Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Nucleotide Receptor P2x

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_50

Historical Background

The first evidence of purinergic signaling was described in 1929, when purines were found to underlie physiological responses in the circulatory and digestive system. After 50 years and a wealth of data supporting purine mediated effects in different systems, Burnstock presented the first direct evidence that ATP acts as a transmitter and introduced the concept of purinergic neurotransmission (Burnstock et al. 2010). Thus, ATP was recognized as both an intracellular energy source and an extracellular signaling molecule. Extracellular ATP has been implicated in intercellular communication in a wide variety of cells from different organisms and associated with a diverse array of biological effects. ATP is an ideal molecule for extracellular signaling, it is small, rapidly diffusing, highly unstable due to the presence of extracellular degrading enzymes and not abundant in the extracellular environment at resting conditions (Soto et al. 1997). ATP exerts it actions by binding to cell surface receptors called P2 receptors. P2 receptors are subsequently divided into two different families: P2Y receptors are metabotropic G-protein-coupled receptors while P2X receptors are ligand-gated ion channels (North 2002). Binding of ATP to P2X receptors opens within milliseconds an integral ion channel. While P2X receptors are functional homologues to the Cys-loop and glutamate receptor families of ligand-gated ion channels, they form a structurally distinct group of membrane receptors. Purinergic signaling via P2X receptors has a remarkably wide range of action, influencing epithelia and endocrine cell secretion, immune and inflammatory processes, cardiovascular performance, skeletal and smooth muscle contraction, and glial and neuronal function (Surprenant and North 2009). This chapter is devoted to the molecular and functional properties of P2X receptors and their involvement in physiological and pathological processes.

Molecular Properties of P2X Subunits

The first two P2X subunits (P2X1 and P2X2) were isolated from rat vas deferens smooth muscle and from PC12 cells by expression cloning. Based on sequence similarity, five additional subunits were identified in the rat and shortly after in human and mouse tissues (North 2002). The sequence identity between subunits (approx. 30–50%) and the lack of similarity to other cloned ligand-gated ion channels indicated they constitute a new family of membrane receptors. P2X subunits have been isolated and characterized from additional vertebrate classes (e.g., aves) and are present in all vertebrate species. They have also been found in fish, protozoa, trematode, fungi, and algae. In contrast, no P2X subunits have been identified in the genomes of the nematode worm (Caenorhabditis elegans), the fruit fly (Drosophila melanogaster), or in prokaryotes (Fountain and Burnstock 2009). Mammalian P2X receptor subunits are 379–595 amino acids long. Multiple splice variants of the originally cloned P2X subunits have been described, showing different amino acid lengths and properties. It was predicted using hydrophobicity plots that each subunit has two transmembrane domains linked by an extracellular loop comprising between 50% and 70% of the total protein length (Fig. 1). This extracellular loop contains 10 cysteine residues conserved in all cloned vertebrate P2X subunits. Both N- and C- terminus were suggested to be intracellular, with the length of the C-terminal domain being the main source of structure variation between the different subunits. The predicted membrane topology has been confirmed using multiple approaches, including mutating the extracellular domain, use of extracellular antibodies, concatemers and chimeric constructs (North 2002). The proposed membrane topology differs from that of the members of Cys-loop receptors superfamily and glutamate ionotropic receptors family but closely resembles that of the degenerins/ENaC/ASIC family (Fig. 1) (North 2006). P2X receptors are trimeric combinations of P2X subunits. Questions about the membrane topology of P2X subunits as well as the quaternary structure of P2X receptors were recently and definitely answered by X-ray crystallography (Kawate et al. 2009). The authors solved the crystal structure of zebrafish P2X4 receptor in its closed stated, as a symmetrical assembly of three P2X subunits surrounding a central ionic channel pore. This study confirms the proposed membrane structure and will further the understanding about the protein domains involved in P2X receptor function.
Nucleotide Receptor P2x, Fig. 1

Ligand-gated receptor membrane topology and stoichiometry. The membrane topology of a single subunit belonging to the three main families of ligand-gated ion channels is shown. The place of agonist interaction for the three types of receptors is shown in purple. In the lower part of the figure, the arrangement of subunits around the channel pore to form a functional receptor is depicted. TM transmembrane domain. The membrane topology and stoichoimetry is shared between the P2X receptor family and the degenerins/ENaC/ASIC family

