Biomedical subjects

R A North

Publications and source records attributed to R A North.

At least 127 records · Page 7Linked to original sources

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↗

PubMed1992

Two types of neurone in the rat ventral tegmental area and their synaptic inputs.

1. Intracellular recordings were made from 241 ventral tegmental neurones in slices of rat midbrain. Seventy-seven per cent of neurones were hyperpolarized by dopamine (principal cells); 16% were hyperpolarized by opioid peptides (secondary cells). 2. Most principal cells fired spontaneously (1-3 Hz) with a threshold of -53 mV; most secondary cells did not fire spontaneously. Action potentials of principal cells were longer (0.9 ms) than those of secondary cells (0.5 ms). 3. Focal electrical stimulation within the ventral tegmental area evoked a biphasic synaptic potential, depolarization followed by hyperpolarization, with a duration of about 200 ms. Experiments with receptor antagonists showed that the depolarizing component resulted from activation of both N-methyl-D-aspartate (NMDA) and non-NMDA receptors and the hyperpolarizing component resulted from activation of GABAA receptors. 4. A later hyperpolarizing synaptic potential developed after a latency of 50 ms, reached its peak in 250 ms and had a duration of about 1 s. It reversed polarity at -108 mV (external potassium concentration was 2.5 mM), was blocked by phaclofen (30 microM-1 mM) or 2-hydroxysaclofen (100-300 microM). In some cells, a phaclofen-resistant component remained that was increased by cocaine and blocked by sulpiride (1 microM). 5. It is concluded that the ventral tegmental area contains two types of neurone having properties similar to those in the substantia nigra. The cells receive synaptic inputs mediated by excitatory amino acids acting at NMDA and non-NMDA receptors, GABA acting at GABAA and GABAB receptors, and dopamine acting at D2 receptors.

Action Potentials↗

PubMed1992

Regulation by second messengers of the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes.

1. Voltage-clamp recordings of membrane current were made from Xenopus oocytes that had been injected with RNA which had been transcribed in vitro from a cloned complementary DNA. 2. Depolarization from -80 mV evoked outward potassium currents that developed very slowly. At -20 mV the time constant for activation was about 50 s, and at +40 mV about 6 s. 3. The potassium current was increased by the calcium ionophore A23187 or by intracellular injection of inositol 1,4,5-trisphosphate (IP3), each of which should increase the intracellular calcium concentration ([Ca2+]i). The current was decreased by injection of BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The current was also reduced by phorbol esters; this effect was blocked by staurosporine. 4. In oocytes that had also been injected with RNA encoding the 5-hydroxytryptamine (5-HT2) receptor, 5-HT increased the potassium current. After caffeine pretreatment, to block the release of intracellular calcium, 5-HT decreased the current; this decrease was prevented by staurosporine. 5. It is concluded that the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes is increased by increases in [Ca2+]i and is decreased by activation of protein kinase C. Stimulation of 5-HT2 receptors can have both these effects, but the former normally predominates.

Animals↗

PubMed1992

Muscarine increases cation conductance and decreases potassium conductance in rat locus coeruleus neurones.

1. Whole-cell patch-clamp recordings were made from rat locus coeruleus neurones in slices of brain tissue in vitro. Muscarine (30 microM) caused an inward current of about 100 pA in neurones voltage clamped at -60 mV. 2. In about 75% of cells, the current elicited by muscarine was independent of potential in the range -60 to -120 mV and had no associated conductance change. 3. In about 25% of cells, the current became smaller with hyperpolarization, was associated with a decreased conductance, and reversed polarity between -100 and -140 mV. The reversal potential changed with the logarithm of the extracellular potassium concentration. Barium and caesium blocked inward rectification and also prevented reversal of the muscarine current. 4. When potassium ions of the intracellular and extracellular solutions were replaced by caesium, the current evoked by muscarine became smaller with depolarization at reversed polarity at +9 mV. This current was associated with an increase in conductance, and was greatly reduced when the extracellular sodium concentration was reduced to 20 mM. 5. The results could be quantitatively accounted for by a model in which muscarine both increases a voltage-independent cation conductance and decreases the inward rectifier potassium conductance.

