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C B Gundersen

Publications and source records attributed to C B Gundersen.

At least 37 records · Page 2Linked to original sources
PubMed1994

Molecular cloning of a putative vesicular transporter for acetylcholine.

Classical neurotransmitters such as acetylcholine (ACh) require transport into synaptic vesicles for regulated exocytotic release. The Caenorhabditis elegans gene unc-17 encodes a protein with homology to mammalian transporters that concentrate monoamine neurotransmitters into synaptic vesicles. Mutations in unc-17 protect against organophosphorus toxicity, indicating a role in cholinergic neurotransmission. Using the relationship of unc-17 to the vesicular amine transporters, we first isolated a related sequence from the electric ray Torpedo californica [Torpedo vesicular ACh transporter (TorVAChT)] that is expressed by the electric lobe but not by peripheral tissues. Using the relationship of the Torpedo sequence to unc-17, we then isolated the cDNA for a rat homologue (rVAChT). Northern blot analysis shows expression of these sequences in the basal forebrain, basal ganglia, and spinal cord but not cerebellum or peripheral tissues. In situ hybridization shows expression of rVAChT mRNA in all cholinergic cell groups, including those in the basal forebrain, brainstem, and spinal cord that previously have been shown to express choline acetyltransferase mRNA. The human VAChT gene also localizes to chromosome 10 near the gene for choline acetyltransferase. Taken together, these observations support a role for rVAChT in vesicular ACh transport and indicate its potential as a novel marker for cholinergic neurons.

Acetylcholine↗
PubMed1994

Extensive lipidation of a Torpedo cysteine string protein.

Cysteine string proteins are relatively low mass components of synaptic vesicle membranes. Structurally, their primary sequence is distinguished by a remarkable, cysteine-rich motif. Investigations revealed an unprecedented degree of lipidation of these cysteine residues. At least 11 of the 13 cysteines of the Torpedo protein were modified, principally by palmitoyl moieties. This fatty acylation creates a prominent hydrophobic domain flanked by polar amino and carboxyl termini. An amphipathic structure of this type is uniquely suited to mediate events at membrane interfaces. Thus, cysteine string proteins are candidates to participate in exocytotic membrane fusion.

Animals↗
PubMed1994

Cysteine string proteins: a potential link between synaptic vesicles and presynaptic Ca2+ channels.

Presynaptic calcium channels are key regulators of neurotransmitter release. Oocyte expression studies suggest that cysteine string proteins are essential subunits or modulators of these channels. Subcellular fractionation revealed that cysteine string proteins copurify with synaptic vesicles. An average vesicle had eight protein monomers with both the amino and carboxyl termini detected on the cytoplasmic face. Thus, docked synaptic vesicles may regulate presynaptic calcium channels and neurotransmitter release.

Animals↗
PubMed1994

Presynaptic dysfunction in Drosophila csp mutants.

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

Animals↗
PubMed1994

Antipeptide antibodies against a Torpedo cysteine-string protein.

An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo cysteine-string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.

Amino Acid Sequence↗
PubMed1994

Glia of the cholinergic electromotor nucleus of Torpedo are the source of the cDNA encoding a GAT-1-like GABA transporter.

A PCR-based strategy was used to clone DNAs encoding Na(+)- and Cl(-)-dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter-1 (GAT-1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT-1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT-1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.

Amino Acid Sequence↗
PubMed1992

Expression of Ca2+ receptors in Xenopus oocytes injected with poly(A)+ mRNA from a rat calcitonin-secreting cell line.

Poly(A)+ mRNA extracted from rat calcitonin-secreting cells (rMTC 44-2) was injected into Xenopus oocytes. In mRNA-injected oocytes the intracellular Ca2+ concentration ([Ca2+]i), measured with the Ca2+ indicator dye, fura2, increased in response to an elevation of the extracellular Ca2+ ions ([Ca2+]o). In some oocytes [Ca2+]i transiently increased in high [Ca2+]o but it did not respond to the subsequent alterations of [Ca2+]o. The addition of 10 microM carbonyl cyanide m-chlorphenylhydrazone (CCCP) to the extracellular medium restored the dependence of [Ca2+]i on [Ca2+]o in such cells. It was concluded that rMTC 44-2 cells possessed a receptor which recognizes changes in [Ca2+]o and that these receptors can be functionally expressed by microinjection of messenger RNA from rMTC 44-2 cells into Xenopus oocytes.

Animals↗
PubMed1992

Suppression cloning of the cDNA for a candidate subunit of a presynaptic calcium channel.

