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R A North

Publications and source records attributed to R A North.

At least 19 recordsLinked to original sources
PubMed1992

Cooperative interactions among subunits of a voltage-dependent potassium channel. Evidence from expression of concatenated cDNAs.

Four copies of the coding sequence for a voltage-dependent potassium channel (RBK1, rat Kv1.1) were ligated contiguously and transcribed in vitro. The resulting RNA encodes four covalently linked subunit domains ([4]RBK1). Injection of this RNA into Xenopus oocytes resulted in the expression of voltage-dependent potassium currents. A single amino acid substitution, Tyr-->Val, located within the outer mouth of the pore, introduced into the equivalent position of any of the four domains, reduced affinity for external tetraethylammonium by approximately the same amount. In constructs containing 0, 1, 2, 3, or 4 Tyr residues the free energy of binding tetraethylammonium was linearly related to the number of Tyr residues. A different amino acid substitution, Leu-->Ile, located in the S4 region, was made in the equivalent position of one, two, three, or four domains. The depolarization required for channel activation increased approximately linearly with the number of Ile residues, whereas models of independent gating of each domain predict marked nonlinearity. Expression of this concatenated channel provides direct evidence that voltage-dependent potassium channels have four subunits positioned symmetrically around a central permeation pathway and that these subunits interact cooperatively during channel activation.

Animals↗
PubMed1992

Effects of ecotropic murine retroviruses on the dual-function cell surface receptor/basic amino acid transporter.

The widely expressed Na(+)-independent transporter for basic amino acids (system y+) is the cell surface receptor (ecoR) for ecotropic host-range mouse retroviruses (murine leukemia viruses (MuLVs)), a class of retroviruses that naturally infects only mice or rats. Accordingly, expression of mouse ecoR cDNA in mink CCL64 fibroblasts yields cells (CEN cells) that have y+ transporter activity above the endogenous background and that bind and are infected by ecotropic MuLVs. The effect of ecotropic MuLV infection on expression of y+ transporter was analyzed in mouse and in mink CEN fibroblasts. Chronic infection with ecotropic MuLVs caused 50-70% loss (down-modulation) of mouse y+ transporter in plasma membranes, detected as a reduced Vmax for uptake and outflow of L-[3H]arginine with no effect on Km values. Down-modulation was specific for mouse y+ and did not affect other transporters or the endogenous mink y+, suggesting that it results from specific interaction between mouse y+ and the viral envelope glycoprotein gp70 in the infected cells. Because this partial loss of mouse y+ from cell surfaces is insufficient to explain the complete interference to superinfection that occurs in cells chronically infected with ecotropic MuLVs, alternative explanations for interference are proposed. In contrast to the y+ down-modulation caused by chronic infection, binding of extracellular envelope glycoprotein gp70 at 37 degrees C resulted in noncompetitive inhibition of amino acid import by mouse y+ but had no effect on export through this same transporter or on any transporter properties of mink y+. The effects of gp70 on transport kinetics suggest that it slows the rate-limiting step of the amino acid import cycle, a conformational transition of the empty transporter in which the binding site moves from the inside back to the outside of the cell, and that gp70 has no effect on the rate-limiting step of the amino acid export cycle. Infected cells retain substantial y+ activity. Moreover, the virus binding site on ecoR is in a mobile region that changes conformation during the amino acid transport cycle.

3T3 Cells↗
PubMed1992

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.

Animals↗
PubMed1992

Cloning and expression of a rat somatostatin receptor enriched in brain.

