Diclofenac does not decrease renal blood flow or glomerular filtration in elderly patients undergoing orthopedic surgery

P2X receptors for adenosine 5'-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1-7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-meATP) and 2-methyl-thioadenosine 5'-triphosphate (2-meSATP), and using the antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2',5' disulphonic acid (PPADS). ATP (10 microM) evoked inward currents in approximately 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, approximately 4% showed a sustained response and approximately 10% showed a combination of transient and sustained components. Rapid application of ATP, alpha,beta-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-beta,gamma-methyleneadenosine 5'-triphosphate (1-beta,gamma-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.     

Extracellular nucleotide signaling in the inner ear

Extracellular nucleotides, particularly adenosine 5'-triphosphate (ATP), act as signaling molecules in the inner ear. Roles as neurotransmitters, neuromodulators, and as autocrine or paracrine humoral factors are evident. The diversity of the signaling pathways for nucleotides, which include a variety of ATP-gated ion channels (assembled from different subtypes of P2X-receptor subunit) and also different subtypes of G protein-coupled nucleotide receptors (P2Y receptors) supports a major physiological role for ATP in the regulation of hearing and balance. Almost invariably both P2X and P2Y receptor expression is apparent in the complex tissue structures associated with the inner-ear labyrinth. However P2X-receptor expression, commonly associated with fast neurotransmission, is apparent not only with the cochlear and vestibular primary afferent neurons, but also appears to mediate humoral signaling via ATP-gated ion channel localization to the endolymphatic surface of the cochlear sensory epithelium (organ of Corti). This is the site of the sound-transduction process and recent data, including both electrophysiological, imaging, and immunocytochemistry, has shown that the ATP-gated ion channels are colocalized here with the mechano-electrical transduction channels of the cochlear hair cells. In contrast to this direct action of extracellular ATP on the sound-transduction process, an indirect effect is apparent via P2Y-receptor expression, prevalent on the marginal cells of the stria vascularis, a tissue that generates the standing ionic and electrical gradients across the cochlear partition. The site of generation of these gradients, including the dark-cell epithelium of the vestibular labyrinth, may be under autocrine or paracrine regulation mediated by P2Y receptors sensitive to both purines (ATP) and pyrimidines such as UTP. There is also emerging evidence that the nucleoside adenosine, formed as a breakdown product of ATP by the action of ectonucleotidases and acting via P1 receptors, is also physiologically significant in the inner ear. P1-receptor expression (including A1, A2, and A3 subtypes) appear to have roles associated with stress, acting alongside P2Y receptors to enhance cochlear blood flow and to protect against the action of free radicals and to modulate the activity of membrane conductances. Given the positioning of a diverse range of purinergic-signaling pathways within the inner ear, elevations of nucleotides and nucleosides are clearly positioned to affect hearing and balance. Recent data clearly supports endogenous ATP- and adenosine-mediated changes in sensory transduction via a regulation of the electrochemical gradients in the cochlea, alterations in the active and passive mechanical properties of the cells of the sensory epithelium, effects on primary afferent neurons, and control of the blood supply. The field now awaits conclusive evidence linking a physiologically-induced modulation of extracellular nucleotide and nucleoside levels to altered inner ear function.     

Detection of polymorphic triplet repeats in the genomes of patients suffering from bipolar affective disorder

P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X2 and P2X3 have been shown to combine to form a unique heteromeric channel. Data from in situ hybridization studies suggest that P2X1 and P2X5 may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X1 and P2X5 receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X1 receptor, 30 microM alpha,beta-methylene ATP (alpha,beta-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas alpha,beta-me-ATP failed to evoke current in cells expressing the homomeric P2X5 receptor. By contrast, alpha, beta-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC50 for alpha,beta-me-ATP was greater in cells expressing both P2X1 and P2X5 than in cells expressing P2X1 alone (5 and 1.6 microM, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X1 and P2X5 subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.     