Animals↗

PubMed1992

Pre- and postsynaptic inhibition by opioids in rat striatum.

The physiological role of opioid peptides in the rat striatum was sought by intracellular recording in vitro. Excitatory synaptic potentials (mediated by glutamate or aspartate) and inhibitory synaptic potentials (mediated by GABA) were isolated pharmacologically and/or by positioning the stimulation electrode over the external capsule. Opioid agonists and antagonists selective for mu-, delta-, and kappa-receptors were applied by superfusion. Two main actions of opioids were observed. First, mu- and delta-selective opioids presynaptically inhibited the excitatory postsynaptic potential, whereas only delta-selective opioids decreased the inhibitory synaptic potential. Second, a small subpopulation of cells (not medium spiny neurons) were hyperpolarized by delta-selective agonists. The results indicate that the main action of opioids on striatal neurons is presynaptic inhibition of the corticostriate excitatory synaptic input.

Action Potentials↗

PubMed1992

Opioids excite dopamine neurons by hyperpolarization of local interneurons.

Increased activity of dopamine-containing neurons in the ventral tegmental area is necessary for the reinforcing effects of opioids and other abused drugs. Intracellular recordings from these cells in slices of rat brain in vitro showed that opioids do not affect the principal (dopamine-containing) neurons but hyperpolarize secondary (GABA-containing) interneurons. Experiments with agonists and antagonists selective for opioid receptor subtypes indicated that the hyperpolarization of secondary cells involved the mu-receptor. Most principal cells showed spontaneous bicuculline-sensitive synaptic potentials when the extracellular potassium concentration was increased from 2.5 to 6.5 or 10.5 mM; these were prevented by TTX and assumed to result from action potentials arising in slightly depolarized local interneurons. The frequency of these synaptic potentials, but not their amplitudes, was reduced by opioids selective for mu-receptors. It is concluded that hyperpolarization of the interneurons by opioids reduces the spontaneous GABA-mediated synaptic input to the dopamine cells. In vivo, this would lead to excitation of the dopamine cells by disinhibition, which would be expected to contribute to the positive reinforcement seen with mu-receptor agonists such as morphine and heroin.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1992

5-hydroxytryptamine1B receptors block the GABAB synaptic potential in rat dopamine neurons.

Intracellular recordings were made from presumed dopamine-containing neurons in slices cut from the midbrain of the rat. Focal electrical stimulation produced a hyperpolarizing synaptic potential that was reduced by 75-95% by the GABAB-receptor antagonist 2-hydroxysaclofen (300 microM). 5-HT (3-100 microM) reduced the amplitude of the GABAB synaptic potential by 20-74%, with a 50% reduction at 10 microM, but did not reduce the amplitude of synaptic potentials mediated by GABAA receptors. 5-HT acted presynaptically because hyperpolarizations produced by exogenously administered GABA (1 mM) in picrotoxin (100 microM) were not affected by 5-HT (30 microM). (+/-)-Cyanopindolol (100 nM), a 5-HT1B antagonist, blocked the effect of 5-HT (10 microM); spiperone (1 microM), which is an antagonist at 5-HT1A and 5-HT2 receptors, had no effect. The amplitude of the GABAB synaptic potential was reduced by the 5-HT1B receptor agonists 1-[3-(trifluoromethyl)-phenyl]-piperazine (300 nM) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline (1 microM), but not by the 5-HT1A agonist N,N-dipropyl-5-carboxamidotryptamine (1 microM) or the 5-HT2 agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (10 microM). We conclude that 5-HT activates presynaptic 5-HT1B receptors that inhibit the release of GABA onto GABAB but not GABAA receptors.

Animals↗

PubMed1991

Current inactivation involves a histidine residue in the pore of the rat lymphocyte potassium channel RGK5.

