Cavernous chambers, intricate passages, a gate with a curious lock — the structure of an ATP-activated ion channel reveals its architecture. And this intriguing interior design is found in another type of ion channel too.
Ion channels are membrane-protein complexes that allow the passage of ions into and out of cells. One type of ion channel, the P2X receptor channel1,2, is activated by extracellular ATP, the nucleotide more commonly known for providing cells with energy. Binding of ATP to the P2X receptor triggers opening of a transmembrane pore, allowing sodium, potassium and calcium ions3 to flow down their electrochemical gradients, changing membrane voltage or activating intracellular signalling cascades, mediating processes as diverse as pain perception and inflammation1,2. Elegant biochemical studies4 had suggested that P2X ion channels are trimers, distinguishing them from the better-studied tetrameric and pentameric ion-channel families. On page 592 of this issue, Kawate and colleagues5 confirm these predictions in their report of the first X-ray crystal structure of a type of P2X receptor, P2X4. In an accompanying paper (page 599), Gonzales and colleagues6 describe the structure of another trimeric ion channel — an acid-sensing ion channel (ASIC) — which reveals unanticipated similarities with P2X receptors.
For those of us interested in the function of ion channels, an X-ray structure is a game-changer, offering crucial information about operational mechanisms and providing a firm grounding for further investigations. Obtaining structures of membrane proteins is an arduous task, and P2X was no exception, requiring seven years to come to fruition. The structure shows that P2X's three dolphin-shaped subunits wrap around each other to form the trimer, and that two transmembrane α-helices (TM1 and TM2) from each subunit span the cell membrane (Fig. 1a). The structure was solved in the absence of ATP, and the region of the protein that lies within the membrane lacks a pathway large enough for ions to cross the membrane, suggesting that the structure represents a closed state.
The structure of the P2X receptor5 is fascinating for many reasons. First, it reveals insight about the region of the protein that forms the transmembrane ion-conducting pore. Previous work had suggested that amino-acid residues in the second transmembrane (TM2) segment influence the type of ion that can pass through the channel3 and that the three TM2 segments probably line the pore3,7. In the P2X receptor structure, the TM2 segments are steeply angled relative to the membrane plane, giving the pore an hourglass appearance consisting of chambers (vestibules) on either side of an extended plug of residues near the middle of the membrane (Fig. 1b). Residues in the region of the plug are inaccessible to ions on the outside of the membrane when the pore is closed, but are highly accessible in the open state7. This indicates that the plug functions as a gate that opens when ATP binds to the receptor. The extracellular vestibule above the gate opens to the outside through fenestrations (Fig. 1a; white asterisk), providing a passageway for ions to enter or exit the extracellular side of the pore — a feature that is reminiscent of the fenestrations seen in some potassium channels8,9 and in the acetylcholine receptor10.
Another striking feature5 is the presence of two outer electronegative (acidic) vestibules located within the extracellular domain (Fig. 1b). The proximity of the central vestibule to the pore, and its negative charge, make one wonder whether this central chamber attracts positively charged ions to the channel. Cavities of similar dimensions have been found in a range of proteins, in which they are proposed to facilitate movement between domains by decreasing the contact surface, or to serve as binding sites for cofactors or regulatory compounds11. Indeed, Kawate and colleagues5 found gadolinium (Gd3+), a heavy metal used to solve the structure, in the central vestibule of the P2X receptor, and showed that Gd3+ inhibits P2X function. If the outer two vestibules serve as binding sites for regulatory ions, how do such ions get into these chambers, and how do they affect the function of the ion channel? One possibility is that ions access the vestibules through a pathway along the P2X central axis that dilates when ATP binds.
So where does ATP bind to open the ion channel? Because the P2X structure was solved in the absence of ATP, the binding site isn't visible. But experiments in which amino acids were mutated12 implicate specific basic and other polar (hydrophilic) residues that line a pocket on the outer surface of the receptor at the interface between each subunit5 (Fig 1a). Although the structure of this site is unlike that of conventional ATP-binding motifs13, such motifs are notoriously diverse and the residues lining the P2X pocket are appropriate for ATP binding. It will be interesting to find out whether ATP binds to this region, to determine whether magnesium ions are required for ATP binding, as it is in many other proteins, and to compare the chemistry of ligand binding with that observed in other ATP-binding proteins. Answering these questions will be highly informative for designing new drugs targeting P2X receptors.
The P2X receptor is not the first trimeric ion- channel structure to be solved. A structure had been reported for an acid-sensing ion channel (ASIC)14, but the protein yielding this structure was non-functional and the transmembrane segments adopted a non-native conformation. The new ASIC structure reported by Gonzales and colleagues6 was solved from a functional protein, and so provides several novel details. Although P2X receptors and ASICs have unrelated amino-acid sequences and open in response to different ligands (ATP versus protons), their transmembrane regions are remarkably similar. The pore of the ASIC is closed by an extended plug that is similar to that in the P2X receptor. And although the extracellular domains of P2X receptors and ASICs are structurally unrelated, both have the acidic central and upper vestibules. By soaking ASIC crystals in different ions, the authors6 located an ion-binding site in the extracellular vestibule, providing a first glimpse of how trimeric channels might coordinate (and thereby select for) different permeating ions.
These two papers5,6 are wonderful examples of how protein structures help to frame a wealth of fascinating questions. How does ligand binding to the extracellular domains trigger opening of the pore, and how does the structure of the pore change during opening? What is the role of the two acidic vestibules in the extracellular domains? Do the similarities in the pore regions of the two channels mean that they gate using similar mechanisms, or are there simply limited ways to construct an ion-conducting pore from so few transmembrane helices? Answering these fundamental questions will undoubtedly keep nerds busy for years to come.
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