A novel strategy, termed suppression cloning, was used to identify a 7.4 kb cDNA encoding a putative subunit of the calcium channels that regulate transmitter release at nerve endings of Torpedo californica. The 585 nt open reading frame of this cDNA encodes a polypeptide of about 21.7 kd that is essential for the expression in frog oocytes of omega-conotoxin-sensitive, dihydropyridine-resistant, calcium channels. Sequence analysis reveals that this protein is closely related to two cloned cysteine string proteins of undertermined function that were recently localized to Drosophila nerve terminals using monoclonal antibodies.

Amino Acid Sequence↗
PubMed1990

Alpha-latrotoxin triggers an increase of ionized calcium in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes are injected with rat brain mRNA, they acquire the ability to respond to bath applied alpha-latrotoxin. This spider venom toxin is normally highly selective for nerve endings, where its binding is associated with a high-frequency, quantal discharge of neurotransmitter. By 'transplanting' toxin acceptor sites to Xenopus oocytes, we have observed both a toxin-mediated rise in cellular ionized Ca along with the triggering of a calcium-dependent chloride channel in these cells. This approach may contribute both to a better understanding of the mechanism of action of this toxin and to efforts to clone the cDNA for this binding site.

Animals↗
PubMed1989

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non-quantal release.

1. Acetylcholine (ACh) release from enzymatically exposed frog motor nerve terminals has been measured directly with closely apposed outside-out clamped patches of Xenopus myocyte membrane, rich in ACh receptor channels. When placed close to the synaptic surface of the terminal, such a membrane patch detects both nerve-evoked patch currents (EPCs) and spontaneous quantal 'miniature' patch currents (MPCs), from a few micrometres length of the terminal, in response to ACh release from the nearest three to five active zones. 2. Chemical measurements of ACh efflux from whole preparations revealed a spontaneous release rate of 4.1 pmol (2 h)-1, and no significant difference in resting efflux between enzyme-treated and control preparations. The ratio of enzyme-treated to contralateral control muscle efflux averaged 1.17, indicating that enzyme treatment did not affect spontaneous ACh release. Vesamicol (1.7 microM), which blocks the ACh transporter in synaptic vesicles, decreased the spontaneous release of ACh to 67% of control. 3. In the absence of nerve stimulation, the frequency of single-channel openings recorded by outside-out patch probes adjacent to nerve terminals was very low (1-2 min-1), and little different at a distance of hundreds of micrometres, suggesting that if ACh was continually leaking from the terminal in a non-quantal fashion, the amount being released near active zone regions on the terminal was below the limit of detection with the patches. 4. Direct measurements of the sensitivity of the patches, coupled with calculated ACh flux rates, lead to the conclusion that the amount of ACh released non-quantally from the synaptic surface of the frog nerve terminal is less than one-tenth the amount expected if all non-quantal release is from this region of the terminal membrane. 5. Following a series of single nerve shocks or a 50 Hz train of nerve stimuli, the frequency of asynchronous single-channel openings increased for several seconds. This transient increase in channel openings was not sensitive to movement of the patch electrode a significant distance (4 microns) away from the active sites, or to manipulations previously reported to block non-quantal transmitter leakage, including addition of 10 mM-Ca2+ or 1.7 microM-vesamicol to the bath. These channel openings appear to be due to an accumulation of ACh which originated from many evoked quanta, and not the effect of locally increased non-quantal ACh release due to nerve stimulation. 6. We conclude that transmitter leakage at adult frog terminals is either localized to a source other than the synaptic surface of the nerve terminal, or released in a widespread and diffuse fashion from many sources, which may include the nerve terminal.

Acetylcholine↗
PubMed1989

Mercuric ions are potent noncompetitive antagonists of human brain kainate receptors expressed in Xenopus oocytes.

Kainate receptors are one of the major subtypes of excitatory amino acid receptors in the vertebrate central nervous system. Using Xenopus oocytes injected with RNA from human temporal cortex, it is possible to detect electrophysiologically the expression of this receptor subtype in these cells. Ions of the group IIb elements, particularly mercuric ions, are highly potent, noncompetitive inhibitors of these human brain kainate receptors. Mercury-containing sulfhydryl reagents are also very effective, irreversible blockers of the kainate-gated currents of these oocytes. The recovery of kainate-activated currents after washout of Hg2+ is slow and incomplete relative to that seen after treatment either with Cd2+ or Zn2+. Cysteine or dithiothreitol can accelerate this recovery of kainate-inducible currents after Hg2+ inhibition. Besides the toxicological implications of these results, mercury compounds may be useful for future studies of the structure and physiology of the kainate receptor-channel complex.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1988

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle.

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.

Acetylcholine↗
PubMed1988

Barbiturates depress currents through human brain calcium channels studied in Xenopus oocytes.

