Eric A. Barnard

Quaternary Structure of the Native GABAA Receptor Determined by Electron Microscopic Image Analysis

Abstract: In the transmitter‐gated ion channel class of receptors, the members of which are all believed to be heterooligomers, the number and arrangement of the subunits are only known with any certainty for the nicotinic acetylcholine receptor from Torpedo electric fish. That receptor has been shown to possess a pentameric rosette structure, with five homologous subunits (α2βγδ) arranged to enclose the central ion channel. The data were obtained by electron image analysis of two‐dimensional receptor arrays, which form as a consequence of that receptors exceptionally high abundance in the Torpedo membranes and are therefore not attainable for other receptors. We have applied another direct approach to determine the quaternary structure of native ionotropic GABA receptors. We have purified those receptors from porcine brain cortex and analysed the rotational symmetry of isolated receptors visualized by electron microscopy. The results show the receptor to have a pentameric structure with a central water‐filled pore, which can now be said to be characteristic of the entire superfamily.

Molecular Cloning of a Novel P2 Purinoceptor from Human Erythroleukemia Cells

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 ± 6 nM), much less for UTP and ADP (~1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

Molecular Cloning and Characterization of the Rat P2Y4 Receptor

Abstract: Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full‐length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y4 receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2‐methylthio‐ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y4 receptor but with this significant difference in agonist pharmacology.

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase

1 BIO cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2 The radioligand [35S]‐dATPαS bound with high affinity (Kd = 9.8nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg−1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]‐dATPαS led to the rank order of affinity: dATPαS (Ki 3.4 nM) > 2‐chloroATP (2‐ClATP) (13 nM), ATP (22 nM) > ATPγS (43 nM) > 2‐methylthioATP (2‐MeSATP) (88 nM) > ADP (368 nM) > > UTP, L‐β,γ‐methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid 4‐sodium (PPADS) was able to displace only 20% of [35S]‐dATPαS binding at a concentration of 100 μm 3 2‐ClATP (EC50 = 0.22 μm), 2‐MeSATP (0.54 μm), ADP (7.9 μm) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 μm 4 A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6‐2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y‐type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2‐MeSATP has been shown to increase phospholipase C activity. 5 Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2‐MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y2 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

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.

Dual coupling of heterologously-expressed rat P2Y6 nucleotide receptors to N-type Ca2+ and M-type K+ currents in rat sympathetic neurones.

The P2Y6 receptor is a uridine nucleotide‐specific G protein‐linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro‐injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N‐type Ca2+ (ICa(N)) and M‐type K+ (IK(M)) currents. In P2Y6 cRNA‐injected neurones, UDP or UTP produced a voltage‐dependent inhibition of ICa(N) by ∼53% in whole‐cell (disrupted‐patch) mode and by ∼73% in perforated‐patch mode; no inhibition occurred in control cells. Mean IC50 values (whole‐cell) were: UDP, 5.9±0.3 nM; UTP, 20±1 nM. ATP and ADP (1 μM) had no significant effect. Pertussis toxin (PTX) substantially (∼60%) reduced UTP‐mediated inhibition in disrupted patch mode but not in perforated‐patch mode. Uridine nucleotides also inhibited IK(M) in P2Y6 cRNA‐injected cells (by up to 71% at 10 μM UTP; perforated‐patch). Mean IC50 values were: UDP, 30±3 nM; UTP, 115±12 nM. ATP (10 μM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX‐resistant. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides.

Ionotropic glutamate receptors: new types and new concepts

The unique importance in the CNS of the ion-channel receptors gated by glutamate, which is responsible for so many functions and plasticities there, creates an urgent need for full information on the range of subtypes of these receptors, before we can begin to understand all of their roles. There is a need to identify unequivo- cally all of the subunits that build the subtypes, to differentiate the indi- vidual subunit characteristics and to recognize how those characteristics may change in the native subunit co-assemblies. This report describes how the number of known sub- classes of ionotropic glutamate receptors has been increased in unexpected ways, by some recent identifications of further subunits and their heteromeric complexes. NMDA

Differential Expression of Syntrophins and Analysis of Alternatively Spliced Dystrophin Transcripts in the Mouse Brain

Expression of syntrophin genes, encoding members of the dystrophin‐associated protein complex, was studied in the mouse brain. In the hippocampal formation there is distinctive co‐localization of specific syntrophins with certain dystrophin isoforms in neurons, e.g. α1,‐syntrophin with the C‐dystrophin in CA regions and β2‐syntrophin with the G‐dystrophin in the dentate gyrus. Expression of the al‐syntrophin is predominant in CA regions and the olfactory bulb and it is also present in the cerebral cortex and the dentate gyrus. The β2‐syntrophin mRNA is most abundant in the dentate gyrus and is also evident in the pituitary, the cerebral cortex and in Ammons horn and in traces in the caudate putamen. The choroid plexus was labelled by both α1 and β2‐syntrophin‐specific probes. The expression of syntrophins in the brain correlates with expression of dystrophins and dystroglycan. There are brain areas such as the cerebral cortex where several different syntrophins and dystrophins are expressed together. Syntrophin expression co‐localizes with utrophin in the choroid plexus and caudate putamen. Finally, no syntrophin was detected in the cerebellar Purkinje cells where the specific dystrophin isoform (P‐type) is present. This specific distribution of syntrophins in the brain is particularly interesting, as muscle syntrophin interacts with neuronal nitric oxide synthase. This may suggest that the dystrophin‐associated protein complex may be involved in synaptic organisation and signal transduction machinery in both muscle and neurons. The dystrophin isoform, with exons 71–74 spliced out and hence lacking syntrophin binding sites, had been believed to be predominant in the brain, but our analyses using in situ hybridization, S1 nuclease protection and the semi‐quantitative polymerase chain reaction revealed that this alternatively spliced mRNA is a minor, low abundance form in the brain.

Distribution of [35S]dATPαS binding sites in the adult rat neuraxis

Abstract Highly abundant, saturable and specific binding sites for [35S]2′-deoxyadenosine 5′-0-(1-thio) triphosphate ([ 35 S]dATPαS, K d : 9 ± 2nM; B max : 39 ± 8 pmol/mg protein ) are present in adult rat brain membranes and have characteristics consistent with those expected for a P2Y1 receptor. The anatomical distribution of these binding sites in the brain and spinal cord was examined using in vitro autoradiography. The [35S]dATPαS binding sites showed a widespread distribution throughout the brain and spinal cord. They could be displaced by a large excess (100 μM) of 2-methylthioATP (2MeS-ATP) but not by uridine-5′-triphosphate (UTP) or α,β-methyleneATP (α,β-meATP). Within the cortical regions labelling was of equal medium density. However, discrete structures and nuclei within the olfactory bulb, subcortical telencephalon, hippocampal complex, thalamic regions and mesencephalon displayed a variety of densities. Within the spinal cord, gray matter was labelled at a greater density than the funiculi. The present study clarifies the anatomical distribution of P2Y1 and closely related receptors within the central nervous system of rat and extends the evidence that those receptors are abundant and widely distributed within the neuraxis.

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5′‐triphosphate (UTP) or adenosine 5′‐triphosphate (ATP) inhibits the N‐type voltage‐gated calcium current by ∼65%, with an IC50 of 0.5 μM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.