
−1), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.In chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (
1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (
2,
3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (
4,
5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (
4,
6).The P2X receptors are a family of cation channels gated by extracellular ATP (
1,
7–
9) and are involved in many physiological and pathophysiological processes (
10–
12). Seven subtypes of the P2X receptors have been identified in mammals (
13), and they share ∼40% sequence identity. The P2X
4 receptor is involved in the pathogenesis of chronic neuropathic, inflammatory pain and the endothelial cell-mediated control of vascular tone (
11,
14,
15). Compared with ATP, α,β-methylene ATP (α,β-meATP), in which the oxygen atom linking the α- and β-phosphorous atoms of ATP is replaced by a methylene group (), reportedly induces a lower maximum current in cells expressing the mouse, rat, and human P2X
4 receptors and other P2X receptors (
16,
17).
Open in a separate windowCharacterization of the P2X
4 receptor. (
A) Chemical structures of ATP and α,β-meATP. (
B and
C) TEVC recordings of ATP- and α,β-meATP-evoked currents from rat P2X
4 receptor expressed in
Xenopus oocytes, respectively. In
B, the currents were evoked twice by ATP (30 μM, 1 min, black bar). In
C, the currents were firstly evoked by ATP (30 μM, 1 min, black bar) and subsequently by α,β-meATP (300 μM, 1 min, black bar). (
D) TEVC recording of the ATP-evoked current (30 μM, 30 s, black bar) from the N-terminally EGFP-tagged ΔzfP2X
4–A′ construct expressed in
Xenopus oocytes. (
E) Size exclusion chromatogram of purified EGFP-tagged ΔzfP2X
4–A′ in rHDLs. Elution volumes corresponding to 17.0, 12.2, 10.4, and 7.1 nm Stokes diameters were determined by thyroglobulin, ferritin, catalase, and BSA, respectively. V
0 and 1CV are void volume and single column volume, respectively. (
F) SDS/PAGE analyses of purified ΔzfP2X
4–A′ embedded in rHDLs. The samples were analyzed by 12% SDS/PAGE with Coomassie Brilliant Blue staining. (
G) Measurement of [
3H]ATP saturation binding to the purified ΔzfP2X
4–A′ in rHDLs. (
H and
I) Estimation of the effects of deuteration based on the crystal structures of zfP2X
4 (PDB ID code 4DW1) and the deuteration incorporation rates. The plots on the
Left (without deuteration) and the
Right (with deuteration) are the sums of the inverse sixth power of the distances between pseudoatoms centered on the methyl hydrogens of M108, M249, M268, or M325 and each hydrogen atom in the crystal structure of zfP2X
4 (sums of the r
−6) and the sums of the r
−6 multiplied by [1 − (deuterium incorporation rates)] of each hydrogen atom, respectively. The graphs in
H and
I were calculated from the crystal structure in the apo state (PDB ID code 4DW0) and that in the ATP-bound state (PDB ID code 4DW1), respectively. Sums of the r
−6 of each methionine methyl group and Hαβγ of the intraresidue methionine (green), Hαβγ of the interresidue methionine (light green), Hαβ of tyrosine (light violet), Hδεζη of tryptophan (orange), Hαβδεζ of phenylalanine (pink), Hαβγ of valine (blue), Hαβγδ of leucine (light blue), Hαβγδ of isoleucine (cyan), Hαβγ of threonine (light cyan), Hαβ of alanine (red), Hαβγδ of arginine (dark blue), Hα of glycine (dark green), and Hαβ of serine (magenta) residues, and the other hydrogens connected to carbon atoms (other unexchangeable hydrogens, light gray) are shown with colors. Hydrogen atoms connected to nitrogen, oxygen, or sulfur atoms were not considered in these calculations because these hydrogens should be exchanged with deuterium in D
2O. The deuterium incorporation rates of the hydrogen atoms within each methionine residue (intraresidue) and the deuterium incorporation rates of other methionine residues (interresidue) were set to 98% and 85%, respectively, because the methionine residues would be derived from 85% of [α-, β-, γ-98%
2H-, methyl-
13C]-methionine and 15% of nonlabeled methionine in the medium.The crystal structures of zebrafish P2X
4 receptor (zfP2X
4) (
18,
19), together with mutational analyses (
20–
26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X
4 forms a homotrimer (
27,
28), in which the transmembrane region of each subunit is composed of two helices (
19). In the crystal structure of zfP2X
4 in the ATP-bound state, three ATP molecules are bound to the intersubunit nucleotide binding pockets. In addition, the region that connects the ATP-binding site and the transmembrane region, which is referred to as the “lower body” (), is expanded by ∼10 Å in the ATP-bound state, and a pore is formed in the transmembrane region, which is proposed to expand by the iris-like movement of the transmembrane helices (
18). However, the mechanism underlying the partial activation of P2X receptors is unknown because the structures of the P2X receptors have not been examined in the partial agonist-bound states.
Open in a separate windowNMR resonances from the endogenous methionine residues of zfP2X
4 in rHDL. (
A and
B) Distribution of the methionine residues in the ΔzfP2X
4–A′. One subunit from the crystal structure of zfP2X
4 in the apo form (
A) (PDB ID code 4DW0) and one from the ATP-bound form (
B) (PDB ID code 4DW1) are shown in ribbons. The lower body and the right flipper are yellow. The A330 residues, the methionine residues, and the residues in which methionine mutations were introduced, L339 and L351, are depicted by green sticks. ATP is depicted by red sticks. Dummy atoms generated by Orientations of Proteins in Membranes (OPM), which represent membrane boundary planes, are gray. (
C) Overlaid
1H-
13C HMQC spectra of [
2H-11AA, α, β-
2H, methyl-
13C-Met]ΔzfP2X
4-A′, embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). The regions with resonances from methionine residues are shown, and the assigned resonances are indicated. The centers of the resonances are indicated with dots. Cross-sections at lines through the centers of each resonance in the ATP-bound state and the cross-sections of the spectra using [α, β-
2H, methyl-
13C-Met]ΔzfP2X
4-A′ are shown on the top of the overlaid spectra. The intensities of the cross-sections were normalized by the concentration of ΔzfP2X
4-A′ and the conditions of the NMR measurements.The P2X
4 receptor used in the previous crystallographic studies was solubilized by detergents, which are widely used for structural investigations of membrane proteins, but the P2X
4 receptor is embedded in lipid bilayers under physiological conditions. It was recently reported that reconstituted high-density lipoproteins (rHDLs), which are also known as nanodiscs (
29), can accommodate membrane proteins within a 10-nm-diameter disk-shaped lipid bilayer (
30). The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (
31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (
32–
34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (
32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.Here, we used NMR to observe the conformational equilibrium of the alanine and methionine residues of zfP2X
4 bound to α,β-meATP in rHDLs. Based on the conformational equilibrium, we discuss the mechanism underlying the partial activation of P2X receptors.
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