P2X receptors are ligand-gated cation channels for which ATP is the endogenous orthosteric agonist. Seven P2X subunits have been identified and they form trimers to produce at least twelve different receptor subtypes. The tertiary structure of several subtypes have been reported, but they all lack clear information on the conformation of the N- and C-terminal cytoplasmic domains because of the truncated constructs used and the flexibility of these domains. Now, McCarthy et al., (1) report single-particle cryo-EM images of the full-length rat P2X7 receptor in both apo (closed pore) and ATP-bound (open pore) states, which suggest why the affinity of this receptor for ATP is low, indicate how cysteine residues in the C-terminal control desensitisation and reveal a surprising guanine nucleotide binding site in the C-terminal.
The potency of ATP at P2X7 is three orders of magnitude lower than at other P2X subtypes. Whilst these new structures show some differences between the orthosteric ATP binding pocket of P2X7 and P2X3 receptors, these are unlikely to explain the great difference in ATP potency. Notably, however, the entrance to the pocket is much narrower in P2X7 (<11A° orifice) than in P2X3 (17A° orifice) receptors. This and any protein flexibility that opens and closes the entrance would decrease the time ATP spends in the binding pocket, so decreasing its affinity.
A unique feature of P2X7 receptors is an 18-amino acid long cytoplasmic region at the end of TM2 (named the C-cys anchor by the authors) that is cysteine-rich and which links TM2 to the cytoplasmic cap, a structural domain formed by N- and C-terminal residues that determines the rate at which P2X receptors desensitise. The present report shows that the C-cys anchor contains at least four cysteine residues and one serine residue that are palmitoylated and that the aliphatic chains extend into the plasma membrane, anchoring the receptor to the membrane. The authors speculate that this could keep the cytoplasmic cap in place and so limit P2X7 desensitisation. Consistent with this, the receptor, which is normally non-desensitising in the presence of ATP, desensitised rapidly and fully when the C-cys anchor was deleted or the cysteine residues removed by mutation.
A further unique feature of P2X7 receptors compared with other subtypes is the long C-terminal (~200 residues), which the authors term the cytoplasmic ballast. The images show for the first time that each receptor has three globular, wedge-shaped cytoplasmic ballasts, each of which hangs beneath the TM domain of an adjacent subunit. Intriguingly, each cytoplasmic ballast contains a dinuclear zinc ion complex and a high-affinity guanosine nucleotide binding site, the functions of which are unclear.
The P2X7 receptor is of particular therapeutic interest because it is cytotoxic due to its ability to activate the NLRP3 inflammasome and release pro-inflammatory cytokines. This study substantially extends our knowledge and understanding of its pharmacological and biophysical properties and forms the basis of further potential experiments designed to fully characterise how it functions.
Comments by Dr. Charles Kennedy, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde
(1) McCarthy et al. (2019). Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization. Cell. https://doi.org/10.1016/j.cell.2019.09.017. [ScienceDirect: View Article]
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