Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors.

Cholesterol dynamically regulates P2X7 receptor function in both physiological and pathological conditions. Studies suggest that cholesterol suppresses P2X7 receptor activity through direct binding or through indirect effects on the biophysical properties of the membrane. Notably, the palmitoylated C-terminus seems to counteract the action of cholesterol to make it less inhibitory. However, the mechanism underlying cholesterol-dependent regulation of P2X7 receptor remains unclear. Here we describe detailed methods that facilitate the quantification of P2X7 channel activity while controlling the amount of cholesterol in the system. We will first describe the use of methyl-ß-cyclodextrin (MCD), a cyclic oligosaccharide consisting of seven glucose monomers, to decrease or increase plasma membrane cholesterol levels. We will then describe protocols for the reconstitution of purified P2X7 in proteoliposomes of defined lipid composition. These methods can be combined with commonly used techniques such as dye-uptake assays or electrophysiology. We also describe a fluorescence assay to measure cholesterol-binding to P2X7. These approaches are complementary to cryo-EM or molecular dynamics simulations, which are also powerful tools for investigating cholesterol-P2X7 interactions. An improved understanding of the mechanisms of action of cholesterol on P2X7 may contribute to elucidate the roles of this receptor in ageing, inflammation, and cancer, whose progression correlates with the level of cholesterol.

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.

A challenge finding P2X1 and P2X4 ligands.

Identifying novel small-molecule P2X1 and P2X4 ligands with sub-type specificity and high-affinity remains a pharmacological challenge. Here we use computational methods, electrophysiology and fluorescent microplate assays to screen for ligand candidates acting at these receptors. Modelling and docking identified 80 compounds for testing at P2X4 receptors, and 20 of these showed >50% inhibition in fluorescence-based assays, making them appealing for further SAR studies. Confirmation of activity by two-electrode voltage clamp, followed by their elaboration resulted in only minor improvements in potency, with the highest IC50 being 295 µM. Testing on P2X1 receptors, resulted in a series of biguanide compounds that yielded a maximum IC50 of 100 µM, but no consistent SAR could be found. Potencies of established antagonists gave expected results, although the measured potencies varied between techniques and no antagonism could be found for compounds such as paroxetine, carbamazepine, 9(10H)-acridanone, acridinol and phenoxazine-type heterocycles. This study highlights the challenge of identifying P2X4 and P2X1 ligands and suggests that a combination of complimentary approaches is needed if we are to be confident of ligand activities at these receptors.

P2X4 and lysosome fusion.

Similar to other members of the P2X receptor family, the P2X4 receptor at the plasma membrane forms a highly Ca2+ permeable, non-selective cation channel that is activated by extracellular ATP. Yet, P2X4 differs from the other subtypes, as it is predominantly localized on late endosomal, lysosomal and/or lysosome-related organelles. It is targeted there by virtue of tyrosine-based and di-leucine like trafficking motifs contained within its C-terminal and N-terminal regions respectively. The physiological role of the stable intracellular expression of P2X4 in acidic compartments has been a long-standing puzzle. Recent evidence, however, points to a dual role in the regulation of ion fluxes across lysosomal membranes to control lysosome membrane fusion and in the re-sensitization of receptors exposed to extracellular ATP.

Regulation of P2X Purinergic Receptor Signaling by Cholesterol.

P2X receptors are cation-selective channels that are activated by the binding of extracellular ATP. They have a high permeability to Ca2+, Na+, and K+ and are expressed widely throughout the nervous, immune, cardiovascular, skeletal, gastrointestinal, respiratory, and endocrine systems. Seven mammalian subtypes of P2X receptor subunits have been identified, P2X1-7, and those that function as homotrimeric receptors (P2X1, 2, 3, 4, and 7) are targeted to lipid rafts, although they show limited resistance to solubilization by Triton X-100. Recent crystal structures of P2X3 and P2X4 receptors have provided considerable high-resolution information about the architecture of this family of receptors and yet the molecular details of how they are regulated by cholesterol are unknown. Currents mediated by the P2X1-4 receptors are either inhibited or relatively insensitive to cholesterol depletion, but there is no clear evidence to support the direct binding of cholesterol to these receptors. In contrast, the activity of the low-affinity, proinflammatory P2X7 receptor is potentiated by cholesterol depletion and regions within the proximal C-terminus play an important role in coupling changes in cholesterol to the gating of the pore. Based upon our understanding of the lipid signaling events that are triggered downstream of P2X7 receptor activation, a change in the levels of cholesterol may contribute to the sensitization of receptor currents and the dilation of the pore that occurs following prolonged, high-level stimulation. This chapter focuses on the regulation of P2X7 receptor signaling by cholesterol and our current understanding of the mechanisms that underlie this.

Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1.

Sigma1 receptors (σ1Rs) are expressed widely; they bind diverse ligands, including psychotropic drugs and steroids, regulate many ion channels, and are implicated in cancer and addiction. It is not known how σ1Rs exert such varied effects. We demonstrate that σ1Rs inhibit store-operated Ca(2+)entry (SOCE), a major Ca(2+)influx pathway, and reduce the Ca(2+)content of the intracellular stores. SOCE was inhibited by expression of σ1R or an agonist of σ1R and enhanced by loss of σ1R or an antagonist. Within the endoplasmic reticulum (ER), σ1R associated with STIM1, the ER Ca(2+)sensor that regulates SOCE. This interaction was modulated by σ1R ligands. After depletion of Ca(2+)stores, σ1R accompanied STIM1 to ER-plasma membrane (PM) junctions where STIM1 stimulated opening of the Ca(2+)channel, Orai1. The association of STIM1 with σ1R slowed the recruitment of STIM1 to ER-PM junctions and reduced binding of STIM1 to PM Orai1. We conclude that σ1R attenuates STIM1 coupling to Orai1 and thereby inhibits SOCE.

A fluorescent approach for identifying P2X1 ligands.

There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 µM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization.

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-ß-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na(+) and N-methyl-D-glucamine (NMDG(+)) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-ß-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry.

Depletion of Ca(2+) from the endoplasmic reticulum (ER) lumen triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca(2+) store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.

The trafficking and targeting of P2X receptors.

The functional expression of P2X receptors at the plasma membrane is dependent on their trafficking along secretory and endocytic pathways. There are seven P2X receptor subunits, and these differ in their subcellular distributions because they have very different trafficking properties. Some are retained within the endoplasmic reticulum (ER), while others are predominantly at the cell surface or within endosomes and lysosomes. Changes in recruitment of receptors to and from the plasma membrane provides a way of rapidly up- or down-regulating the cellular response to adenosine triphosphate (ATP). An additional layer of regulation is the targeting of these receptors within the membranes of each compartment, which affects their stability, function and the nature of the effector proteins with which they form signaling complexes. The trafficking and targeting of P2X receptors is regulated by their interactions with other proteins and with lipids and we can expect this to vary in a cell-type specific manner and in response to changes in the environment giving rise to differences in receptor activity and function.

Splice variants of the P2X7 receptor reveal differential agonist dependence and functional coupling with pannexin-1.

P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.

The Kv7.2/Kv7.3 heterotetramer assembles with a random subunit arrangement.

Voltage-gated K(+) channels composed of Kv7.2 and Kv7.3 are the predominant contributors to the M-current, which plays a key role in controlling neuronal activity. Various lines of evidence have indicated that Kv7.2 and Kv7.3 form a heteromeric channel. However, the subunit stoichiometry and arrangement within this putative heteromer are so far unknown. Here, we have addressed this question using atomic force microscopy imaging of complexes between isolated Kv7.2/Kv7.3 channels and antibodies to epitope tags on the two subunits, Myc on Kv7.2 and HA on Kv7.3. Initially, tsA 201 cells were transiently transfected with equal amounts of cDNA for the two subunits. The heteromer was isolated through binding of either tag to immunoaffinity beads and then decorated with antibodies to the other tag. In both cases, the distribution of angles between pairs of bound antibodies had two peaks, at around 90° and around 180°, and in both cases the 90° peak was about double the size of the 180° peak. These results indicate that the Kv7.2/Kv7.3 heteromer generated by cells expressing approximately equal amounts of the two subunits assembles as a tetramer with a predominantly 2:2 subunit stoichiometry and with a random subunit arrangement. When the DNA ratio for the two subunits was varied, copurification experiments indicated that the subunit stoichiometry was variable and not fixed at 2:2. Hence, there are no constraints on either the subunit stoichiometry or the subunit arrangement.

A pore residue of the KCNQ3 potassium M-channel subunit controls surface expression.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons.

A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 knock-out mice.

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-D-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7(-/-) mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7(-/-) strains has important implications for our understanding of the role of this receptor in health and disease.

Assembly and trafficking of P2X purinergic receptors (Review).

P2X receptors are cation selective ion channels gated by the binding of extracellular ATP. Seven subtypes have been identified and they have widespread and overlapping distributions throughout the body. They form homo- and heterotrimeric complexes that differ in their functional properties and subcellular localization. They form part of larger signalling complexes, interacting with unrelated ion channels and other membrane and cytosolic proteins. Up- or down-regulation of their expression is associated with several disease states. This review aims to summarize recent work on the assembly and trafficking of this family of receptors.

Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis.

The P2X(4) receptor has a widespread distribution in the central nervous system and the periphery, and plays an important role in the function of immune cells and the vascular system. Its upregulation in microglia contributes to neuropathic pain following nerve injury. The mechanisms involved in its regulation are not well understood, although we have previously shown that it is constitutively retrieved from the plasma membrane and resides predominantly within intracellular compartments. Here, we show that the endogenous P2X(4) receptors in cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages are localized predominantly to lysosomes. Lysosomal targeting was mediated through a dileucine-type motif within the N-terminus, together with a previously characterized tyrosine-based endocytic motif within the C-terminus. P2X(4) receptors remained stable within the proteolytic environment of the lysosome and resisted degradation by virtue of their N-linked glycans. Stimulation of phagocytosis triggered the accumulation of P2X(4) receptors at the phagosome membrane. Stimulating lysosome exocytosis, either by incubating with the Ca(2+) ionophore ionomycin, for normal rat kidney (NRK) cells and cultured rat microglia, or the weak base methylamine, for peritoneal macrophages, caused an upregulation of both P2X(4) receptors and the lysosomal protein LAMP-1 at the cell surface. Lysosome exocytosis in macrophages potentiated ATP-evoked P2X(4) receptor currents across the plasma membrane. Taken together, our data suggest that the P2X(4) receptor retains its function within the degradative environment of the lysosome and can subsequently traffic out of lysosomes to upregulate its exposure at the cell surface and phagosome.

Evidence for functional P2X4/P2X7 heteromeric receptors.

The cytolytic ionotropic ATP receptor P2X7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X4, they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X4 and P2X7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X4 and P2X7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X7 although there was no change in overall expression. Coexpression of a dominant-negative P2X4 mutant (C353W) with P2X7, inhibited P2X7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X4S341W with P2X7 produced a current that was potentiated by ivermectin and inhibited by 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X4 and P2X7, which suggests that they associate to form heteromeric receptors.

The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels.

Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing both P2X(2) and P2X(6) subunits. We transfected tsA 201 cells with both P2X(2) and P2X(6) subunits, bearing different epitope tags. We manipulated the transfection conditions so that either P2X(2) or P2X(6) was the predominant subunit expressed. By atomic force microscopy imaging of isolated receptors decorated with antiepitope antibodies, we demonstrate that when expression of the P2X(2) subunit predominates, the receptors contain primarily 2 x P2X(2) subunits and 1 x P2X(6) subunit. In contrast, when the P2X(6) subunit predominates, the subunit stoichiometry of the receptors is reversed. Our results show that the composition of P2X receptor heteromers is plastic and dependent on the relative subunit expression levels. We suggest that this property of receptor assembly might introduce an additional layer of subtlety into P2X receptor signaling.

An uncharged region within the N terminus of the P2X6 receptor inhibits its assembly and exit from the endoplasmic reticulum.

ATP-gated P2X receptors are trimeric complexes formed by the homomeric or heteromeric assembly of seven different subunits. We have shown previously that, unlike all of the other P2X subunits, the P2X6 subunit cannot form homomeric receptors and when expressed alone is retained in the endoplasmic reticulum (ER) in monomeric form (J Biol Chem 280: 107591-10765, 2005). However, other studies have shown that P2X6 can form functional heteromeric receptors with P2X2 and P2X4 subunits. In this study, we used a combination of immunocytochemistry, surface biotinylation, and atomic force microscopy to investigate the assembly and trafficking of the P2X6 subunit, both alone and as part of a heteromer. We show that as a heteromer, it exits the ER and is either stably expressed at the cell surface or constitutively internalized, depending on its partner. Through the use of targeted mutation, we demonstrate that an uncharged region at the N terminus of P2X6 exerts an inhibitory effect on its assembly and export from the ER. When this region is removed, or when charge is added to it, P2X6 forms homotrimeric assemblies, undergoes complex glycosylation and is delivered to the plasma membrane, albeit less efficiently than the P2X2 receptor. The N-terminal mutants were, however, nonfunctional. Substituting the uncharged 14-amino acid N-terminal region for the equivalent region of P2X2 increased ER retention but was not sufficient to prevent the formation of functional homomeric receptors. We propose that the N terminus of the P2X6 subunit contributes to a mechanism that prevents the inappropriate export and plasma membrane expression of nonfunctional P2X receptors.