Genomic Organization and Splicing

The chromosomal localization (obtained from the Ensembl database, www.ensembl.org) of the seven P2X subunit genes is summarized in Table 1. Several P2X genes are localized in the same human chromosome. Thus, P2X4 and P2X7 genes are located in the long arm of chromosome 12. Similarly, P2X1 and P2X5 genes are located within 1 Mb in the short arm of chromosome 17. A co-localization of paralog genes in chromosomes could arise from tandem duplication. However, it might also indicate the formation of a gene cluster, in which the expression of functionally related genes is co-regulated (Makino and McLysaght 2008). This could be the case for P2X subunits since co-expression of P2X4 and P2X7 has been detected in many different tissues and cell types, including microglia, vascular endothelium, ciliated epithelium, and the immune system. Moreover, P2X1 and P2X5 subunits co-express and heteromerize to form the P2X receptor in astrocytes (Surprenant and North 2009).
Nucleotide Receptor P2x, Table 1

Chromosomal location and length of human P2X subunit genes

Gene

Chromosomal location

Gene length (Kb)

P2RX1

17p13.2

20.1

P2RX2

12q24.3

3.7

P2RX3

11q12.1

31.6

P2XR4

12q24.3

24.2

P2XR5

17p13.2

23.9

P2XR6

22q11.2

13.8

P2XR7

12q24.3

53.2

Data obtained from the Emsembl database at http://uswest.ensembl.org/index.html

The number of exons comprising the sequence of P2X subunits varies between 11 for P2X2 and 15 for P2X7 (Cheewatrakoolpong et al. 2005, Nicke et al. 2009), while the remaining P2X genes contain 12 identified exons. Most exon–intron borders are conserved between the different genes, indicating, as expected, a common evolutionary origin. In contrast, intronic length varies largely between genes, as reflected by the differences in gene length listed in Table 1. Splice variants have been identified for all P2X subunits. Many of the isolated splice variants lack transmembrane domains or part of the extracellular domain giving rise to truncated P2X subunits that are not able to assemble in functional receptors. In addition, several subunits present a shorter version of their C-terminal domain due to the use of cryptic splice sites inside exon sequences or of inclusion of intron sequences in the corresponding DNA (Cheewatrakoolpong et al. 2005, Koshimizu and Tsujimoto 2006). The importance of this splicing in the receptor function will be described in the next section in more detail.

Regulation of P2X Receptors via Their C-terminal Domain

The C-terminal domain of P2X subunits is the least conserved part of the protein both in length and amino acid composition (North 2002), indicating that it might confer subunit specific properties. Moreover, the intracellular location of the domain makes it a candidate for interaction with intracellular adaptor and cytoskeletal proteins. Indeed, it has been shown that the P2X2 C-terminus interacts with the adaptor protein Fe65, a cytosolic protein containing several protein-binding domains (Fig. 2). P2X subunits and Fe65 colocalize in brain synapses and Fe65 has been shown to modify the functional properties of P2X2 receptors upon co-expression in heterologous systems (Masin et al. 2006). A splice variant of P2X2 with a 69 amino acid deletion in the C-terminal domain, assemble into a membrane receptor with different kinetic properties, and it does not interact with Fe65 (Masin et al. 2006).
Nucleotide Receptor P2x, Fig. 2

Carboxy terminus of P2X subunits. Representation of the functional and interacting domains present in the C-termini of P2X2, P2X4, and P2X7 subunits. The surface stabilization sequence YXXK present in all P2X subunits C-terminus proximal domain is represented only for P2X4

A role for the cytosolic C-terminal domain of the P2X4 subunit in trafficking of the receptor in and out of the plasma membrane has been described. Thus, a tyrosine-based non-canonical sorting signal in the C-terminal tail of P2X4 subunits (YXXGL, Fig. 2) that directly binds to the AP2 adaptor protein has been identified. When the interaction was disrupted by mutations in the sorting signal, an increase in the surface expression of P2X4 receptors in transfected neurons was observed (Murrell-Lagnado and Qureshi 2008). In addition, another tyrosine-containing domain (YXXXK) present in the proximal C-terminus of all P2X subunits was shown to be involved in membrane stabilization of P2X receptors (Murrell-Lagnado and Qureshi 2008).

