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PubMed2003

Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms.

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.

Amides↗

PubMed2003

The unique hexokinase of Kluyveromyces lactis. Molecular and functional characterization and evaluation of a role in glucose signaling.

The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.

Amino Acid Sequence↗

PubMed2005

The diacylgylcerol-sensitive TRPC3/6/7 subfamily of cation channels: functional characterization and physiological relevance.

Among the "classical" or "canonical" transient receptor potential (TRPC) family, the TRPC3, -6, and -7 channels share 75% amino acid identity and are gated by exposure to diacylglycerol. TRPC3, TRPC6, and TRPC7 interact physically and coassemble to form functional tetrameric channels. This review focuses on the TRPC3/6/7 subfamily and describes their functional properties and regulation as homomers obtained from overexpression studies in cell lines. It also summarizes their heteromultimerization potential in vitro and in vivo and presents initial data concerning their physiological functions analyzed in isolated tissues with downregulated channel activity and gene-deficient mouse models.

Animals↗

PubMed2005

Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development caused by mutations in the TCOF1 gene, which encodes the nucleolar phosphoprotein treacle. We previously reported a function for mammalian treacle in ribosomal DNA gene transcription by its interaction with upstream binding factor. As an initial step in the development of a TCS model for frog the cDNA that encodes the Xenopus laevis treacle was cloned. Although the derived amino acid sequence shows a poor homology with its mammalian orthologues, Xenopus treacle has 11 highly homologous direct repeats near the center of the protein molecule similar to those present in its human, dog and mouse orthologues. Comparison of their amino acid compositions indicates conservation of predominant specific amino acid residues. Antisense-mediated down-regulation of treacle expression in X. laevis oocytes resulted in inhibition of rDNA gene transcription. The results suggest evolutionary conservation of the function of treacle in ribosomal RNA biogenesis in higher eukaryotes.

Amino Acid Sequence↗

PubMed2000

NMR structure determination and structure-based functional characterization of conserved hypothetical protein MTH1175 from Methanobacterium thermoautotrophicum.

The solution structure of MTH1175, a 124-residue protein from the archaeon Methanobacterium thermoautotrophicum has been determined by NMR spectroscopy. MTH1175 is part of a family of conserved hypothetical proteins (COG1433) with unknown functions which contains multiple paralogs from all complete archaeal genomes and the archaeal gene-rich bacterium Thermotoga maritima. Sequence similarity indicates this protein family may be related to the nitrogen fixation proteins NifB and NifX. MTH1175 adopts an alpha/beta topology with a single mixed beta-sheet, and contains two flexible loops and an unstructured C-terminal tail. The fold resembles that of Ribonuclease H and similar proteins, but differs from these in several respects, and is not likely to have a nuclease activity.

Amino Acid Sequence↗

PubMed1998

The mouse GalR2 galanin receptor: genomic organization, cDNA cloning, and functional characterization.

The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity (K(D) = 0.47 nM) and saturable binding with 125I-galanin (Bmax = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin approximately = M40 approximately = M15 approximately = M35 approximately = C7 approximately = galanin(2-29) approximately = galanin(1-16) >> galanin(10-29) approximately = galanin(3-29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.

Amino Acid Sequence↗

PubMed2001

Expression and functional characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) belonging to a subclass unique to apicomplexan organisms.

We have obtained a full-length P type ATPase sequence (PfATP4) encoded by Plasmodium falciparum and expressed PfATP4 in Xenopus laevis oocytes to study its function. Comparison of the hitherto incomplete open reading frame with other Ca(2+)-ATPase sequences reveals that PfATP4 differs significantly from previously defined categories. The Ca(2+)-dependent ATPase activity of PfATP4 is stimulated by a much broader range of [Ca(2+)](free) (3.2-320 micrometer) than are an avian SERCA1 pump or rabbit SERCA 1a (maximal activity < 10 micrometer). The activity of PfATP4 is resistant to inhibition by ouabain (200 micrometer) or thapsigargin (0.8 micrometer) but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 microM). We used a quantitative polymerase chain reaction to assay expression of mRNA encoding PfATP4 relative to that for beta-tubulin in synchronized asexual stages and found variable expression throughout the life cycle with a maximal 5-fold increase in meronts compared with ring stages. This analysis suggests that PfATP4 defines a novel subclass of Ca(2+)-ATPases unique to apicomplexan organisms and therefore offers potential as a drug target.

Amino Acid Sequence↗

PubMed2002

Functional characterization of the 11 non-ATPase subunit proteins in the trypanosome 19 S proteasomal regulatory complex.

