Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X(2) receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys(69) and Lys(71)). We substituted cysteines in this region (Asp(57) to Asp(71)) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile(67) contribute to the ATP-binding site.     

Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor

The first hydrophobic segment of the rat P2X(2) receptor extends from residue Leu(29) to Val(51). In the rat P2X(2) receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp(15) through Thr(60)) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 microM). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as alphabeta-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val(48) at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.     

Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating

P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed. We have used cysteine-scanning mutagenesis to determine the contribution of residues Glu(52)-Gly(96) to human P2X1 receptor properties. ATP potency was reduced for the mutants K68C, K70C, and F92C. The efficacy of the partial agonist BzATP was also reduced for several mutants forming the back of the proposed agonist binding site. Molecular docking in silico of both ATP and BzATP provided models of the agonist binding site consistent with these data. Individual cysteine mutants had no effect or slightly increased antagonism by suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate. Mutants at the entrance to and lining the upper vestibule were unaffected by cysteine-reactive methanethiosulfonate (MTS) reagents, suggesting that it does not contribute to ionic permeation. Mutants that were sensitive to modification by MTS reagents were predominantly found either around the proposed ATP binding pocket or on the strands connecting the binding pocket to the transmembrane region and lining the central vestibule. In particular, ATP sensitivity and currents were increased by a positively charged MTS reagent at the G60C mutant at the interface between the central and extracellular vestibule. This suggests that dilation of the base of the central vestibule contributes to gating of the receptor.     

Identification of residues involved in neurotensin binding and modeling of the agonist binding site in neurotensin receptor 1

The neurotensin receptor 1 (NTR1) subtype belongs to the family of G protein-coupled receptors and mediates most of the known effects of the neuropeptide including modulation of central dopaminergic transmission. This suggested that nonpeptide agonist mimetics acting at the NTR1 might be helpful in the treatment of Parkinson's disease and schizophrenia. Here, we attempted to define the molecular interactions between neurotensin-(8-13), the pharmacophore of neurotensin, and the rat NTR1. Mutagenesis of the NTR1 identified residues that interact with neurotensin. Structure-activity studies with neurotensin-(8-13) analogs identified the peptide residues that interact with the mutated amino acids in the receptor. By taking these data into account, computer-assisted modeling techniques were used to build a tridimensional model of the neurotensin-(8-13)-binding site in which the N-terminal tetrapeptide of neurotensin-(8-13) fits in the third extracellular loop and the C-terminal dipeptide binds to residues at the junction between the extracellular and transmembrane domains of the receptor. Interestingly, the agonist binding site lies on top of the previously described NTR1-binding site for the nonpeptide neurotensin antagonist SR 48692. Our data provide a basis for understanding at the molecular level the agonist and antagonist binding modes and may help design nonpeptide agonist mimetics of the NTR1.     

Identification of amino acid residues contributing to the pore of a P2X receptor.

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

Direct identification of the agonist binding site in the human brain cholecystokininB receptor.

In investigating the agonist binding site of the human brain cholecystokininB receptor (CCKBR), we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfated cholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed by purification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, and with high affinity (KD = 1.2 nM) to the CCKBR and has full agonistic activity. As the starting material for receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCKBR. Covalent labeling of the WGA-lectin-enriched receptor revealed a 70-80 kDa glycoprotein with a protein core of about 50 kDa. Identification of the agonist binding site was achieved by the application of subsequent chemical and enzymatical cleavage to the purified receptor. A radiolabeled peptide was identified by Edman degradation amino acid sequence analysis combined with MALDI-TOF mass spectrometry. The position of the radioactive probe within the identified peptide was determined using combined tandem electrospray mass spectrometry and peptide mapping. The probe was covalently attached within the sequence L52ELAIRITLY61 that represents the transition between the N-terminal domain and predicted transmembrane domain 1. Using this interaction as a constraint to orientate the ligand within the putative receptor binding site, a model of the CCK-8s-occupied CCKBR was constructed. The hormone was found to be placed in a binding pocket built from both extracellular and transmembrane domains of CCKBR with its N-terminus mainly interacting with residues Arg57 and Tyr61.     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.     