The tetradecapeptide somatostatin (SRIF) is a hormone release-inhibiting substance that mediates diverse effects in brain and peripheral organs via specific receptors. A cDNA encoding a rat SRIF receptor was identified by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed in rat brain. The complete cDNA encodes a protein of 391 amino acids with seven potential transmembrane domains. Expression of the cDNA product in transfected COS-7 cell lines provides the same high affinity of binding to [125I-Tyr11]SRIF-14 as that of rat cerebral cortex tissues. However, the binding of [125I-Tyr11]SRIF-14 to cloned rat SRIF receptor is not displaced by MK678, a SRIF analog that partially displaces [125I-Tyr11]SRIF-14 binding sites in membranes of rat cerebral cortex. Northern analysis and in situ hybridization indicate that mRNA (4.0 kilobases) for cloned rat SRIF receptor is preferentially expressed in rat brain regions such as cerebral cortex and hippocampus with no detectable expression in most peripheral organs. This pattern contrasts with the exclusive peripheral expression of a recently cloned human SRIF receptor. The cDNA probe of rat receptor detects mRNA from mouse brain but not from human cerebral cortex and cerebellum.

Amino Acid Sequence↗
PubMed1992

Burst firing in dopamine neurons induced by N-methyl-D-aspartate: role of electrogenic sodium pump.

Dopamine-containing neurons of the mammalian midbrain are required for normal behavior and movements. In vivo they fire action potentials in bursts, but in vitro they discharge regularly spaced action potentials. Burst firing in vitro has now been shown to be robustly induced by the glutamate agonist N-methyl-D-aspartate (NMDA) although not by the non-NMDA agonists kainate or quisqualate. The hyperpolarization between bursts of action potentials results from electrogenic sodium ion extrusion by a ouabain-sensitive pump. This mechanism of burst generation in mammalian neurons may be important in the pathophysiology of schizophrenia and Parkinson's disease.

Action Potentials↗
PubMed1992

Hypotonic solution increases the slowly activating potassium current IsK expressed in xenopus oocytes.

A slowly activating potassium current was expressed in Xenopus oocytes by injection of RNA transcribed from a rat kidney cDNA clone. Hypotonic solutions (160 mOsmol/l; control was 220 mOsmol/l) increased the current by increasing the rate of activation and by decreasing the depolarization needed to activate the current. This effect of hypotonicity was not observed in calcium-free solution, but was unaffected by staurosporine or the calmodulin antagonist W7. Cytochalasin D reduced the current and prevented the increase by hypotonic solution. The results suggest that the increase in this potassium current by hypotonic solution might result from calcium entry and changes in the actin network.

Animals↗
PubMed1992

An amino acid mutation in a potassium channel that prevents inhibition by protein kinase C.

A slowly activating, voltage-dependent potassium channel protein cloned from rat kidney was expressed in Xenopus oocytes. Two activators of protein kinase C, 1-oleoyl-2-acetyl-rac-glycerol and phorbol 12,13-didecanoate, inhibited the current. This inhibition was blocked by the kinase inhibitor staurosporine. Inhibition of the current was not seen in channels in which Ser103 was replaced by Ala, although other properties of the current were unchanged. These results indicate that inhibition of the potassium current results from direct phosphorylation of the channel subunit protein at Ser103.

Amino Acid Sequence↗
PubMed1992

Synaptic inputs to GABAA and GABAB receptors originate from discrete afferent neurons.

gamma-Aminobutyric acid (GABA) inhibits neurons by acting at GABAA and GABAB receptors but it is not known whether the two receptors are associated with discretely separate afferent inputs or whether GABA released from a single presynaptic neuron activates both receptors. Intracellular recordings were used to show that, in the lateral amygdala and ventral tegmental area of the rat, distinct sets of GABA-containing neurons provide the synaptic input to GABAA and GABAB receptors. Synaptic potentials resulting from GABAA receptor activation (blocked by bicuculline) and from GABAB receptor activation (blocked by 2-hydroxysaclofen) occurred spontaneously but as unrelated events. Furthermore, the two components of evoked synaptic potentials were differentially inhibited by agonists acting presynaptically (muscarine and 5-hydroxytryptamine). The finding that GABA acting at GABAA and GABAB receptors originates from distinct sets of presynaptic fibers suggests that two groups of GABA-containing neurons might be generally distinguishable in the mammalian nervous system.

2-Amino-5-phosphonovalerate↗
PubMed1992

Cloning, functional expression, and developmental regulation of a neuropeptide Y receptor from Drosophila melanogaster.