Arguably, the best studied C-terminal domain is that of P2X7 receptors. In the immune system, P2X7 receptors mediate ATP-induced inflammatory responses by activating caspase-1 and the subsequent release of interleukins. Macrophages of a mouse line in which the C-terminal domain of P2X7 has been deleted by knock-in methods lack the immune response elicited by ATP. Moreover, efficient caspase-1 activation by ATP requires priming by bacterial membrane lipopolysaccharides (LPS) and an LPS binding domain has been identified in the distal C-terminal sequence (Surprenant and North 2009) (Fig. 2). P2X7 receptors have been shown to interact with many intracellular proteins, including pannexin-1, protein tyrosin-phosphatase β, heat shock, and epithelial membrane proteins (Surprenant and North 2009); however, the domains mediating the interaction are not known. In addition to playing a role in protein interaction and intracellular signaling, the C-terminal domain is modulating the channel properties of P2X7 receptors. Thus, the increases in permeability to big cations or fluorescent dyes of P2X7 receptors upon repetitive application of ATP also depend on the C-terminal domain (North 2002).

Pharmacology, Tissue Expression, and Physiological Roles of P2X Receptors

In considering the pharmacology and P2X receptors (P2XR), a number of considerations are needed. First, P2X receptors are ligand-gated ion channels, and as such serve as both cell-surface receptors and ion channels. The natural agonist is extracellular ATP or adenine nucleotides. A number of nucleotide and non-nucleotide antagonists have been developed with selectivity at each of the seven P2X receptors. Most of the agonists are relatively nonselective while the antagonists with a high degree of selectivity have been developed. Second, the endogenous or native P2XR may be homomeric or heteromeric. Each P2X channel is a subunit of the trimeric channel. Each P2X receptor is capable of complexing with itself to form a homotrimeric channel or with another P2X receptor as a heterotrimer. A total of seven heterotrimers have been demonstrated. P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6, P2X4/7. Pharmacology of heterologously expressed P2XR subtype in oocytes or HEK293 cells is often different from that of P2XR in native tissues. Third, agonist and antagonist selectivity is different at homomeric vs. heteromeric receptors. Individual P2X subunit of a heterotrimeric P2XR retains some of its selective characteristic response to agonist, antagonist, extracellular zinc, pH, or desensitization kinetics. Fourth, heteromers can exhibit unique pharmacology of agonist and/or antagonist actions or other properties that are different from those for homomeric receptors. In some heteromers, the individual characteristic properties specific to one of its subunit can become dominant and make the heteromer more like the homomeric assembly of the “dominant” subunit. Table 2 summarizes the EC50s of known agonists and IC50 of antagonists at both homomeric and heteromeric receptors (Ralevic and burnstock 1998; Gever et al. 2006; North 2002; Jarvis and Khakh 2009; Roberts et al. 2006; Burnstock and Knight 2004). Table 3 summarizes the tissue expression pattern of both homomeric and heteromeric P2X receptors (Ralevic and burnstock 1998; Gever et al. 2006; North 2002; Burnstock and Knight 2004; Surprenant and North 2009). Each homomeric or heteromeric receptor will be discussed in the following sections.
Nucleotide Receptor P2x, Table 2

Agonists and antagonists at P2X receptors

Agonists

P2X1

P2X2

 

P2X3

P2X4

P2X5

P2X6

P2X7

P2X1/2

P2X1/4

P2X1/5

P2X2/3

P2X2/6

P2X4/6

P2X4/7

ATP

≤1

2–10

 

≤1

7–10

5–10

10

≥100

0.5–0.6

10

1–5

0.7–2

30

6

>300

2-meSATP

≤1

≤2

 

≤1

10

10

9

100

0.07

 

1

1

35

7

 

α, β-meSATP

≤1

>300

 

≤1

>300

>300

>100

>300

0.1

10

3

5

>100

12

 

BZ-ATP

0.003

0.8

 

0.08

7

>500

 

20

0.003

  

0.8

  

9

Antagonists

               

Suramin

1–5

1–10

 

3–5

>500, 180a

4

>100

>300

   

10

   

PPADS

1

1–2

 

1

>100, 28a

3

>100

3–4

       

TNP-ATP

0.001–0.006

≥1

 

0.001

15

>10

 

>30

 

0.5

0.4

0.007

  

1

NF 449

0.0003, 0.0005a

>50

 

2

>100

  

>100, 40a

  

0.0007

0.12

   

NF 023

0.2

>50

 

30

>100

          

NF 279

0.02, 0.05a

0.8–1

 

2

>300

  

10–20, 3a

       

RO 0437626

3

>100

 

>100

       

>100

   

NF 110

0.08

4

 

0.01

>300

          

RO −3

>10

>10

 

0.01–0.1

>10

>10

 