Probing the effects of residues located outside the agonist binding site on drug-receptor selectivity in the nicotinic receptor

The nicotinic acetylcholine receptors (nAChRs) are a family of closely related but pharmacologically distinct neurotransmitter-gated ion channels. They are therapeutic targets for a wide range of neurological disorders, and a key issue in drug development is selective targeting among the more than 20 subtypes of nAChRs that are known. The present work evaluates a proposed hydrogen bonding interaction involving a residue known as the "loop B glycine" that distinguishes receptors that are highly responsive to ACh and nicotine from those that are much less so. We have performed structure-function studies on the loop B site, including unnatural amino acid mutagenesis, in three different nAChR subtypes and found that the correlation between agonist potency and this residue is strong. Low potency receptor subtypes have a glycine at this key site, and mutation to a residue with a side chain converts a low potency receptor to a high potency receptor. Innately high potency receptors have a lysine at the loop B site and show a decrease in potency for the reverse mutation (i.e., introducing a glycine). This residue lies outside of the agonist binding site, and studies of other residues at the agonist binding site show that the details of how changes at the loop B glycine site impact agonist potency vary for differing receptor subtypes. This suggests a model in which the loop B residue influences the global shape of the agonist binding site rather than modulating any specific interaction.     

Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase

Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.     

An intersubunit zinc binding site in rat P2X2 receptors

P2X receptors are ATP-gated ion channels made up of three similar or identical subunits. It is unknown whether ligand binding is intersubunit or intrasubunit, either for agonists or for allosteric modulators. Zinc binds to rat P2X2 receptors and acts as an allosteric modulator, potentiating channel opening. To probe the location of this zinc binding site, P2X2 receptors bearing mutations of the histidines at positions 120 and 213 were expressed in Xenopus oocytes. Studies of H120C and H213C mutants produced five lines of evidence consistent with the hypothesis that the residues in these positions bind zinc. Mixing of subunits containing the H120A or H213A mutation generated receptors that showed zinc potentiation, even though neither of these mutant receptors showed zinc potentiation on its own. Furthermore, expression of trimeric concatamers with His --> Ala mutations at some but not all six positions showed that zinc potentiation correlated with the number of intersubunit histidine pairs. These results indicate that zinc potentiation requires an interaction across a subunit interface. Expression of the H120C/H213C double mutant resulted in the formation of ectopic disulfide bonds that could be detected by changes in the physiological properties of the receptors after treatment with reducing and oxidizing agents. Immunoblot analysis of H120C/H213C protein separated under nonreducing conditions demonstrated that the ectopic bonds were between adjacent subunits. Taken together, these data indicate that His120 and His213 sit close to each other across the interface between subunits and are likely to be key components of the zinc binding site in P2X2 receptors.     

Identification of a tubulin binding motif on the P2X2 receptor

To isolate proteins interacting with P2X receptors, GST fusion proteins containing the intracellular C terminal tail of P2X(2), P2X(5), or P2X(7) were used as bait to screen detergent extracts of rat brain synaptosomes. By SDS-PAGE combined with mass spectrometry, two interacting proteins were identified: betaIII tubulin and myelin basic protein. While myelin basic protein bound to all three P2X subunits, betaIII tubulin interacted exclusively with the P2X(2) subunit. The tubulin binding domain could be confined to a proline-rich segment (amino acids 371-412) of the P2X(2) subunit. Our results suggest a role for microtubules in the cellular localisation of the P2X(2) receptor.     

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma-1 ligands, [3H](+)pentazocine and [3H]NE-100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di-Leu105,106di-Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti-type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.     

Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding

Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.     

Genetics of the P2X7 receptor and human disease

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.     