Barbiturates have had wide use as sedatives, anesthetics and anticonvulsants. Among the sites implicated in the membrane action of barbiturates are the gamma-aminobutyric acidA receptor, receptors for excitatory amino acids and Ca and potassium channels. The expression in Xenopus oocytes of various ligand- and voltage-gated channels offers the opportunity for more-detailed studies of such neuroactive substances as the barbiturates. Using RNA from human temporal cortex, we obtained the expression of an omega-conotoxin-sensitive, dihydropyridine-resistant Ca channel in Xenopus oocytes. Under voltage clamp, barbiturates depressed both the peak current and the steady-state current through this Ca channel. Barbiturates had no effect on the shape of the current-voltage relation, nor did they cause a shift in the voltage-dependence of channel activation. However, both the rate of inactivation of open Ca channels, as well as the proportion of channels inactivated at steady state were increased by barbiturates. The IC50 for these effects was about 0.25 mM for the more potent barbiturates tested. These results are consistent with the hypothesis that sedative and anesthetic effects of barbiturates can be mediated in part by an action to depress Ca currents.

Animals↗
PubMed1987

Expression of an omega-conotoxin-sensitive calcium channel in Xenopus oocytes injected with mRNA from Torpedo electric lobe.

Xenopus laevis oocytes were injected with poly(A)+ RNA isolated from the electric lobe of Torpedo californica. Six to nine days after mRNA injection of the oocytes a cadmium-sensitive inward current could be detected in oocytes bathed in a calcium- and chloride-free solution containing 40 mM barium. This inward current could be distinguished from the native barium current of control oocytes by its high sensitivity to blockade by cadmium ions and its inhibition by omega-conotoxin, a peptide neurotoxin from Conus geographicus. Neither the current of control cells nor that of injected cells was detectably affected by nisoldipine (1 microM) or nitrendipine (1 microM). However, the barium current of control oocytes showed appreciably more inactivation (in the barium solution used for recording) than the omega-conotoxin-sensitive current that develops in mRNA-injected oocytes. Culturing of mRNA-injected oocytes in medium containing actinomycin D failed to prevent the appearance of the omega-conotoxin-sensitive current. These results support the conclusion that mRNA from Torpedo electric lobe is translated to produce an additional calcium channel in Xenopus oocytes. The features of this channel suggest that it may be the same type of calcium channel that controls transmitter release at nerve endings in Torpedo electroplax.

Animals↗
PubMed1987

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl alpha-D-[14C]glucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (Vmax) of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/glucose cotransporter was found in a single fraction of approximately 2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.

Animals↗
PubMed1986

Actions of pentobarbital on rat brain receptors expressed in Xenopus oocytes.

Functional receptor channels activated by GABA and other neurotransmitters were "transplanted" from rat brain to Xenopus oocytes by injecting the oocytes with total poly(A)+ mRNA isolated from rat or chick brain. Membrane currents elicited in the oocyte by GABA inverted polarity at about the chloride equilibrium potential (ca. -25 mV). Pentobarbital potentiated the GABA-activated currents, without appreciably changing the reversal potential or form of the current-voltage relationship. At low (less than 10(-5) M) concentrations of GABA, pentobarbital (100 microM) potentiated the responses by a factor of 10 or more, but responses to high (ca. 1 mM) concentrations of GABA were almost unchanged. Half-maximal activation of the response was obtained with about 3 X 10(-5) M GABA when applied alone and with about 4 X 10(-6) M GABA when applied together with 100 microM pentobarbital. At low doses of GABA, the size of the current increased as the 1.4th power of GABA concentration, but this relationship became nearly linear in the presence of pentobarbital. The potentiation of the GABA response increased linearly with concentrations of pentobarbital up to about 300 microM, reaching a maximum of about 50-fold. At higher concentrations of pentobarbital, the response to GABA declined. Relaxations of GABA-activated currents following voltage steps became slower in the presence of pentobarbital, suggesting that the open life-time of the channels was prolonged. In addition to actions on GABA-activated currents, pentobarbital itself elicited a small membrane current that inverted polarity at a potential (-10 mV) more positive than the GABA-activated current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

On the orientation of foreign neurotransmitter receptors in Xenopus oocytes.

Xenopus oocytes can be made to incorporate into their membrane foreign neurotransmitter receptors and voltage-activated sodium channels. In their original location the receptors are normally activated by the extracellular action of transmitter substances. Tests were made to see if some of the newly synthesized foreign receptors were inserted in the oocyte membrane with their active site facing inwards. Since intracellular injections of acetylcholine, gamma-aminobutyric acid, serotonin and kainic acid and tetrodotoxin into the oocyte failed to elicit a response, we conclude that very few, or none, of the receptor molecules expressed in the oocyte by the exogenous mRNA are inserted with the wrong orientation in the membrane.

Animals↗