The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived and dysfunctional proteins in eukaryotes. The recently demonstrated presence of a functional 26 S proteasome in Trypanosoma brucei led to the identification and isolation of genes encoding all 11 non-ATPase (Rpn) subunit proteins in the trypanosome 19 S regulatory complex. Using the technique of RNA interference, expression of individual RPN genes was disrupted in the procyclic form of T. brucei, resulting, in each case, in intracellular accumulation of polyubiquitinated protein, cell arrest at the G2/M phase, and eventual cell death. With the exception of Rpn10, depletion of individual Rpn proteins disrupted also trypanosome 19 S complex formation, with the complex virtually depleted in the cell lysate. This functional and structural essentiality of 10 of the 11 Rpn proteins in T. brucei differs significantly from that observed in other organisms. When Rpn10 was deficient in trypanosomes, a 19 S complex without Rpn10 was still formed, whereas cell growth was arrested. This structural dispensability but functional indispensability of Rpn10 may constitute another unique aspect of the proteasomes in T. brucei.

Adenosine Triphosphatases↗

PubMed2004

Structural and functional characterization of gene products encoded in the human genome by homology detection.

Availability of the human genome data has enabled the exploration of a huge amount of biological information encoded in it. There are extensive ongoing experimental efforts to understand the biological functions of the gene products encoded in the human genome. However, computational analysis can aid immensely in the interpretation of biological function by associating known functional/structural domains to the human proteins. In this article we have discussed the implications of such associations. The association of structural domains to human proteins could help in prioritizing the targets for structure determination in the structural genomics initiatives. The protein kinase family is one of the most frequently occurring protein domain families in the human proteome while P-loop hydrolase, which comprises many GTPases and ATPases, is a highly represented superfamily. Using the superfamily relationships between families of unknown and known structures we could increase structural information content of the human genome by about 5%. We could also make new associations of domain families to 33 human proteins that are potentially linked to genetically inherited diseases.

Databases, Genetic↗

PubMed1999

Topological and functional characterization of the N-glycans of soybean (Glycine max) agglutinin.

Soybean agglutinin (SBA), is a noncovalently bound tetramer comprised of four identical subunits having a single N-glycan chain, Man9GlcNAc2, that is known to be essential for regeneration of the functional tetrameric structure from unfolded subunits. In this study, SBA was found to have strong affinity for concanavalin A, indicating that the N-glycans are extensively solvent-exposed. The susceptibilities of the N-glycans to alpha-mannosidase and endo-beta-N-acetylglucosaminidase revealed that their distal areas have nonreducing ends embedded among the subunits, whereas their proximal regions are solvent-exposed. Endo-beta-N-acetylglucosaminidase-digested SBA was unable to retain its conformation and gradually unfolded. Periodate-oxidized SBA, whose N-glycans closely correspond to the invariant pentasaccharide core, tended to dissociate into the subunits, but permitted to stay as folded monomers. This SBA species was capable of refolding from unfolded subunits but unable to form the functional tetramer. It seems probable that the proximal regions of the N-glycans function in the formation and stabilization of the subunit conformation, whereas the branches outside the invariant cores stabilize the tetrameric structure.

Carbohydrate Conformation↗

PubMed1986

Phenotypic and functional characterization of human Leu1 (CD5) B cells.

Leu1 B cells have been observed in high numbers in CLL patients, fetal spleen, and in bone marrow transplant recipients. We studied these cells in normal peripheral blood, spleen, and tonsil. They were phenotypically and functionally similar to conventional B cells. Leu1 B cells in the spleen and blood displayed a mature phenotype similar to the Leu1 B cells in the fetus, some expressing high levels of both sIgM and sIgD, and some expressing high levels of sIgD but little sIgM. Most Leu1 B cells in the tonsil also expressed high levels of both sIgM and sIgD, although some tonsil Leu1 B cells had high levels of sIgM but little sIgD. The significance of the Leu1 B cell is unknown, although it may be involved in the humoral immunoincompetence of the fetus, of CLL patients, and of transplant recipients. In our data there is no evidence to suggest that these cells are activated, immature, or capable of an immunoregulatory function.

Antibodies, Monoclonal↗

PubMed2006

Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRalphaR477H and hGRalphaG679S associated with generalized glucocorticoid resistance.