Neuropeptide Y, peptide YY, and pancreatic polypeptide are homologous 36-amino acid peptides that differ from most other peptide transmitters by having a relatively rigid conformation in aqueous solutions, defined as the pancreatic polypeptide fold, and a critical C-terminal tyrosine amide. These peptides serve as gastrointestinal hormones and neurotransmitters. A cDNA encoding a novel G protein-coupled receptor activated by neuropeptide Y was cloned from Drosophila by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed early in embryogenesis. The cDNA encodes a protein of 449 amino acids with the characteristics of a G protein-coupled receptor and shares significant amino acid identity with mammalian tachykinin receptors. When expressed in Xenopus oocytes, the PR4 protein is activated by mammalian neuropeptides in the order: peptide YY greater than neuropeptide Y much greater than pancreatic polypeptide. Northern analysis showed that PR4 receptor is expressed at equivalent levels in adult Drosophila head and body and that the expression of the PR4 receptor is regulated during development. The molecular characterization of this receptor should lead to a better understanding of the functional role of this important family of hormone receptors in adult organisms and during development.

Amino Acid Sequence↗
PubMed1992

The inward rectifier potassium conductance in rat basophilic leukemia cells.

Whole-cell recordings were made from rat basophilic leukemia cells. The properties of the cells in the potential range negative to -20 mV could be accounted for by two potassium conductances, a leakage conductance (0.54 +/- 0.01 nS, mean +/- s.e.m., n = 6) and an inward rectifier conductance. The inward rectifier conductance activated at a potential close to EK and had a maximal value of 2.7 +/- 0.22 nS (n = 6). Cesium (2 mM, n = 6) decreased the inward current at every potential. Strontium (10 mM, n = 7) and rubidium (2 mM, n = 3) had a similar effect, but they also increased the slope conductance between -63 and -103 mV. Barium (1-100 microM) blocked selectively the inward rectifying current without affecting the leakage current; the effect was use-, voltage-, and temperature-dependent. TEA decreased the current; the concentration giving 50% inhibition was 37.5 mM. The current was unaffected by phorbol esters as well as several hormones and transmitters that were tested.

Animals↗
PubMed1992

Neurobiology of opiate abuse.

Opiates interact with cell surface receptors on neurons involved in the transmission of information along neural pathways that are related to behaviours essential for the life of the self and of the species. Opiates are provided with powerful and multifaceted rewarding properties that are fundamental for the acquisition, maintenance and relapse of opiate addiction. Gaetano Di Chiara and Alan North argue that both dopaminergic and non-dopaminergic systems are involved in opiate reward, and that opiate addiction results from adaptive and learning processes involving both positive reinforcing mechanisms related to the rewarding properties of opiates and negative reinforcing mechanisms related to the aversive properties of withdrawal in dependent subjects.

Animals↗
PubMed1992

Substance P opens cation channels and closes potassium channels in rat locus coeruleus neurons.

Whole-cell recordings were made from neurons of the rat locus coeruleus in a tissue slice removed from rat brain. Substance P caused an inward current in cells voltage-clamped at -60 mV. The effect of substance P was concentration-dependent (30 nM-3 microM) and was mimicked by similar concentrations of substance K and neuromedin K. The inward current resulted predominantly from an increase in membrane cation conductance; in potassium-free solutions it reversed polarity at about 12 mV. Substance P also reduced the conductance of an inwardly rectifying potassium current; this action was studied with low external sodium concentration. It is concluded that substance P excites rat locus coeruleus neurons by activating an intracellular transduction pathway leading to both cation conductance increase and potassium conductance decrease.

Animals↗
PubMed1992

5-hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala.

A fast excitatory synaptic potential mediated by 5-hydroxytryptamine (5-HT) was recorded in rat lateral amygdala neurons in brain slices. The synaptic potential has brief duration (tens of milliseconds), is mimicked by 5-HT, is potentiated by a 5-HT uptake inhibitor, and is blocked by selective 5-HT3 receptor antagonists. The underlying synaptic current reversed polarity at about 0 mV. This is an example of fast neurotransmission in the mammalian brain mediated by an amine rather than an amino acid. The antiemetic, anxiolytic, and perhaps antipsychotic actions of 5-HT3 antagonists might result from blockade of such synapses.

