P2 Receptors: P2X Ion Channel Family

Once known as P2 purinoreceptors, surface receptors for extracellular nucleotides are now called P2 receptors. This subtle change in nomenclature reflects the more varied nature of nucleotidic ligands, other than those containing a purine moiety, that are capable of activating these surface receptors. The current nomenclature system for P2 receptors is also based on their molecular structure and signal transduction mechanisms, so defining a family of ionotropic P2 receptors (P2X Ligand-Gated Ion Channels, LGICs) and another family of metabotropic P2 receptors (P2Y G Protein-Coupled Receptors, GPCRs).

The seven subunits forming P2X receptors show a common topology of i) intracellular N- and C-termini which possess consensus binding motifs for protein kinases, ii) two transmembrane spanning regions (TM1 and TM2) which line the ion pore, iii) a large extracellular loop, possibly folded into a β-sheet, with 10 conserved cysteine residues forming a series of disulphide bridges, iv) a hydrophobic H5 region close to the pore vestibule, for receptor/channel modulation by extracellular cations (e.g. magnesium, calcium, zinc, copper and hydrogen ions), and v) an ATP-binding site, which involves amino acid residues in the extracellular loop immediately adjacent to TM1 and TM2. The P2X_1-7 receptor subunits show 30-50% sequence identity at the peptide level. The stoichiometry of assembled P2X_1-7 receptors is thought to involve three subunits, arranged as a stretched trimer.

Triplets of identical P2X subunits form homomeric assemblies and produce the P2X_1-7 receptors. Additionally, some P2X receptors exist as heterotrimeric assemblies (e.g. P2X_1/2 in SCG neurons, P2X_2/3 in sensory ganglia, P2X_2/6 and P2X_4/6 in CNS neurons, P2X_1/5 in some blood vessels). Members of this extended P2X family of homomeric and heteromeric assemblies show many pharmacological and operational differences. For example, the potency orders for nucleotidic agonists vary significantly between P2X receptor subtypes. With blocking agents, some P2X subtypes (P2X₄ and P2X_4/6) are relatively insensitive to potent antagonists of other P2X receptor subtypes. Agonism and antagonism at some P2X receptors, notably P2X₂ and P2X_2/3, are affected by extracellular H⁺ ions. The kinetics of activation, inactivation and deactivation vary considerably among P2X receptor subtypes. Calcium permeability is high for some P2X receptor subtypes, amounting to some 6-10% of the ionic current carried. The P2X₇ receptor converts from an ion channel to a pore and, in some cases, this conversion brings about cell death. Other P2X receptors (P2X₂, P2X_2/3, P2X₄ and P2X_2/6) show reversible and time-dependent changes in the ion permeability properties of their intrinsic ion channel but here, this phenomenon is not as profound as seen with P2X₇.

The loci of the P2X_1-7 genes have been defined in the genome of human, rat and, importantly, mice. Some P2X receptors also have been identified from cDNA libraries of chimp, dog, guinea-pig, frog and zebrafish, as well from invertebrates such as Schistosoma mansoni. Notably, the zebrafish genome contains nine P2X genes of which two are paralogues for P2X₃ and P2X₄ receptors. All P2X genes show a complex organization, with the encoding sequences interrupted by up to 14 introns. Occasionally, errors occur in P2X gene transcription, which yield splice variants that are either dominant negative, modulatory or functionless. Using mice, gene deletion has been achieved for P2X₁, P2X₂, P2X₃ and P2X₇ receptors. None are lethal, but P2X₁-null homozygote males are infertile. Disruption of either P2X₂ or P2X₃ genes causes hyporeflexia, whereas knock-out of the P2X₇ gene has a profound effect on neuropathic pain.

The Tables below contains accepted modulators and additional information. For a list of additional products, see the Materials section below.