>10

   

1–2

>10

  

A 317491

>10

>100

 

0.1

>100

 

>100

>100

   

0.1

   

5 -BDBD

    

0.5

          

A 740003

>100

>100

 

>100

>100

 

>100

0.02–0.05

       

A 438079

>100

>100

 

>100

>100

 

>100

0.06–0.07

       

A 804598

>100

>100

 

>100

>100

 

>100

0.01

       

GSK314181A

>10

>10

 

>10

>10

  

0.1

       

AZ11645373

>10

>10

 

>10

>10

>10

 

0.1

       

IP5I

0.003

>300

 

3–7

Potentiation

      

3

   

KN −62

       

0.3

       

Value of EC50 (agonists) or EC50 (antagonists) are μM. Most of the data are taken from Antonio et al. (2009), Aschrafi et al. (2004), Ase et al. (2010), Burnstock and Knight (2004), Burnstock et al. (2010). Additional data are taken from Donnelly-Roberts et al. (2008), Evans et al. (1995), Rettinger et al. (2000), Klapperstück et al. (2000), Rettinger et al. (2005), Hechler et al. (2005), Hülsmann et al. (2003), Ford et al. (2006), Honore et al. (2006), Donnelly-Roberts et al. (2009), King (2007), Sneddon et al. (2000), Soto et al. (1999), Hausmann et al. (2006), Kassack et al. (2004), King et al. (2004), Jaime-Figueroa et al. (2005)

ATP adenosine 5′-triphosphate, 2meSATP 2-methythioadenosine 5′-triphosphate, α,β-meATP 2-methythioadenosine 50-triphosphate, BzATP 2,3-O-(4-benzoylbenzoyl)-ATP, PPADS pyridoxal-5′-phosphate-6-azophenyl-20 0,40 0-disulphonic acid, TNP-ATP 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate, IP5I diinosine pentaphosphate, KN-62 1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, A-317491 5-({(3-phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino}carbonyl)-1,2,4-benzenetricarboxylic acid, RO-3 5-(2-isopropyl-4,5-dimethoxybenzyl)pyrimidine-2,4-diamine, A-740003 N-[1-(N0 0-cyano-N′-quinolin-5-ylcarbamimidamido)-2,2-dimethylpropyl]-2-(3,4-dimethoxyphenyl)acetamide, A-438079 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine, A-804598 2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine, MRS2179 20 0-deoxy-N6-methyl adenosine 3′ 0,5′ 0diphosphate, NF279 8,80-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(naphthalene)-1,3,5-trisulfonic acid, NF449 4-[({3-[({3,5-bis[(2,4-disulfophenyl)carbamoyl]phenyl}carbamoyl)amino]-5-[(2,4-disulfophenyl)carbamoyl]phenyl}carbonyl)amino]benzene-1,3-disulfonic acid, GSK314181A 5-{[(3R)-3-aminopyrrolidin-1-yl]methyl}-2-chloro-N-(tricyclo[3.3.1.13,7]dec-1-ylmethyl)benzamide, AZ11645373 3-(1-(30-nitrobiphenyl-4-yloxy)-4-(pyridine-4-yl)butan-2-yl)thiazolidine-2,4-dione, Cibacron Blue 1-amino-4-(4-(4-chloro-6-(2-sulfophenylamino)-1,3,5-triazin-2-ylamino)-3-sulfophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid.

aHuman P2XR

Nucleotide Receptor P2x, Table 3

Tissue distribution

 

P2X1

P2X2

P2X3

P2X4

P2X5

P2X6

P2X7

P2X1/2

P2X1/4

P2X1/5

P2X2/3

P2X2/4

P2X2/6

P2X4/7

Skeletal muscle

+

  

+

+

+

       

Cardical muscle

+

+

+

+

+

        

Smooth muscle

              

Urinary bladder

+

+

 

+

+

+

+

       

Gut

+

+

    

+

       

Blood vessels

+

+

+

+

+

 

+

 

+

     

vas Deferens

+

+

 

+

  

+

       

Ureter

+

+

  

+

+

        

Overy

 

+

            

Epithelial cell

              

Nasal mucosa

 

+

  

+

         

Gut

 

+

  

+

 

+

       

Bladder

  

+

 

+

+

+

       

Ureter

   

+

+

+

+

       

Skin

    

+

 

+

       

Bronchial

     

+

        

Salivary gland

   

+

          

Overy

+

+

   

+

        

Thymus

 

+

+

  