2-Amino-5-phosphonovalerate↗
PubMed1992

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.

Amino Acid Sequence↗
PubMed1992

Multiple subunits of a voltage-dependent potassium channel contribute to the binding site for tetraethylammonium.

RNAs encoding a wild-type (RBK1) and a mutant (RBK1(Y379V,V381T); RBK1*) subunit of voltage-dependent potassium channels were injected into Xenopus oocytes. When expressed separately, they made homotetrameric channels that differed about 100-fold in sensitivity to tetraethylammonium (TEA). Mixtures of channels having one, two, or three low affinity subunits were expressed by injecting various proportions of RBK1 and RBK1* RNAs. The affinity for TEA of these three channel species was deduced by fitting concentration-response curves for the inhibition of potassium currents. DNAs were also concatenated to construct a sequence that encoded two connected subunits, and channels that contained four, two, or no TEA-sensitive subunits were expressed. The results suggest that bound TEA interacts simultaneously with all four subunits.

Amino Acid Sequence↗
PubMed1992

Characterization and functional expression of genomic DNA encoding the human lymphocyte type n potassium channel.

Voltage-gated potassium channels play important functional roles in the development and maintenance of human lymphocyte functions. One such channel, known as the type n channel, has been well defined in human T cells and exhibits unique functional properties that distinguish it from other species of potassium channels. We report the characterization of a human genomic DNA clone, HGK5, encoding a 523-amino-acid potassium channel protein encoded by an open reading frame on a single exon. RNA transcribed in vitro from HGK5 genomic DNA directs expression of functional voltage-dependent potassium currents in Xenopus oocytes. The functional characteristics of the expressed channels are strikingly similar to those of the type n channel on human T lymphocytes. This, together with the presence of significant levels of HGK5 mRNA in human T lymphocytes, supports the notion that HGK5 encodes the human type n voltage-gated potassium channel. The effects of concanavalin A treatment on HGK5 mRNA levels in cultured human T lymphocytes was also examined. Mitogenic concentrations of concanavalin A induced a time-dependent decrease in HGK5 mRNA levels, suggesting that previously observed increases in potassium current density following concanavalin A treatment of human T lymphocytes are not due to increased transcriptional activity of the type n potassium channel gene.

Amino Acid Sequence↗
PubMed1992

Potassium channels opened by noradrenaline and other transmitters in excised membrane patches of guinea-pig submucosal neurones.

1. Unitary potassium currents were recorded in outside-out patches of membrane from guinea-pig submucosal neurones. The actions of alpha 2-adrenoceptor agonists, somatostatin and [Met5]enkephalin were studied. 2. Three main groups of background potassium channels were active. At -70 mV with 160 mM-potassium on both sides of the membrane, they had conductances of 30-65 (small), 120-160 (intermediate) and 220-260 pS (large). 3. The open channel current-voltage relation showed only constant-field rectification. Extracellular barium (2 mM) and caesium (2 mM) decreased inward but not outward currents. Tetraethylammonium (10 mM) had no effect. 4. Noradrenaline, somatostatin and [Met5]enkephalin each increased the open probability of all three classes of channel when two or more unitary amplitude channels were active in the membrane patch. Agonists were ineffective when no channel, or a single channel, was discernible in the patch. Agonists did not cause the appearance of unitary currents distinct from those seen prior to their application. 5. The effect of the agonists required intracellular guanosine 5'-triphosphate. 6. The results show that the hyperpolarization of submucosal plexus neurones by noradrenaline, somatostatin and [Met5]enkephalin results from the increased opening of at least three types of background potassium channel, and that the coupling from the receptors to the channels is maintained in excised membrane patches.

Adrenergic alpha-Agonists↗