Footnotes

a) P2X₁ resembles the P2X receptor in smooth muscle (SM) cells.

b) P2X₂ resembles the P2X receptor in neurons (N).

c) P2X₇ is the cytolytic P2Z receptor.

d) Length of individual subunits in amino acid residues (aa). Functional channels require three subunits, and all subunits can form functional homotrimeric assemblies in expression systems (Xenopus oocytes, HEK293 cells, CHO cells). Endogenous P2X receptors also can exist as heterotrimeric assemblies comprising two or three different subunits.

e) P2X₆ subunits cannot efficiently form a homomultimeric receptor as these subunits are mostly retained in the ER, but they can readily form heteromultimeric ion channels with other subunits (e.g. P2X_2/6 and P2X_4/6).

f) There are no truly selective agonists for P2X receptors which are all activated by ATP, although each subtype can be distinguished by potency ratios for ATP and two or more nucleotide agonists. Agonist potency is enhanced under acidic conditions at P2X₂ receptors, but reduced at P2X_1,3,4,7 receptors.

g) There are no truly selective antagonists for P2X subtypes, except for P2X₇ which is blocked by KN-62 and hexamethylene amiloride (HMA). Human P2X₄ receptors are relatively insensitive to known P2 receptor antagonists, and rat P2X₄ even more so, although hP2X₄ can be blocked by BBG. Suramin is particularly effective at P2X₂ receptors under acidic conditions (pH 5.5).

h) KN-62, KN-04 and HMA are more potent at human than rat P2X₇ receptors, and vice versa for BBG.

i) P2X_1-7 receptors form intrinsic cation channels that are permeable to Na⁺, K⁺ and Ca²⁺ ions. The permeability properties of some P2X receptors change during prolonged activation. The P2X₇ converts from an ion channel to a pore that is permeable to large molecules (400-900 Da). P2X receptor subtypes can be distinguished by their desensitization rates.

j) [³⁵S]ATPγS should be used in the absence of divalent cations.

k) Loss-of-function polymorphism of P2X₇ receptors in macrophages abolishes ATP-mediated killing of Mycobacterium tuberculosis.

Abbreviations

A-317491: [3-Phenoxybenzyl-(1,2,3,4-tetrahydro-napthalene-1-yl)-carbamoyl]-benzene-1,2,4-tricarboxylic acid
Ap₄A: Diadenosine tetraphosphate
Ap₅A: Diadenosine pentaphosphate
ATP: Adenosine 5´-triphosphate
ATPγS: Adenosine 5´-O-(3-thiotriphosphate)
BBG: Brilliant blue G
BzATP: 3'-Benzoylbenzoyl adenosine 5´-triphosphate
CTP: Cytidine 5´-triphosphate
GTP: Guanosine 5´-triphosphate
HMA: Hexamethylene amiloride
HT-AMP: 2-Hexylthioadenosine 5´-monophosphate
Ip₅I: Diinosine pentaphosphate
isoPPADS: Pyridoxal-5-phosphate-6-azophenyl-2',5'-disulphonic acid
KN-62: 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine
2-MeSATP: 2-Methylthioadenosine 5'-triphosphate
α,β-MeATP: α,β-Methylene-adenosine-5'-triphosphate
D-β,γ-MeATP: D-β,γ-Methylene-adenosine-5'-triphosphate
L-β,γ-MeATP: L-β,γ-Methylene-adenosine-5'-triphosphate
MRS 2159: Pyridoxal-5-phosphate-6-azophenyl-4'-carboxylate
MRS 2500: (N)-Methanocarba-N6-methyl-2-iodo-2'-deoxyadenosine-3',5'-bisphosphate
NF023: 8,8'-(Carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-napththalenetrisulfonic acid)
NF279: 8,8'-(Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)bis(1,3,5-napththalenetrisulfonic acid)
NF449: 8,8'-(Carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))tetrakis-benzene-1,3-disulphonic acid)
PAPET-ATP: 2-[2-(4-Aminophenyl)ethylthio]adenosine 5´-triphosphate
Phenol red: Phenolsulfonphthalein sodium salt
Phenolphthalein: 3,3-bis[4-Hydroyphenol]-1(3H)-isofuranone
PPADS: Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid
PPNDS: Pyridoxal-5-phosphate-6-(2´-naphthylazo-6-nitro-4´,8´-disulphonate)
RB-2: Reactive blue 2
TNP-ATP: 2',3'-O-(2,4,6-Trinitrophenyl) adenosine triphosphate
UTP: Uridine 5´-triphosphate