TheP2X4 receptor is thehomotrimer composed of three P2X4 monomers.[5] They are nonselective cation channels with highcalcium permeability, leading to thedepolarization of the cell membrane and the activation of various Ca2+-sensitive intracellular processes.[9][10][11] The P2X4 receptor is uniquely expressed onlysosomal compartments as well as thecell surface.[12]
Ribbon structures of (A) the P2X4 homotrimeric receptor and (B) the subunit monomer
P2X receptors are composed of three subunits that can be homomeric or heteromeric by nature. In mammals, there are seven different subunits, each encoded in a different gene (P2RX1-P2RX7).[5] Each subunit has twotransmembranealpha helices (TM1 and TM2) linked by a large extracellular loop.[5][12][19] Analysis ofx-ray crystallographic structures revealed a 'dolphin-like'tertiary structure, where the 'tail' is embedded in thephospholipid bilayer and the upper and lowerectodomains form the 'head' and 'body' respectively.[12][19][20] Adjacent interfaces of the subunits form a deep binding pocket for ATP.[12][19] ATP binding to theseorthosteric sites causes a shift inconformation opening the channel pore.
The P2X4 subunits can form homomeric or heteromeric receptors.[21] In 2009, the first purinergic receptor crystallized was the closed state homomericzebrafish P2X4 receptor.[22][19] Although truncated at itsN- andC- termini, thiscrystal structure resolved and confirmed that these proteins were indeedtrimers with an ectodomain rich withdisulfide bonds.[5][12]
Schematic of the P2X4 receptor conformational states
P2X receptors have three confirmed conformational states: ATP-unbound closed, ATP-bound open, and ATP-bound desensitized.[12][19] Imaging of the human P2X3 and rat P2X7 receptors has revealed structural similarities and differences in their cytoplasmic domains. In the ATP-bound state, both receptor types formbeta sheet structures from N- and C- termini of adjacent subunits.[12][19] These newly foldedsecondary structures come together to form a 'cytoplasmic cap' that helps stabilize the open pore. Crystal structures of the desensitized receptor no longer exhibit the cytoplasmic cap.[12][19]
Electrophysiology studies have revealed differences in the rates of receptor desensitization between different P2X subtypes.[5][12]Homotrimers P2X1 and P2X3 are the fastest, with desensitization observed milliseconds after activation, while P2X2 and P2X4 receptors are on the timescale of seconds. Notably, the P2X7 receptor uniquely does not undergo desensitization.[12] Mutational studies working with the rat P2X2 and P2X3 receptors have identified threeresidues in the N-terminus that majorly contribute to these differences. By changing theamino acids in the P2X3 to match the analogous P2X2, the desensitization rate slowed down. Conversely, changing residues of P2X2 to match P2X3 increased the desensitization rate.[19] In combination with the open state crystal structures, it was hypothesized that the cytoplasmic cap was stabilizing the open pore conformation.[12][19]
Additionally, structural analysis of the open P2X3 receptor revealed transient changes in TM2, the transmembrane alpha helix lining the pore. While in the open state conformation, a small mid-region of TM2 develops into a310-helix.[12][19] This helical structure disappears with desensitization and instead TM2 reforms as a complete alpha helix repositioned closer to the extracellular side.[12]
The helical recoil model uses the observed structural changes in TM2 and the transient formation of the cytoplasmic cap to describe a possible mechanism for the desensitization of P2X receptors. In this model, it is theorized that the cytoplasmic cap fixes the intracellular end of the TM2 helix while stretching its extracellular end to allow ion influx.[19] This would induce the observed 310-helix. The cap then disassembles and releases its hold on TM2 causing the helix to recoil towards the outer leaflet of the membrane.[12][19]
In support of this theory, the P2X7 uniquely has a large cytoplasmic domain withpalmitoylatedC-cysteine anchor sites.[5][12][19] These sites further stabilize its cytoplasmic cap by anchoring the domain into the surrounding inner leaflet. Mutations of the associated palmitoylation site residues cause observed atypical desensitization of the receptor.[12]
P2X4 receptors are functionally expressed on both the cell surface and in lysosomes.[20] Although preferentially localized and stored inlysosomes, P2X4 receptors are brought to the cell surface in response to extracellular signals.[23] These signals includeIFN-γ,CCL21,CCL2.[24][25][26]Fibronectin is also involved in upregulation of P2X4 receptors through interactions withintegrins that lead to the activation ofSRC-family kinase member,Lyn.[27] Lyn then activatesPI3K-AKT andMEK-ERK signaling pathways to stimulate receptor trafficking.[28] Internalization of P2X4 receptors isclathrin- anddynamin-dependentendocytosis.[29]
The main pharmacological distinction between the members of thepurinoceptor family is the relative sensitivity to the antagonistssuramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). The product of this gene has the lowest sensitivity for these antagonists[8]
The P2X4 receptor has been linked toneuropathic pain mediated bymicrogliain vitro andin vivo.[30][31] P2X4 receptors are upregulated following injury.[32] This upregulation allows for increased activation ofp38 mitogen-activated protein kinases, thereby increasing the release of brain-derived neurotrophic factor (BDNF) from microglia.[33] BDNF released from microglia induces neuronal hyperexcitability through interaction with theTrkB receptor.[34] More importantly, recent work shows that P2X4 receptor activation is not only necessary for neuropathic pain, but it is also sufficient to cause neuropathic pain.[35]
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^Kawano A, Tsukimoto M, Noguchi T, Hotta N, Harada H, Takenouchi T, et al. (March 2012). "Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages".Biochemical and Biophysical Research Communications.419 (2):374–380.doi:10.1016/j.bbrc.2012.01.156.PMID22349510.
^Solini A, Santini E, Chimenti D, Chiozzi P, Pratesi F, Cuccato S, et al. (May 2007). "Multiple P2X receptors are involved in the modulation of apoptosis in human mesangial cells: evidence for a role of P2X4".American Journal of Physiology. Renal Physiology.292 (5):F1537 –F1547.doi:10.1152/ajprenal.00440.2006.hdl:11573/412000.PMID17264311.S2CID18668753.
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^Tsuda M, Tozaki-Saitoh H, Masuda T, Toyomitsu E, Tezuka T, Yamamoto T, Inoue K (January 2008). "Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after peripheral nerve injury".Glia.56 (1):50–58.doi:10.1002/glia.20591.PMID17918263.S2CID8834339.
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