BACKGROUND: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor alpha (hGRalpha) gene, which impair one or more of hGRalpha's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRalphaR477H and hGRalphaG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. METHODS AND RESULTS: In transient transfection assays, hGRalphaR477H displayed no transcriptional activity, whereas hGRalphaG679S showed a 55% reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGRalpha. Neither hGRalphaR477H nor hGRalphaG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRalphaR477H preserved normal affinity for the ligand, whereas hGRalphaG679S displayed a 2-fold reduction compared with hGRalpha. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRalphaR477H (25 min) and hGRalphaG679S (30 min) into the nucleus than the wild-type hGRalpha (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRalphaR477H did not bind to glucocorticoid-response elements, whereas hGRalphaG679S preserved its ability to bind to glucocorticoid-response elements. Finally, in glutathione-S-transferase pull-down assays, hGRalphaG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRalphaR477H and hGRalpha, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. CONCLUSIONS: The natural mutants hGRalphaR477H and hGRalphaG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.

Amino Acid Sequence↗

PubMed1994

Functional characterization of mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking short domains within the large, lumen-exposed loop of the chlorophyll protein CP47 in photosystem II.

Several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions or a single-site mutation within the large, lumen-exposed loop (loop E) of the chlorophyll a-binding photosystem II core protein, CP47, are analyzed for their functional properties by measuring the flash-induced pattern of thermoluminescence, oxygen yield, and fluorescence quantum yield. A physiological and biochemical characterization of these mutant strains has been given in two previous reports [Eaton-Rye, J.J., & Vermaas, W.F.J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J.J., Renger, G., & Vermaas, S. F.J. (1993) Biochemistry 32, 4444-4454]. The results of the present study show that deletion of charged and conserved amino acids in a region roughly located between residues 370 and 390 decreases the binding affinity of the extrinsic PS II-O protein to photosystem II. Marked differences with PSII-O deletion mutants are observed with respect to Ca2+ requirement and the flash-induced pattern of oxygen evolution. Under conditions where a sufficient light activation is provided, the psbB mutants assayed in this study reveal normal S-state parameters and lifetimes. The results bear two basic implications: (i) the manganese involved in water oxidation can still be bound in a functionally normal or only slightly distorted manner, and (ii) the binding of the extrinsic PS II-O protein to photosystem II is impaired in mutants carrying a deletion in the domain between residues 370 and 390, but the presence of the PS II-O protein is still of functional relevance for the PS II complex, e.g., for maintenance of a high-affinity binding site for Ca2+ and/or involvement during the process of photoactivation.

Binding Sites↗

PubMed1999

Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120.

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.

Amino Acid Sequence↗

PubMed2004

Generation, purification and functional characterization of three C3a anaphylatoxins in rainbow trout: role in leukocyte chemotaxis and respiratory burst.

Activation of the complement system leads to cleavage of the C3 molecule into C3b and C3a fragments. The C3a fragment plays a major role in immunity by inducing chemotaxis of eosinophils and mast cells and stimulating the respiratory burst in leukocytes. Although this anaphylotoxin has been well studied in mammals, there is currently a lack of information about the structure and function of C3a anaphylotoxins in non-mammalian vertebrate species. Therefore, in the present study, we have isolated and characterized three different C3a anaphylatoxin molecules from rainbow trout, a teleost fish. C3a was generated from the trout C3-1, C3-3, and C3-4 isoforms by incubating each individual C3 molecule with purified trout factor B/C2 and factor D in the presence of Mg2+, then purifying the resulting C3a molecules by gel filtration. SDS-PAGE, N-terminal sequence and mass spectrometric analysis demonstrated the high degree of purity and expected molecular masses of the three C3a molecules. We showed that although activated trout serum was able to induce complement-dependent chemotaxis in trout head kidney leukocytes, none of the three isolated C3a molecules induced chemotaxis in the same cells. In contrast, all three C3a molecules strongly stimulated the respiratory burst of head kidney leukocytes in a dose-dependent manner. When the carboxy-terminal Arg was removed from all three C3a molecules, their ability to induce the respiratory burst was lost. These studies, therefore, provide strong evidence for the existence of three functional C3a molecules in a non-mammalian vertebrate species and suggest that some of the basic mechanisms of action of the C3a molecule have been conserved for more than 300 million years.

Anaphylatoxins↗

PubMed2005

Molecular cloning and functional characterization of the dopamine transporter from Eloria noyesi, a caterpillar pest of cocaine-rich coca plants.