+

+

       

Blood vessels

+

+

+

+

+

 

+

       

Organs

              

Lung

+

 

+

+

         

Trachea

   

+

  

+

       

Spleen

+

            

Liver

 

 

+

          

Kidney

+

+

+

+

+

+

+

       

Thymus

+

  

+

 

+

+

       

Testis (Leydig)

 

+

+

+

  

+

+

   

+

  

Salivary gland

   

+

  

+

       

Adrenal gland

 

+

+

+

+

+

+

       

Pancrease

   

+

          

Uterus

     

+

        

CNS

              

Brain

+

+

+

+

+

+

+

  

+

    

Spinal cord

+

+

+

+

 

+

+

       

Cortical astrocytes

+

   

+

    

+

    

Neuron ganglion

              

Sensory

+

+

+

+

 

+

+

   

+

+

  

Dorsal root

+

+

+

+

 

+

     

+

  

Trigeminal

+

+

+

+

+

+

        

Coeliac

+

+

+

+

 

+

+

       

Autonomic

+

+

+

+

+

+

+

  

+

   

Esophagus myenteric

              

Enteric sensory neurones

            

+

 

Otic neurones

 

+

+

       

+

   

Blood cells

              

Plateletes

+

             

Lymphocytes

+

+

 

+

  

+

       

Granulocytes

      

+

       

Monocytes

      

+

       

Macrophage

      

+

      

+

Neutrophils

+

  

+

+

 

+

       

+ Detected

– Not present

Homomeric P2X1R

P2X1 mRNA and protein are detected in a fairly broad range of tissues, such as urinary bladder, smooth muscle of small arteries and vas deferens, brain, spinal cord and several neuron ganglions, and platelets. Low levels of P2X1 are also found in lungs, spleen, and heart. Notably, P2X1R is the significant P2X subtypes in smooth muscle of blood vessels and other hollow organs including bladder, intestine, and vas deferens. Studies demonstrated that ATP or α,β-meATP could elicite an inward current and membrane depolarization, induce the contraction in a variety of smooth muscle tissues. These effects of that ATP or α,β-meATP in smooth muscle are eliminated or reduced in P2X1R knockout (KO) mice, confirming the significant role of P2X1R in the regulation of native smooth muscle contractility (Ralevic and burnstock 1998; Gever et al. 2006).

P2X1R can also regulate platelet functions as the main ATP-gated ion channel in platelets and megakaryocytes. The endogenous platelet P2XR has a similar pharmacology as that of the heterologously expressed recombinant P2X1R. In P2X1R KO mice, functions of platelets, such as aggregation, secretion, adhesion, and thrombus formation, are impaired. Vascular disease superimposed on P2X1R KO mice showed reduced mortality in the presence of systemic thromoembolism and laser-induced vessel injury. On the other hand, hypersensitive ex vivo platelet response and increased mortality in vivo secondary to increased thromboembolism were observed in transgenic mice overexpressing human P2X1R in megakaryocytic cell line (Gever et al. 2006).

Homomeric P2X2R

P2X2R is expressed throughout the central and peripheral nervous (CNS) systems as well as other non-neuron cell types, such as in bladder, adrenal medulla, endothelial and epithelial cells, skeletal, cardiac and smooth muscles, lymphocytes, intestine, and vas deferens (see Table 3). In the heart, P2X2R mRNA was detected in smooth muscle cells of coronary artery and only in atrium myocardium. Homemeric P2X2R appears to play a significant role in ATP-mediated fast synaptic transmission at both nerve terminals and interneuronal synapses. Thus, P2X2R is likely involved in memory, learning, motor function, autonomic coordination, and sensory integration in CNS as well as afferent and efferent signal pathway in peripheral nervous system (PNS). P2X2R is also expressed in many other non-neuron tissues in which its function is still not clear, although it may have a role in autocrine/paracrine hormone release, exocytosis/endocytosis, smoother muscle contractility, and pacemaker activity.

