Molecular Assembly of the Extracellular Domain of P2X2, an ATP-Gated Ion Channel

doi:10.1006/bbrc.1997.7713

Biochemical and Biophysical Research Communications

Volume 240, Issue 3, 26 November 1997, Pages 618-622

https://doi.org/10.1006/bbrc.1997.7713 Get rights and content

Abstract

We have produced the putative extracellular domain (ECD) of the ATP-gated ion channel, P2X₂, in a bacterial expression system. The hexahistidine-tagged protein was purified by immobilized metal affinity chromatography and refolded by sulfitolysis and dialysis. We demonstrate that P2X₂-ECD forms a stable tetramer in solution by gel filtration chromatography, dynamic light scattering and analytical sedimentation centrifugation. [α-³²P]ATP has been covalently crosslinked by UV irradiation to the P2X₂-ECD and this binding is specific and competable by antagonists suramin and cibacron blue. These results indicate that the binding affinity among P2X₂-ECD subunits is appreciably stronger than 3.4 μM (0.1 mg/ml), implying that the extracellular domain of P2X₂is primarly responsible for tetramerization of whole P2X₂and thus probably plays a role in determining homo- and heteromerization specificity of P2X channel subunits.

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We have learned various data on the role of purinoceptors (P2X4, P2X7, P2Y6 and P2Y12) expressed in spinal microglia and several factors that presumably activate microglia in neuropathic pain after peripheral nerve injury. Purinergic receptor-mediated spinal microglial functions make a critical contribution to pathologically enhanced pain processing in the dorsal horn. Microglial purinoceptors might be promising targets for treating neuropathic pain. A predicted therapeutic benefit of interfering with microglial purinergic receptors may be that normal pain sensitivity would be unaffected since expression or activity of most of these receptors are upregulated or enhanced predominantly in activated microglia in the spinal cord where damaged sensory fibers project.
A consensus segment in the M2 domain of the hP2X <inf>7</inf> receptor shows ion channel activity in planar lipid bilayers and in biological membranes
2012, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Recent X-ray crystallographic studies of P2X4R indicated trimeric functional organization [21]. When expressed heterologously, most P2X receptors (P2XR) isoforms form channels with distinct pharmacological and kinetic properties [15,22,23]. Their primary sequences contain between 379 (P2X1R) and 595 (P2X7R) amino acids, and a sequence alignment of the first 400 residues reveals more than 30% homology [9].
The P2X₇ receptor (P2X₇R) is an ATP-gated, cation-selective channel permeable to Na⁺, K⁺ and Ca²⁺. This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X₇R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X₇R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X₇R. The conserved part of the M2 domain of P2X₇R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.
Pain and purinergic signaling
2010, Brain Research Reviews
A growing body of evidence indicates that extracellular nucleotides play important roles in the regulation of neuronal and glial functions in the nervous system through P2 purinoceptors. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (seven types; P2X1–P2X7) contain intrinsic pores that open by binding with ATP, and P2Y receptors (eight types; P2Y1, 2, 4, 6, 11, 12, 13 and 14) are activated by nucleotides and couple to intracellular second-messenger systems through heterotrimeric G-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons in physiological and pathophysiological conditions. Studies have shown that microglia, a type of glial cells known as resident macrophages in the CNS, express several subtypes of P2X and P2Y receptors, and these receptors play a key role in pain signaling in the spinal cord under pathological conditions such as by peripheral nerve injury (called neuropathic pain). Within the spinal dorsal horn, peripheral nerve injury leads to a progressive series of changes in microglia including morphological hypertrophy of the cell body and proliferation, which are considered indicative of activation. These activated microglia upregulate expression of P2X/Y receptors (e.g., P2X4 and P2Y12). Importantly, pharmacological, molecular and genetic manipulations of the function or expression of these microglial molecules strongly suppress neuropathic pain. We expect that further investigation to determine how ATP signaling via P2X receptors participates in the pathogenesis of chronic pain will lead to a better understanding of the molecular mechanisms of pathological pain and provide clues for the development of new therapeutic drugs.