ATP stimulates sympathetic transmitter release via presynaptic P2X purinoceptors

ATP is a fast transmitter in sympathetic ganglia and at the sympathoeffector junction. In primary cultures of dissociated rat superior cervical ganglion neurons, ATP elicits noradrenaline release in an entirely Ca2+-dependent manner. Nevertheless, ATP-evoked noradrenaline release was only partially reduced (by approximately 50%) when either Na+ or Ca2+ channels were blocked, which indicates that ATP receptors themselves mediated transmembrane Ca2+ entry. An "axonal" preparation was obtained by removing ganglia from explant cultures, which left a network of neurites behind; immunostaining for axonal and dendritic markers revealed that all of these neurites were axons. In this preparation, ATP raised intraaxonal Ca2+ and triggered noradrenaline release, and these actions were not altered when Ca2+ channels were blocked by Cd2+. Hence, Ca2+-permeable ATP-gated ion channels, i.e., P2X purinoceptors, are located at presynaptic sites and directly mediate Ca2+-dependent transmitter release. These presynaptic P2X receptors displayed a rank order of agonist potency of ATP >/= 2-methylthio-ATP > ATPgammaS > alpha,beta-methylene-ATP approximately beta,gamma-methylene-L-ATP and were blocked by suramin or PPADS. ATP, 2-methylthio-ATP, and ATPgammaS also evoked inward currents measured at neuronal somata, but there these agonists were equipotent. Hence, presynaptic P2X receptors resemble the cloned P2X2 subtype, but they appear to differ from somatodendritic P2X receptors in terms of agonist sensitivity. Suramin reduced depolarization-evoked noradrenaline release by up to 20%, when autoinhibitory mechanisms were inactivated by pertussis toxin. These results indicate that presynaptic P2X purinoceptors mediate a positive, whereas G-protein-coupled P2Y purinoceptors mediate a negative, feedback modulation of sympathetic transmitter release.     

Purinoceptors: are there families of P2X and P2Y purinoceptors?

There has been an exponential growth in interest in purinoceptors since the potent effects of purines were first reported in 1929 and purinoceptors defined in 1978. A distinction between P1 (adenosine) and P2 (ATP/ADP) purinoceptors was recognized at that time and later, A1 and A2, as well as P2x and P2y subclasses of P1 and P2 purinoceptors were also defined. However, in recent years, many new subclasses have been claimed, particularly for the receptors to nucleotides, including P2t, P2z, P2u(n) and P2D, and there is some confusion now about how to incorporate additional discoveries concerning the responses of different tissues to purines. The studies beginning to appear defining the molecular structure of P2-purinoceptor subtypes are clearly going to be important in resolving this problem, as well as the introduction of new compounds that can discriminate pharmacologically between subtypes. Thus, in this review, on the basis of this new data and after a detailed analysis of the literature, we propose that: (1) P2X(ligand-gated) and P2Y(G-protein-coupled) purinoceptor families are established; (2) four subclasses of P2X-purinoceptor can be identified (P2X1-P2X4) to date; (3) the variously named P2-purinoceptors that are G-protein-coupled should be incorporated into numbered subclasses of the P2Y family. Thus: P2Y1 represents the recently cloned P2Y receptor (clone 803) from chick brain; P2Y2 represents the recently cloned P2u (or P2n) receptor from neuroblastoma, human epithelial and rat heart cells; P2Y3 represents the recently cloned P2Y receptor (clone 103) from chick brain that resembles the former P2t receptor; P2Y4-P2Y6 represent subclasses based on agonist potencies of newly synthesised analogues; P2Y7 represents the former P2D receptor for dinucleotides. This new framework for P2 purinoceptors would be fully consistent with what is emerging for the receptors to other major transmitters, such as acetylcholine, gamma-aminobutyric acid, glutamate and serotonin, where two main receptor families have been recognised, one mediating fast receptor responses directly linked to an ion channel, the other mediating slower responses through G-proteins. We fully expect discussion on the numbering of the different receptor subtypes within the P2X and P2Y families, but believe that this new way of defining receptors for nucleotides, based on agonist potency order, transduction mechanisms and molecular structure, will give a more ordered and logical approach to accommodating new findings. Moreover, based on the extensive literature analysis that led to this proposal, we suggest that the development of selective antagonists for the different P2-purinoceptor subtypes is now highly desirable, particularly for therapeutic purposes.     