Heteromeric P2X1/2R

Phenotypically, heteromeric P2X1/2R is identical to homomeric P2X1R except the different sensitivity to pH changes. Furthermore, Aschrafi et al. found that assembly of heteromeric P2X1//P2X2R is favored over the respective homomeric P2X1R. It suggested that ATP-stimulated currents originally attributed to homomeric P2X1R in native tissue may actually be mediated by heteromeric P2X1/2R (Aschrafi et al. 2004). Using pharmacology profile and specific P2X subunit gene knockout mice, Calvert JA et al. confirmed existence of functional heteromeric P2X1/2R in sympathetic neurons from the superior cervical ganglion and it is implied that heteromeric P2X1/2R may have a broad role in regulation of the neuron function. Although the dominant phenotype of endogenous P2XR in sympathetic neurons is P2X2-like, a subpopulation of neurons showed P2X1 property such as α,β-meATP responsiveness that was reduced in P2X1R KO mice. P2X2-like means slow desensitization, sensitivity to blockade by antagonists, potentiation by acidic pH (this property is unique to P2X2 receptor) and extracellular zinc, and partial inhibition by high extracellular calcium. It is of interest that the α,β-meATP responsiveness was abrogated by high extracellular calcium and alkaline pH; the latter two properties are more characteristic of P2X2R. Thus, a presumed heteromeric P2X1/2 native receptor includes properties of both subunits (Calvert and Evans 2004).

Homomeric P2X3R

Homomeric P2X3R has restricted distribution and is only expressed in peripheral terminals of unmyelinated C-fiber and thinly myelinated afferent neurons, such as trigeminal and dorsal root ganglions. These receptors are mainly expressed on nociceptive sensory neurons. P2X3R can be activated by both α,β-meATP and 2-methylthioATP and are sensitive to blockade by suramin, PPADS, and TNP-ATP and is selectively antagonized by NF023. The functional role of homomeric P2X3R in these neurons is to mediate the sensory neurotransmission.

Heteromeric P2X2/3R

These heteromeric receptors have a mixed property that incorporates that of both P2X2 and P2X3 receptors. Thus, they can be activated by α,β-meATP and high sensitivity by TNP-ATP (P2X3-like), and show slow desensitization and potentiation by acidic pH (P2X2-like). A feature unique to this heteromer is that diinosine pentaphosphate is a much more potent blocker at homomeric P2X3 than at heteromeric P2X2/3R. This feature is useful in characterizing whether any of the native tissue P2XR is a P2X2/3R heteromer. These heteromers are expressed in subpopulations of sensory neurons, sympathetic ganglion cells, and brain neurons. They are thought to be important in initiating sensory signaling in pathways for taste, chemocreception, visceral distension, and neuropathic pain (Ralevic and Burnstock 1998). Heteromeric P2X2/3R is the first heteromeric channel to be studied following gene knockout confirmed the role of heteromeric P2X2/3R in several sensory signaling (Roberts et al. 2006).

Homomeric P2X4R

Homomeric P2X4R is not activated by α,β-meATP but is responsive to ATP and 2-methylthioATP. Of all the P2X receptors, it is the only receptor that can be potentiated by ivermectin via allosteric enhancement. Another unique feature is its lack of sensitivity to blockade by suramin or PPADS (North 2002). It has a desensitization kinetics intermediate between that for P2X1 and P2X2 receptors. It may be the most widely distributed of all the P2X receptors with expression in brain and spinal cord, automatic and sensory ganglions, arterial smooth muscle, osteoclasts, parotid acinar cells, kidney, lung, heart, bladder, thymus, colon, pancreas, and B lymphocytes (Gever et al. 2006; Burnstock and Knight 2004). However, the functional roles in some of these tissues are not clearly defined yet. However, recent studies showed that P2X4R are expressed in microglial cells and their activation can mediate neuropathic pain. P2X4R are also expressed in the endothelium in which activation can mediate nitric oxide-mediated vasorelaxation/vasodilatation. Global P2X4R KO mice showed hypertension and smaller arteries. Recent studies have also implicated P2X4 receptor as an important subunit of the endogenous cardiac myocyte P2X receptors. Increased expression of cardiac P2X4R by cardiac myocyte-specific transgenic overexpression or by stimulation with hydrolysis-resistant P2X agonist can confer a protected phenotype in models of both ischemic and non-ischemic heart failure (Zhou et al. 2010). Expression of a P2X4-like contractile phenotype in human atrial myocardium was recently described. P2X4R may also interact with P2X7 receptors in inflammatory responses such as pain signaling (see P2X7 receptors).

Heteromeric P2X1/4R

Both the P2X1R and P2X4R are expressed in the smooth muscle in renal resistance arteries. α,β -meATP evoked a spike-like membrane depolarization followed by a sustained depolarization which could be partially blocked by nanomolar P2X1 selective antagonist, NF279. The residual current could further be blocked by millimolar NF279, consistent with the existence of heteromeric P2X1/4R. Thus, heteromeric P2X1/4R showed both agonist and antagonist pharmacology that are more P2X1-like. TNP-ATP can block this heteromer with affinity that is higher than but closer to that for the homomeric P2X1 receptor. The P2X1/4R heteromer participates in the sympathetic control and paracrine regulation of renal blood flow (Harhun et al. 2010).