A truncated P2X<inf>7</inf> receptor variant (P2X<inf>7-j</inf>) endogenously expressed in cervical cancer cells antagonizes the full-length P2X<inf>7</inf> receptor through hetero-oligomerization
2006, Journal of Biological Chemistry
Citation Excerpt :
The molecular mechanism by which single P2X7 molecules oligomerize is unclear. The present results support the speculation that ectodomains of the P2X receptor are essential to carry out the oligomerization (47). However, our data do not support the speculation that residues in or near the second membrane-spanning segment are critical for multimerization of P2X receptors (50) because the P2X7-j could interact with the full-length P2X7 despite lacking the second transmembrane stretch.
A truncated naturally occurring variant of the human receptor P2X₇ was identified in cancer cervical cells. The novel protein (P2X_7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X₇ receptor. The P2X_7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X₇ receptor agonist benzoyl-ATP. The P2X_7-j interacted with the full-length P2X₇ in a manner suggesting heterooligomerization and blocked the P2X₇-mediated actions. Interestingly, P2X_7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but fulllength P2X₇ immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X₇ immunoreactivity suggesting lack of P2X₇ homo(tri)-oligomerization. These results identify a novel P2X₇ variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.
Chapter 9 P2X Receptors in Sensory Neurons
2006, Current Topics in Membranes
Citation Excerpt :
One functional P2X receptor channel is presumably formed by a number of P2X subunits, as is considered to be the case in other ligand‐gated ion channels. The number of subunits of a P2X receptor has been proposed to be three (Nicke et al., 1998; Stoop et al., 1999; Jiang et al., 2003; Nicke et al., 2003) or four (Kim et al., 1997; Ding and Sachs, 2000). It appears that all three or four subunits are either identical, namely homomeric receptors, or not identical, namely heteromeric receptors.
This chapter focuses on the developments of the mechanisms by which P2X receptors on sensory neurons participate in the pathogenesis of chronic pain. It also summarizes the accumulated evidence from immunohistochemical, electrophysiological, and behavioral studies. Adenosine triphospahte (ATP) might be an important player for tissue injury‐induced pain and pathological chronic pain by P2X receptors, expressed on peripheral and/or central terminals or on the cell body of sensory neurons. Efforts focusing on the distribution and electrophysiological properties of P2X receptors in sensory neurons reveal that P2X₃ and P2X_2/3 receptors might be the predominant functional P2X receptors. Activating P2X receptors at sensory nerve endings at both the periphery and the dorsal horn produces action potentials and an increase in sensory synaptic strength, respectively, both of which have important implications in pain generation and modulation. Electrophysiological studies using acutely dissociated and cultured primary sensory neurons from the dorsal root ganglia (DRG), trigeminal ganglia (TG), and nodose ganglia (NG) have shown that functional P2X receptors are expressed.
Purification and aqueous phase atomic force microscopic observation of recombinant P2X<inf>2</inf> receptor
2005, European Journal of Pharmacology
Recombinant P2X₂ receptor was observed by atomic force microscope in the aqueous phase. The P2X₂ receptor was expressed in an insect cell line, and recombinant proteins were prepared under native conditions. The membrane fractions were extracted, and histidine-tagged receptor protein was purified from the fractions by column chromatography. When the purified protein fraction was diluted with water and served for atomic force microscopy, dispersed particles of about 3 nm in height were observed. In the presence of 1 mM ATP, the assembly-like images of the particles were obtained. More densely assembled images of the particles were achieved when the protein was dissolved in a Tris buffer containing 1 mM ATP. Under this condition, imaging of the surface of the particles exhibited a circular structure with a diameter of about 10 nm having a pore-like structure. These results suggest that atomic force microscopy provides structural information about P2X₂ receptor in aqueous phase.

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Regular ArticleMolecular Assembly of the Extracellular Domain of P2X2, an ATP-Gated Ion Channel

Abstract

Pharmacol. Ther.

FEBS Lett.

Biochem. Biophy. Res. Comm.

Anal. Biochem.

Anal. Biochem.

Biophys. J.

Pharmacol. Rev.

Annu. Rev. Cell Dev. Biol.

Nature

Nature

Nature

Regular Article
Molecular Assembly of the Extracellular Domain of P2X₂, an ATP-Gated Ion Channel