RGK5 is a rat genomic DNA clone that encodes the n-type potassium channel found in T-lymphocytes and other cells. Current through this channel declines (inactivates) over a period of hundreds of milliseconds during a maintained depolarizing pulse, whether in lymphocytes or when expressed in Xenopus oocytes. Here we demonstrate that an amino acid residue near the outer pore of the channel, histidine401, is involved in the inactivation process. Replacement of this residue by tyrosine, the amino acid found in the equivalent position of the homologous but non-inactivating channel RBK1, reduced inactivation of RGK5 over a 5 s depolarizing pulse from 84.3 +/- 1.9% to 18.3 +/- 1.1%. Conversely, replacement of this tyrosine in RBK1 (Tyr379) by histidine increased its inactivation from 21.6 +/- 1.1% to 42.3 +/- 1.5%. These results suggest a mechanism of channel inactivation distinct from that previously described for the A-type potassium channel.

Cloning, Molecular↗

PubMed1991

Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter.

The complementary DNA sequence encoding the cell-surface receptor for ecotropic host-range murine retroviruses (ecoR) shows that it contains 622 amino acids and 14 hydrophobic potentially membrane-spanning sequences. Because this receptor occurs on many or all murine cells and is probably essential for viability of cultured fibroblasts, its normal function might be to transport an essential metabolite. We expressed ecoR in Xenopus laevis oocytes by injecting RNA transcribed from the cloned cDNA. These oocytes specifically bound the gp70 envelope glycoprotein from an ecotropic murine leukaemia virus. An inward current was recorded electrophysiologically when a mixture of amino-acids was applied: this resulted from a stereoselective, saturable uptake of lysine, arginine and ornithine; it was independent of sodium and not substantially altered by gp70. Cysteine and homoserine were also taken up, but sodium was necessary for their transport. These properties of ecoR correspond to those of the y+ amino-acid transporter. Our results demonstrate the subversion of a ubiquitous cell membrane protein, in this case a basic amino acid transporter, for use as a retroviral receptor.

Animals↗

PubMed1991

Transmitter regulation of voltage-dependent K+ channels expressed in Xenopus oocytes.

Voltage-dependent K+ channels (RBK1, RBK2 and RGK5) were co-expressed in Xenopus oocytes with 5-hydroxytryptamine (5-HT2) receptors. K+ currents measured 2-4 days later were inhibited by 5-HT (100 nM-10 microM, 20-30 s application) by up to 90%. The effect of 5-HT was mimicked by intracellular injection of Ins(1,4,5)P3. Increasing the Ca2+ concentration at the inner surface of excised membrane patches did not decrease the K+ current.

Animals↗

PubMed1991

Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels.

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel.

Amino Acid Sequence↗

PubMed1991

Distinct muscarinic receptors inhibit release of gamma-aminobutyric acid and excitatory amino acids in mammalian brain.

Intracellular recordings were made from neurons of rat lateral amygdala, nucleus accumbens, and striatum in vitro. Synaptic potentials mediated by gamma-aminobutyric acid and by excitatory amino acids were isolated pharmacologically by using receptor antagonists, and their amplitudes were used as a measure of transmitter release. Muscarine and acetylcholine inhibited the release of both gamma-aminobutyric acid and excitatory amino acids, but measurements of the dissociation equilibrium constants for the antagonists pirenzepine, 11-(2-[(diethylamino)methyl]-1-piperidinyl)acetyl-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one, methoctramine, and hexahydrosiladifenidol indicated clearly that different muscarinic receptors were involved (M1 and probably M3, respectively). The differential localization of distinct muscarinic receptor subtypes on terminals releasing the major inhibitory and excitatory transmitters of the brain could be exploited therapeutically in some movement disorders and Alzheimer disease.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1991

Cloning, heterologous expression and developmental regulation of a Drosophila receptor for tachykinin-like peptides.