Homomeric P2X5R

Homomeric P2X5R is expressed in brain, spinal cord, heart, and eye. These homomeric receptors have pharmacology and desensitization kinetics similar to those of P2X2 receptors. The current mediated by rat P2X5R has a smaller amplitude than that induced by P2X1, P2X2, P2X3, or P2X4 receptors. P2X5 channels have a uniquely high chloride conductance. Recently, high levels of homomeric P2X5R are found in differentiating tissues, such as skeletal muscle, epithelial cells of nasal mucosa, gut, bladder, utter, and skin. Activation of homomeric P2X5R inhibits proliferation while increases the differentiation of rat skeletal muscle satellite cells. Homomeric P2X5R may be also involved in the regulation of proliferation and differentiation of certain type of cancer cells in skin and prostate.

Heteromeric P2X1/5R

P2X1R and P2X5R can assemble into a heteromeric P2X1/5R. A defining property of this heteromer is its activation by α,β-meATP, which cannot activate P2X5R. Other characteristic properties of this heteromer is a greater sensitivity to activation by ATP, a biphasic response to ATP with a transient peak current followed by a sustained plateau current, and a sensitivity to TNP-ATP intermediate between the sensitive homomeric P2X1 and the insensitive homomeric P2X5 receptors. Further, this heteromer does not appear to dilate to a larger pore on prolonged ATP exposure. Although the physiological role of heteromeric P2X1/5R is not defined, it has been postulated that it may mediate excitatory junction potentials at arterial neuroeffector junctions in guinea pig (Gever et al. 2006; North 2002). Recently, Ase et al. identified the heteromeric P2X1/5R in astrocytes from mouse brain and implied that it might participate in the astroglial Ca2+ signaling and excitability (Ase et al. 2010).

Homomeric P2X6R

P2X6R is expressed mainly in CNS. However, P2X6R is the only P2X receptor that usually does not form the functional homomeric P2X6R without extensive glycosylation. When such functional homomeric P2X6R are formed (after glycosylation), the heteromer has significantly higher responsiveness to α,β-meATP. Since P2X4R and P2X2R are the other two P2X subunits that usually coexist with P2X6R, the physiological role of the P2X6R is mediated either by the heteromeric P2X2/6R or heteromeric P2X4/6R (Gever et al. 2006; North 2002).

Heteromeric P2X2/6R

The P2X2R and P2X6R can be co-immunoprecipitated after co-expression in HEK293 cells or oocytes. The P2X2/6R heteromer showed a hybrid sensitivity to blockade by suramin at pH 6.5. There was a bi-phasic inhibition with a high sensitivity P2X2-like component and a lower sensitivity portion that is more P2X6-like. In general, the pharmacology of P2X2/6R heteromer is similar to that of P2X2 receptors (much less like that of homomeric P2X6 receptors). A subtle difference is that the P2X2/6R heteromer has a greater sensitivity to pH and to α,β-meATP. This heteromer is expressed by respiratory neurons in the brain stem (Gever et al. 2006; North 2002).

Heteromeric P2X4/6R

P2X4 receptors can also form a heteromer with P2X6 receptors. The pharmacology of this heteromer is similar to that of the homomeric P2X4 receptor. The heteromer, like the homomeric P2X4 receptor, is relatively insensitive to blockade by PPADS or suramin or reactive blue-2. The heteromeric P2X4/6R is similar to homomeric P2X4 receptors in its potentiation by the P2X4-specific allosteric enhancer and by zinc. In fact, the P2X4/6R heteromer appears more sensitive to the potentiation effect of ivermectin. The incorporation of P2X6 receptor in this heteromer also modestly increases the sensitivity to 2-meSATP and α,β-meATP(Gever et al. 2006; North 2002). On tissue expression and function of P2X2/6R and P2X4/6R heteromers, Antonio et al. confirmed the presence of the heteromeric P2X2/6R and P2X4/6R in mouse Leydig cells using immunofluorescence and pharmacologic profiles. Both heteromers are involved in regulating testosterone secretion (Antonio et al. 2009). Heteromeric P2X2/6R and P2X4/6R are also found in rat dorsal root ganglion neuron where they contribute to the transmission of nociceptive message, especially under inflammatory condition (De Roo et al. 2003).