The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1 receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1 receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+ influx/mobilization demonstrated that the platelet P2X1 receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.     

Early expression of a novel nucleotide receptor in the neural plate of Xenopus embryos

Extracellular ATP functions as a neurotransmitter and neuromodulator in the adult nervous system, and a signaling molecule in non-neural tissue, acting either via ligand-gated ion channels (P2X) or G-protein-coupled receptors (P2Y). ATP can cause an increase in intracellular Ca2+ (Ca2+i) in embryonic cells and so regulate cell proliferation, migration, and differentiation. We have isolated a Xenopus cDNA encoding a novel P2Y receptor, XlP2Y, which is expressed abundantly in developing embryos. Recombinant XlP2Y responds equally to all five naturally occurring nucleoside triphosphates (ATP, UTP, CTP, GTP, and ITP), which elicit a biphasic Ca2+-dependent Cl- current (ICl,Ca) where the second phase persists for up to 60 min. XlP2Y also causes a continuous release of Ca2+i and a low level persistent activation of ICl,Ca in Xenopus oocytes through the spontaneous efflux of ATP. mRNAs for XlP2Y are expressed transiently in the neural plate and tailbud during Xenopus development, coincident with neurogenesis. This restricted pattern of expression and novel pharmacological features confer unique properties to XlP2Y, which may play a key role in the early development of neural tissue.     

Activation of mitogen-activated protein kinase pathways by Mycoplasma fermentans membrane lipoproteins in murine macrophages: involvement in cytokine synthesis

We have investigated aspects of ion selectivity in K+ channels by functional expression of wild-type and mutant heteromultimeric G protein-coupled inward-rectifier K+ (GIRK) channels in Xenopus oocytes. Within the K+ channel pore (P) region signature sequence, a large number of point mutations in GIRK1 and GIRK4 subunits have been made at a key tyrosine residue--the "signature" tyrosine of the GYG. Studies of mutant GIRK1/GIRK4 heteromultimers reveal that the GIRK1 and GIRK4 subunits contribute asymmetrically to K+ selectivity. The signature tyrosine of GIRK1 can be mutated to many different residues while retaining selectivity; in contrast, the analogous position in GIRK4 must be tyrosine for maximum selectivity. Other residues of the P region also contribute to selectivity, and studies with GIRK1/GIRK4 chimeras reveal that an intact, heteromultimeric P region is necessary and sufficient for optimal K+ selectivity. We propose that the GIRK1 and GIRK4 P regions play roles similar to the two P regions of an emerging family of K+ channels whose subunits each have two P regions connected in tandem. We find different consequences between similar mutations in inward-rectifier and voltage-gated K+ channels, which suggests that the pore structures and selectivity mechanisms in the two classes of channel may not be identical. We confirm that GIRK4 subunits alone can form functional channels in oocytes, but we find that these channels are measurably permeable to Na2+ and Ca2+.     

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels

P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. Keywords: blue native PAGE/cross-linking/P2X receptor/quaternary structure.     

P2Y and P2U receptors differentially release intracellular Ca2+ via the phospholipase c/inositol 1,4,5-triphosphate pathway in astrocytes from the dorsal spinal cord

