User:Rjcam2/P2RX4

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The P2RX4 gene encodes for the P2X4 monomer, a subunit of the P2X receptor family. P2X receptors are protein trimers that can be homomeric or heteromeric. These receptors are The P2X₄ receptor is a ligand-gated ion channels that selectively open in response to ATP binding. Each receptor subtype, determined by the subunit composition, varies in its affinity to ATP and desensitization kinetics.

The P2X4 receptor is the homotrimer composed of three P2X4 monomers. They are nonselective cation channels with The P2X₄ receptor has high calcium permeability, leading to the depolarization of the cell membrane and the activation of various Ca²⁺-sensitive intracellular processes. The P2X4 receptor is uniquely expressed on lysosomal compartments as well as the cell surface.

The receptor is found in the central and peripheral nervous systems, in the epithelia of ducted glands and airways, in the smooth muscle of the bladder, gastrointestinal tract, uterus, and arteries, in uterine endometrium, and in fat cells. P2X₄ receptors have been implicated in the regulation of cardiac function, ATP-mediated cell death, synaptic strengthening, and activating of the inflammasome in response to injury.

Receptor structure and kinetics

Ribbon structures of (A) the P2X4 homotrimeric receptor and (B) its 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). Each subunit has two transmembrane alpha helices (TM1 and TM2) linked by a large extracellular loop. Analysis of x-ray crystallographic structures revealed a 'dolphin-like' tertiary structure, where the 'tail' is embedded in the phospholipid bilayer and the upper and lower ectodomains form the 'head' and 'body' respectively. Adjacent interfaces of the subunits form a deep binding pocket for ATP. ATP binding to these orthosteric sites causes a shift in conformation opening the channel pore.

The P2X₄ subunits can form homomeric or heteromeric receptors. The P2X₄ receptor has a typical P2X receptor structure. In 2009, The zebrafish P2X₄ receptor was the first purinergic receptor to be crystallized and have its three-dimensional structure solved, forming the model for the P2X receptor family was the closed state homomeric zebrafish P2X4 receptor. Although truncated at its N- and C- termini, this crystal structure resolved and confirmed that these proteins were indeed trimers with an ectodomain rich with disulfide bonds.

Continued binding leads to increased permeability to N-methyl-D-glucamine (NMDG⁺) in about 50% of the cells expressing the P2X₄ receptor. [Maybe create a section called "Selectivity". This statement would fit better there]

Each subunit varies in binding affinity with ATP and the kinetics of receptor desensitization.

The desensitization of P2X₄ receptors is intermediate when compared to P2X1 and P2X2 receptors.

Gating Mechanism

Schematic of the P2X4 receptor conformational states

P2X receptors have three confirmed conformational states: ATP-unbound closed, ATP-bound open, and ATP-bound desensitized. 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 form beta sheet structures from N- and C- termini of adjacent subunits. These newly folded secondary 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.

'Helical Recoil' Model of Desensitization

Electrophysiology studies have revealed differences in the rates of receptor desensitization between different P2X subtypes. 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. Mutational studies working with the rat P2X2 and P2X3 receptors have identified three residues in the N-terminus that majorly contribute to these differences. By changing the amino 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. In combination with the open state crystal structures, it was hypothesized that the cytoplasmic cap was stabilizing the open pore conformation.

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 a 3₁₀-helix. This helical structure disappears with desensitization and instead TM2 reforms as a complete alpha helix repositioned closer to the extracellular side.

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. 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.

In support of this theory, the P2X7 uniquely has a large cytoplasmic domain with palmitoylated C-cysteine anchor sites. 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.

Receptor trafficking[edit]

P2X4 receptors are functionally expressed on both the cell surface and in lysosomes.

P2X₄ receptors are stored in lysosomes and brought to the cell surface in response to extracellular signals. These signals include IFN-γ, CCL21, CCL2. Fibronectin is also involved in upregulation of P2X₄ receptors through interactions with integrins that lead to the activation of SRC-family kinase member, Lyn. Lyn then activates PI3K-AKT and MEK-ERK signaling pathways to stimulate receptor trafficking. Internalization of P2X₄ receptors is clathrin- and dynamin-dependent endocytosis.