Homomeric P2X7R

Homomeric P2X7R is mainly localized on glia and immune cells such as mast cells, macrophages lymphocytes, erythrocytes, and erythroleumemia. With prolonged exposure to high extracellular ATP concentrations, the homomeric P2X7R becomes open to larger size molecules such as ethidium and YO-PRO-1, leading to cell death. At low ATP concentration, the receptor is a cation channel like the other P2X receptor. Activation of homomeric P2X7R has been associated with the processing and releasing of active interleukin-1β and interleukin-18 from immune cells and glia. Selective P2X7R antagonist KN-62 (known to be selective at human P2X7R) could block the release of interleukin-1 β in macrophages and microglia. This function of P2X7R is further confirmed in experiment in P2X7R KO mice, which fail to release interleukin-1 β when challenged by ATP or BzATP. P2X7R also has a role in mediating the release of cytokine, reactive oxygen species, and neurotransmitter in microglia and astrocytes. Other roles implicated include apoptosis around β-amyloid plaques in Alzheimer’s disease model and neurodegeneration and cell death in models of spine cord injury or cerebral ischemia (Gever et al. 2006; North 2002).

Heteromeric P2X4/7R

Homomeric P2X7R is structurally similar to other P2XR except its significantly longer intracellular C-terminal. Originally, P2X7R was thought not able to heteropolymorize with any other P2XR. However, Guo et al. reported a functional heteromeric P2X4/7R in macrophages, representing the latest example of heteromeric P2X receptors (Guo et al. 2007). The two P2X receptors can be co-immunoprecipitated in detergent extracts from co-transfected HEK293 cells and from murine macrophages that express endogenous P2X4 and P2X7 receptors. A mutant P2X4R lacks ATP-gated channel activity but is capable of trafficking to the plasma membrane. When this mutant P2X4R (Named S341W) was co-expressed with P2X7R, ivermectin was able to potentiate the ATP-induced current in cells co-expressing these receptors. Since ivermectin could not potentiate homomeric P2X7R -mediated current, the potentiation by ivermectin of the co-expressed S341W P2X4R and P2X7R is most likely due to allosteric enhancement of the heteromer via the S341W P2X4 receptor. The overlapping expression of P2X4R and P2X7R have been identified in a number of tissues including some non-excitatory cells such as epithelial cells from salivary glands, exocrine pancreas, airway, leukocytes, microglial cells, and osteoclasts. The possible presence of heteromeric P2X4/7R in these tissues or cells raises the question that heteromeric P2X4/7R may mediate important physiological function(s) in these tissues (Dubyak 2007).

Future Directions

Further systematic examination of the effect of each agonist and antagonist will be needed at heteromeric P2X receptors. This can be performed in cells co-expressing individual P2X subunits using heterologous systems such as HEK293 cells or oocytes. Features including pharmacology, desensitization kinetics, and sensitivity to pH, extracellular calcium, or zinc will all need to be determined. Once characterized, these features should be compared to those obtained in the native tissues to ascertain whether they are similar. Similarity of features would support the particular heteromer as the native tissue receptor. Another possibility is that a heteromer may comprise three different P2X receptor subtypes. If true, this possibility will add further complexity.

Summary

P2X receptors are a family of ligand-gated ion channels. The natural agonist ligands are ATP. There are seven P2X receptors (P2X1–7) and each receptor is a subunit of the trimeric channel. Each subunit can complex with itself to form a homotrimeric channel or with other subunits to form a heterotrimeric channel. Evidence points to existence of both homo- and hetero-trimeric channels as endogenous channels in the tissue. In the heterotrimeric channel, molecular and pharmacological properties of the constituent subunit can both dominate or become masked. Evidence is accumulating to indicate potentially important biological and pathophysiolgical roles of these receptor channels. It is expected that many of these homo- or heterotrimeric channels will become novel therapeutic targets for various diseases. Much more work is needed.

Notes

Acknowledgment

F.S. would like to thank Marianela Masin for help with the Figures.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Jian-Bing Shen
    • 1
  • Bruce T. Liang
    • 1
  • Florentina Soto
    • 2
  1. 1.Calhoun Cardiovascular CenterUniversity of Connecticut Health CenterFarmingtonUSA
  2. 2.Department of Ophthalmology and Visual SciencesWashington University in St. LouisSt. LouisUSA