Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

Abstract

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 cationselective 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.NMR | membrane proteins | ligand-gated ion channels | insect cell expression system | purinergic receptors I n 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 channe...

Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel

“…8a, d), rather than those of the apo, closed hP2X3, zfP2X4, and pdP2X7 structures or the antagonist bound, closed hP2X3 and pdP2X7 structures (Supplementary Fig. 8b, c, e, f, h), consistent with the previous NMR analysis of the zfP2X4 receptor 27 . The transmembrane pore is formed by the TM2 helices with the residues from Asp319 to Leu344 lining the pore, consistent with the previous electrophysiological analysis (Fig.…”

“…6). These structural features are quite consistent with the previous electrophysiological analyses of the P2X1 and P2X4 receptors, in which the head domain is involved in the TNP-ATP recognition 34, 35 , and the NMR analysis of the P2X4 receptor, in which TNP-ATP binding induces the expansion of the extracellular domain, in a similar manner to that observed with ATP-dependent activation 27 . In contrast, in the structure of hP2X3 soaked with TNP-ATP, the overall conformation is essentially identical to the apo, closed state of hP2X3 (Fig.…”

“…The crystal structure of the hP2X3 receptor in complex with TNP-ATP adopted the same conformation as that in the apo, closed state 23 . In contrast, the NMR analysis of the zfP2X4 receptor reconstituted in nanodiscs showed that TNP-ATP binding induces the expansion of the extracellular domain, in a similar manner to that observed with ATP-dependent activation 27 . Therefore, the mechanism by which the competitive antagonist works at P2X receptors remains controversial.…”

X-ray structures define human P2X3 receptor gating cycle and antagonist action

“…There is also concern that the available structure of zfP2X 4 bound to ATP 27 may not represent a physiologic state because the truncated crystallization construct, lacking both terminal domains, might distort pore architecture 12,30–32 . A recent NMR study suggests that TNP-ATP inhibits activation by closing the extracellular fenestrations to ion access, rather than by stabilizing a closed-pore conformation 33 . To understand the molecular mechanisms underlying activation and antagonism of P2X receptors, we crystallized the human P2X 3 (hP2X 3 ) receptor in an apo/resting state, an agonist-bound/open-pore state, an agonist-bound/closed-pore/ desensitized state, and two competitive antagonist-bound states.…”

Isotope-labeling strategies for solution NMR studies of macromolecular assemblies

“…This approach may fail for complexes for which the individual subunits have poor solubility. The LEGO-NMR 78 New constructs and expression of proteins General scheme and example of segmental isotope-labeling. Schematic overview of (a) intein-based and (b) sortase based protein ligation.…”