Contribution of P2X1 receptor intracellular basic residues to channel properties

The intracellular amino and carboxy termini of P2X receptors have been shown to contribute to the regulation of ATP evoked currents. In this study we produced, and expressed in Xenopus oocytes, individual alanine point mutants of positively charged amino acids (eight lysine, seven arginine and one histidine) in the intracellular domains of the human P2X1 receptor. The majority of these mutations had no effect on the amplitude, time-course or rectification of ATP evoked currents. In contrast the mutant K367A was expressed at normal levels at the cell surface however ATP evoked currents were reduced by >99% and desensitised more rapidly demonstrating a role of K367 in channel regulation. This is similar to that previously described for T18A mutant channels. Co-expression of T18A and K367A mutant P2X1 receptors produced larger ATP evoked responses than either mutant alone and suggests that these amino and carboxy terminal regions interact to regulate channel function.     

Isoform specificity of P2X2 purinergic receptor C-terminus binding to tubulin

Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X2 is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X2 binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X2 with a naturally occurring splicing variant named P2X2b. These isoforms differ in their C-terminal regions which contain the proposed beta-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X2 binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X2 C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X2 interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles.     

Molecular dissection of the agonist binding site of an AMPA receptor.

Two discontinuous segments (S1 and S2), separated by membrane-associated domains, in ionotropic glutamate receptor (GluR) subunits show sequence similarity to bacterial periplasmic amino acid-binding proteins, suggesting an evolutionary and structural relationship. Experimental evidence arguing for and against the inferred extracellular location of the S1 and S2 domains in GluRs has been presented. Here, we report that an extracellularly expressed fusion protein consisting of the S1 and S2 domains of alpha-amino-5-methyl-3-hydroxyisoxazolone-4-propionate (AMPA)-selective glutamate receptor GluR-D joined together via a hydrophilic linker peptide specifically reproduces the AMPA-binding properties of GluR-D, whereas the separately expressed segments do not bind ligand. This provides direct evidence that the S1 and S2 segments of GluR-D contain the structural determinants necessary and sufficient for selective agonist binding. Dissection of a functional neurotransmitter binding site as a soluble protein separate from the integral membrane channel will facilitate new approaches to analyse the structure of GluRs.     

Prenatal expression of purinergic receptor P2X3 in human dorsal root ganglion

The dorsal root ganglion (DRG) is consisted of neurons that relay multiple types of spinal sensory stimuli to the central nervous system. Several neuroactive molecules may be involved in sensory modulation especially pain processing at the DRG, including the purinergic receptor P2X3 and calcitonin-gene-related peptide (CGRP). P2X3 receptor has been considered a promising pharmaceutical target for the development of new pain medicine. Currently, litter is known about the expression of P2X3 in the human DRG. The present study characterized the localization of P2X3 in prenatal human DRG obtained from fetuses at 4-8 gestational months, by comparing to CGRP expression as well as binding pattern of isolectin-B4 (IB4), a marker of small DRG neurons presumably relevant to nociception. P2X3 immunoreactivity (IR) appeared in most neuron-like perikarya, with their numerical density reduced during the gestational period studied. P2X3 IR was co-labeled very commonly with IB4 binding and infrequently with CGRP IR and was not colocalized with IR for the gliocyte marker glutamine synthetase. Together, the data show an early and broad expression of P2X3 in prenatal human DRG neurons, pointing to a biological role of purinergic signaling during the development of spinal sensory system.     

Structure of mouse interleukin 3 (IL-3) binding protein (AIC2A). Amino acid residues critical for IL-3 binding.

Despite the high degree of sequence homology between two mouse proteins AIC2A and AIC2B (91% at the amino acid level), only the AIC2A protein binds interleukin 3 (IL-3). Soluble AIC2A protein bound IL-3 with affinity similar to the membrane-bound AIC2A protein, indicating that binding of IL-3 to AIC2A was mediated by the external domain alone. The extracellular domain of the AIC2A protein has two repeats of the common motif shared by members of the cytokine receptor family. Neither one of these repeats alone bound IL-3. Hybrids of AIC2A and AIC2B revealed that the first domain of the cytokine receptor motif could be replaced with the AIC2B sequence without an affinity change, suggesting the importance of the second domain. By changing individual amino acid residues of AIC2A in the second domain which differ from those of AIC2B, we identified several amino acid residues critical for IL-3 binding. All these residues are located at the putative hinge region within the second domain.