In astrocytes, raising intracellular Ca2+ concentration is a principal mechanism for transducing extracellular signals following activation of cell-surface receptors. Receptors that may be activated by purine nucleotides, P2 receptors, are known to be expressed by astrocytes from dorsal spinal cord; these astrocytes express two distinct subtypes of P2 receptor, P2Y and P2U. A main goal of the present study was to determine the intracellular signalling pathways mediating the Ca2+ responses produced by stimulating these receptors. Experiments were done using cultured astrocytes from rat dorsal spinal cord. Ca2+ responses were evoked by 2-methylthio-ATP or UTP, nucleotides previously shown to selectively activate P2Y and P2U receptors, respectively, in these cells. P2Y- and P2U-evoked Ca2+ responses were found not to depend upon extracellular Ca2+ and were blocked by thapsigargin, a Ca2+-ATPase inhibitor known to deplete inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Intracellular application of the inositol 1,4,5-triphosphate-sensitive receptor antagonist, heparin, or of the G-protein inhibitor guanosine 5'-O-(2-thiodiphosphate), blocked the P2Y- and P2U-evoked Ca2+ responses. Moreover, the responses were prevented by the phospholipase C inhibitor, U-73122, but were unaffected by the inactive analogue, U-73343. These results indicate that P2Y and P2U receptors on dorsal spinal astrocytes are linked via G-protein coupling to release of intracellular Ca2+ via the phospholipase C/inositol 1,4,5-triphosphate pathway. When we assessed the releasable pools of intracellular Ca2+, by repeated agonist applications in zero extracellular Ca2+, we found that the pool accessed by activating P2U receptors was only a subpool of that accessed by activating P2Y receptors. This implies that there are separable inositol 1,4,5-triphosphate-releasable pools of Ca2+ in dorsal spinal astrocytes and that these may be differentially released by activating distinct metabotropic P2 receptors. This differential release of Ca2+ may be important for physiological as well as pathophysiological events occurring within the spinal cord.     

ATP-stimulated activation of the mitogen-activated protein kinases through ionotrophic P2X2 purinoreceptors in PC12 cells. Difference in purinoreceptor sensitivity in two PC12 cell lines

Extracellular purine nucleotides elicit a diverse range of biological responses through binding to specific cell surface receptors. The ionotrophic P2X subclass of purinoreceptors respond to ATP by stimulation of calcium ion permeability; however, it is unknown how P2X purinoreceptor activation is linked to intracellular signaling pathways. We report that stimulation of PC12 cells with ATP results in the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 and was wholly dependent upon extracellular calcium ions. Treatment of the cells with adenosine, AMP, ADP, UTP, or alpha,beta-methylene ATP was without effect; however, MAP kinase activation was abolished by pretreatment with suramin and reactive blue 2. The calcium-activated tyrosine kinase, Pyk2, acts as an upstream regulator of the MAP kinases and became tyrosine phosphorylated following treatment of the cells with ATP. We have ruled out the involvement of depolarization-mediated calcium influx because specific blockers of voltage-gated calcium channels did not affect MAP kinase activation. These data provide direct evidence that calcium influx through P2X2 receptors results in the activation of the MAP kinase cascade. Finally, we demonstrate that a different line of PC12 cells respond to ATP through P2Y2 purinoreceptors, providing an explanation for the conflicting findings of purine nucleotide responsiveness in PC12 cells.     

Molecular composition of GABAC receptors

In the central nervous system inhibitory neurotransmission is primarily achieved through activation of receptors for gamma-aminobutyric acid (GABA). Three types of GABA receptors have been identified on the basis of their pharmacology and electrophysiology. The predominant type, termed GABAA and a recently identified type, GABAC, have integral chloride channels, whereas GABAB receptors couple to separate K+ or Ca2+ channels via G-proteins. By analogy to nicotinic acetylcholine receptors, native GABAA receptors are believed to be heterooligomers of five subunits, drawn from five classes (alpha, beta, gamma, delta, epsilon/chi). An additional class, called rho, is often categorized with GABAA receptor subunits due to a high degree of sequence similarity. However, rho subunits are capable of forming functional homooligomeric and heterooligomeric receptors, whereas GABAA receptors only express efficiently as heterooligomers. Intriguingly, the pharmacological properties of receptors formed from rho subunits are very similar to those exhibited by GABAC receptors and rho subunits and GABAC responses have been colocalized to the same retina cells, indicating that rho subunits are the sole components of GABAC receptors. In contrast, the propensity of GABAA receptor and rho subunits to form multimeric structures and their coexistence in retinal cells suggests that GABAC receptors might be heterooligomers of rho and GABAA receptor subunits. This review will summarize our current understanding of the molecular composition of GABAC receptors based upon studies of rho subunit assembly.     

Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors

In the past five years, an extended series (P2Y1-n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y1-n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.     