Cocaine is produced by coca plants as a chemical defense to deter feeding by insects. It has been shown that cocaine sprayed on tomato leaves reduces insect feeding, causes abnormal behaviors at low doses and kills feeding insects at doses equivalent to that in coca leaves [Nathanson, J.A., Hunnicutt, E.J., Kantham, L., Scavone, C., 1993. Cocaine as a naturally occurring insecticide. Proc. Natl. Acad. Sci. U. S. A. 90, 9645-9648.]. Most insects avoid coca leaves except the larvae of Eloria noyesi, a caterpillar pest of coca plants, which feeds preferentially on coca leaves. In the current study, we cloned and characterized the dopamine transporters (DATs) from caterpillars of E. noyesi (enDAT) and the silkworm, Bombyx mori (B. mori, bmDAT). The two insect DATs shared 88% amino acid sequence homology and functional similarity. Although enDAT and bmDAT showed the highest affinity for dopamine among endogenous amines, they were more sensitive to mammalian NET-selective inhibitors than to mammalian DAT-selective inhibitors. Despite a high cocaine content in the food source for E. noyesi, cocaine sensitivity of enDAT was similar to that of bmDAT, suggesting that mechanisms other than DAT insensitivity to cocaine, such as cocaine sequestration, might be responsible for cocaine resistance in this species. Given the significant differences in pharmacological profile from mammalian DATs, invertebrate DATs provide excellent tools for identifying regions and residues in the transporters that contribute to high-affinity binding of psychostimulants and antidepressants.

Amino Acid Sequence↗

PubMed1999

Biochemical and functional characterization of nitric oxide synthase III gene transfer using a replication-deficient adenoviral vector.

Nitric oxide (NO) produced in endothelial cells has been implicated in the regulation of blood pressure, regional blood flow, inhibition of platelet aggregation, and endothelial and vascular smooth muscle cell proliferation. In a variety of cardiovascular disease states, such as atherosclerosis, arterial hypertension, and restenosis, expression of endothelial NO synthase (NOS-III) and endothelial NO production appear to be altered. Thus, NOS-III is an attractive target for cardiovascular gene therapy for which adenoviral vectors are one of the most effective vector systems. Therefore, a recombinant adenoviral vector expressing NOS-III (adenovirus type 5 [Ad5] cytomegalovirus [CMV] NOSIII) was constructed and biochemically and pharmacologically characterized both in vitro and in intact cells. Ad5CMVNOSIII-derived recombinant NOS-III was successfully expressed, as shown by immunoprecipitation and immunocytochemistry, and biologically active, as shown by functional assays in human primary umbilical vein and EA.hy926 endothelial cells, as well as 293 human embryonic kidney and Chinese hamster ovary cells. The Km values for NADPH and L-arginine and the Ka for tetrahydrobiopterin as well as the enzyme's dependency on other cofactors were similar to recombinant reference enzyme and literature values. NOS-III expression levels correlated linearly with the multiplicity of infection with Ad5CMVNOSIII and lasted for at least 8 days. NOS-III transfection inhibited endothelial cell proliferation. In conclusion, adenovirus-mediated gene transfer of Ad5CMVNOSIII to vascular and nonvascular cells resulted in the dose-dependent expression of intact, physiologically regulated, and functionally active NOS-III.

Adenoviridae↗

PubMed2004

Functional characterization of a P2X receptor from Schistosoma mansoni.

The cloning and characterization of a P2X receptor (schP2X) from the parasitic blood fluke Schistosoma mansoni provides the first example of a non-vertebrate ATP-gated ion channel. A number of functionally important amino acid residues conserved throughout vertebrate P2X receptors, including 10 extracellular cysteines, aromatic and positively charged residues involved in ATP recognition, and a consensus protein kinase C site in the amino-terminal tail, are also present in schP2X. Overall, the amino acid sequence identity of schP2X with human P2X(1-7) receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC(50) of 22.1 microM. 2',3'-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was a partial agonist (maximum response 75.4 +/- 4.4% that of ATP) with a higher potency (EC(50) of 3.6 microM) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid blocked schP2X responses to 100 microm ATP with IC(50) values of 9.6 and 0.5 microM, respectively. Ivermectin (10 microM) potentiated currents to both ATP and Bz-ATP by approximately 60% with a minimal effect on potency (EC(50) of 18.2 and 1.6 microM, respectively). The relative permeability of schP2X expressed in HEK293 cells to various cations was determined under bi-ionic conditions. schP2X has a relatively high calcium permeability (P(Ca)/P(Na) = 3.80 +/- 0.29) and an estimated minimum pore diameter similar to that of vertebrate P2X receptors. SchP2X provides a useful comparative model for the better understanding of human P2X receptor function and may also provide an alternative drug target for treatment of schistosomiasis.

Adenosine Triphosphate↗