We identified clones encoding a Drosophila receptor for tachykinin-like peptides by low stringency screening of an embryonic cDNA library with probes from the bovine substance K receptor. The cDNAs encode a seven transmembrane domain protein (DTKR) of 519 amino acids with 40-48% amino acid identity to mammalian tachykinin receptors within transmembrane regions. Xenopus oocytes injected with DTKR cRNAs showed selective responses to vertebrate substance P, its agonists and not to other vertebrate tachykinin peptides. These responses were eliminated by treatment of oocytes with pertussis toxin. In the adult fly, Northern and PCR analysis demonstrated preferential expression of DTKR in the head; in situ hybridization indicated that DTKR is accumulated in the cell bodies of neurons in the adult CNS. The levels of DTKR transcript are regulated during development. Northern and PCR amplification analysis showed that while DTKR transcripts are present at all stages, high levels of expression occur in later stages of embryogenesis (starting at 10-14 h), coinciding with the beginning of major periods of neural development. Whole mount embryo in situ hybridization demonstrated that DTKR is expressed at these later stages of embryogenesis (11-15 h) in the brain and in a specific subset of neurons in each neuromere of the developing ventral ganglion. The gene encoding DTKR was mapped by in situ hybridization to a single location at 99D on the right arm of chromosome 3. These observations demonstrate that the tachykinin family of peptide transmitters and their receptors represent an evolutionarily ancient form of cellular communication within the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Opioid, 5-HT1A and alpha 2 receptors localized to subsets of guinea-pig myenteric neurons.

The expression of mu opioid, alpha 2 and 5-hydroxytryptamine1A (5-HT1A) receptors on guinea-pig myenteric neurons was determined using receptor selective agonists during intracellular recordings in vitro. Agonists known to hyperpolarize myenteric neurons by increasing potassium conductance were tested: noradrenaline and UK 14304 (alpha 2 agonists); 5-HT, 8-hydroxydipropylaminotetralin, 5-carboxamidotryptamine (5-HT1A agonists); normorphine, [Met5]-enkephalin and D-Ala2-Phe4, Gly-ol5 enkephalin (mu agonists). The alpha 2 agonists hyperpolarized 46/67 AH cells; mu agonists hyperpolarized 11/66 AH cells and 5-HT1A agonists inhibited 28/57 AH cells. Hyperpolarizations to both alpha 2 and mu agonists were observed in 11/59 AH cells; hyperpolarizations to both alpha 2 and 5-HT1A agonists were observed in 23/49 AH cells. Hyperpolarizations mediated at alpha 2 receptors were observed in 11/54 S neurons and mu agonists hyperpolarized 17/45 S cells. alpha 2 and mu receptors were localized together on 10/43 S cells tested with receptor selective agonists. 5-HT1A-mediated hyperpolarizations were not observed in 36 S cells. Presynaptic inhibition of fast excitatory post-synaptic potentials (fast e.p.s.p.s., S neurons) was observed in all cells tested with alpha 2 agonists (n = 32); in 14/23 cells tested with 5-HT1A agonists and in 8/22 cells tested with mu agonists. Both alpha 2 and 5-HT1A agonists inhibited fast e.p.s.p.s in 15/23 cells, while alpha 2 and mu agonists both inhibited the fast e.p.s.p. in 8/21 cells. Inhibition of fast e.p.s.p.s by mu and 5-HT1A agonists occurred together in 2/19 cells. Slow non-cholinergic e.p.s.p.s were inhibited by alpha 2 agonists in 19/19 cells and by 5-HT1A agonists in 19/21 cells. alpha 2- and 5-HT1A-mediated inhibition of slow e.p.s.p.s occurred together in 12/14 cells. These data allow AH neurons to be divided into two groups: those expressing alpha 2 and 5-HT1A receptors and those expressing alpha 2 and mu receptors. alpha 2 and mu receptors coexist on S neurons which do not express 5-HT1A receptors. Terminals that release acetylcholine express either alpha 2 and mu or alpha 2 and 5-HT1A receptors, consistent with the idea that they are provided by AH cells. Terminals that release mediators of the slow e.p.s.p. express primarily alpha 2 and 5-HT1A receptors.

Adrenergic alpha-Agonists↗

PubMed1991

Eggs and Salmonella food-poisoning: an evaluation.

Evidence on the extent of the part played by infected hens' eggs in causing salmonella food-poisoning is inconclusive. The role of freshly cooked shell eggs is currently much exaggerated. Prevention should be sought through improved catering practices and kitchen hygiene, and attempts to eradicate salmonellas from laying flocks are likely to be ineffective.

Animals↗