Dynamic epidermal cooling in conjunction with laser-induced photothermolysis of port wine stain blood vessels

Observation that the G protein-coupled P2U receptor (P2Y2 receptor) is activated by UTP as well as ATP provided the first indication that a class of uridine nucleotide-responsive receptors might exist. This hypothesis was confirmed by our identification of a uridine nucleotide-specific receptor on C6-2B rat glioma cells and by the recent cloning of two uridine nucleotide-responsive receptors, the P2Y6 receptor [J. Biol. Chem. 270:26152-26158 (1995)] and the P2Y4 receptor [J. Biol. Chem. 270:30849-30852 (1995) and J. Biol. Chem. 270:30845-30848 (1995)]. The relative nucleotide selectivities of these uridine nucleotide-activated receptors have not been established. Therefore, we cloned and expressed the P2Y6 and P2Y4 receptors in 1321N1 human astrocytoma cells and compared their relative selectivities for UDP, UTP, and other uridine and adenine nucleotides with that of the P2Y2 receptor expressed in the same cells. These comparisons were made by measuring inositol phosphate accumulation under conditions in which the initial purity and stability of agonists were rigidly ensured and quantitatively assessed. The data indicate that the P2Y2 receptor is activated with similar potencies by ATP and UTP but not by ADP or UDP; the P2Y6 receptor is activated most potently by UDP but weakly by UTP, ATP, and ADP; and the P2Y4 receptor is activated most potently by UTP, less potently by ATP, and not at all by nucleotide diphosphates. Furthermore, the P2Y6 receptor, which displays a uridine nucleotide selectivity essentially identical to that of the uridine nucleotide-specific receptor in C6-2B cells, was shown to be natively expressed in C6-2B cells and to account for the uridine nucleotide responses originally identified in these cells. These results define the uridine nucleotide selectivity of three phospholipase C-linked receptors: a receptor that is selectively activated by UDP (P2Y6 receptor), selectively activated by UTP (P2Y4 receptor), and activated by UTP and ATP but not by diphosphate nucleotides (P2Y2 receptor).     

Calcium channels associated with glutamate receptors

Ionotropic glutamate receptors are nowadays classified into two major categories based on the sensitivity to N-methyl-D-aspartic acid(NMDA) in the mammalian central nervous system. The NMDA channel has much higher permeability to Ca2+ than the non-NMDA channels in most situations, while point mutation could lead to increased permeation of Ca2+ across the non-NMDA channels. Opening of the NMDA channel is under control by a variety of endogenous low molecules, including glycine, polyamines, Mg2+, Zn2+ and H+, in addition to modulation by Ca2+/calmodulin, protein kinases and protein phosphatases. Moreover, the NMDA channel contains a redox site modulatory for the opening. Recent molecular cloning studies have revealed that the NMDA channel is a protein complex consisting of different subunits such as NMDAR1 and NMDAR2 with heterologous expression in the brain. Studies on mice knocked out of a particular gene argue in favor of an essential role of the corresponding subunits in molecular mechanisms underlying neuronal network formation and synaptic plasticity.     

Subunit stoichiometry of the epithelial sodium channel

The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.     

Nucleotide receptors in the nervous system. An abundant component using diverse transduction mechanisms

Extracellular nucleotides achieve their role as cell-to-cell communicators by acting at cell surface transmembrane receptors-the P2 receptors. Before molecular cloning led to the isolation of any P2-receptor sequence, a small number of receptor types had been proposed on the basis of pharmacological evidence. The application of molecular biology to this field of receptor research has indicated that a great underestimation of the number of receptor subtypes and of their abundance had occurred. There are now known to be seven characterized P2Y (G protein linked) receptors and the same number again of P2X receptors of the transmitter-gated ion channel type. In this review, we discuss the properties of these cloned receptors, their distribution within the nervous system, and their methods of signal transduction.     

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'.     

Immunisation of cattle in Zimbabwe using Theileria parva (Boleni) without concurrent tetracycline therapy

Nicotinic ACh (nACh) receptors in the CNS are composed of a diverse array of subunits and have a range of pharmacological properties. However, despite the fact that they are ligand-gated cation channels, their physiological functions have not been determined. This has led to increased interest in presynaptic nACh receptors that act to modulate the release of transmitter from presynaptic terminals.