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C B Gundersen

Publications and source records attributed to C B Gundersen.

At least 19 recordsLinked to original sources
PubMed1997

The DnaJ-like cysteine string protein and exocytotic neurotransmitter release.

The fast, tightly regulated release of neurotransmitters from presynaptic nerve terminals is effected by a complex molecular apparatus. The precise roles of the various proteins involved remain largely conjectural. Cysteine string proteins (CSPs) are novel synaptic vesicle components that have been conserved in evolution. They are characterized by an N-terminus 'J'-domain and a central, multiply palmitoylated string of cysteine residues. Vertebrate CSPs have been implicated in a functional interaction of synaptic vesicles with presynaptic Ca2+ channels. Genetic 'knockout' of CSPs in Drosophila results in a temperature-sensitive breakdown of elicited transmitter release. Here we try to integrate these observations into speculative functional models on the role of this new protein family in synaptic vesicle exocytosis.

Animals↗
PubMed1996

Cysteine-string proteins: a cycle of acylation and deacylation?

We used tunicamycin, an inhibitor of protein fatty acylation, to examine the possibility that there is a cycle of acylation and deacylation of cysteine string proteins at nerve terminals. Using both physiological and immunoblot approaches, we obtained no evidence for a cycle of acylation and deacylation that affects these proteins. These data suggest that this lipid modification of cysteine string proteins is relatively more stable than that observed for other nerve ending proteins, like SNAP-25.

Acylation↗
PubMed1995

Cysteine-string proteins as templates for membrane fusion: models of synaptic vesicle exocytosis.

Cysteine-string proteins are relatively small, cysteine-rich components of synaptic vesicle membranes. Recent investigations demonstrated that at least 11 of the 13 cysteine residues of the Torpedo cysteine-string protein are fatty acylated. This exceptional level of fatty acylation occurs along a short stretch (less than 25 residues) of amino acids which are flanked on either side by very polar amino and carboxy termini. This amphipathic structure may have unique capabilities to catalyze events at membrane interfaces. We propose two distinct pathways to explain how these capabilities might subserve membrane fusion and exocytosis.

Animals↗
PubMed1995

The nucleotide and deduced amino acid sequence of a rat cysteine string protein.

Cysteine string proteins are novel, heavily lipidated components of synaptic vesicles. They have previously been studied in Drosophila (insect) and Torpedo (fish). To facilitate further investigation of the structure and function of these proteins in mammals, we isolated and sequenced the cDNA and conducted an initial characterization of a rat cysteine string protein. Nucleotide sequencing reveals that this rat protein is highly homologous to the insect and fish cysteine string proteins. At the amino acid level, the fish and rat proteins are 82% identical. The rat cysteine string protein is encoded by an approximately 5 kb mRNA that is ubiquitously expressed in rat brain. Using antibodies that cross-react with the rat protein, we find that the rat cysteine string protein is predominantly associated with nerve endings and synaptic vesicles. Moreover, like its Torpedo (fish) counterpart, it is extensively fatty acylated. It will be of considerable interest to ascertain the functional correlates of these cross-species similarities of cysteine string proteins.

Amino Acid Sequence↗
PubMed1995

Cysteine string proteins and presynaptic function.

A brief review is presented of investigations of a novel family of synaptic vesicle proteins, the cysteine string proteins (csps). Studies of csp mutants in Drosophila reveal that csps are crucial components of the excitation-secretion machinery at nerve terminals. Current data cannot distinguish between a primary role of csps in modulating calcium ion influx at the nerve terminal versus a more-direct role in the exocytotic cascade. In this context, the remarkable post-translational modification of csps, namely the fatty acylation of as many as 12 of the 13 cysteine residues of the Torpedo protein, suggests that csps may participate more directly in the process of membrane fusion that underlies exocytosis. This would be achieved by using the fatty acyl chains of the csps as templates for 'lipid flow' that would allow the fusion of vesicular and plasma membranes. These hypotheses provide a useful framework for empirical tests of the role of csps in nerve terminal function.

Animals↗
PubMed1995

Cysteine string protein immunoreactivity in the nervous system and adrenal gland of rat.

Cysteine string proteins (csps) are a recently discovered class of cysteine-rich proteins. They have been shown to associate preferentially with synaptic vesicle fractions of Torpedo electric organ or rat brain where they have been implicated in events associated with transmitter secretion. However, to date there has been no information concerning the distribution of csps in rat tissues. We investigated the localization of csps in the rat retina and CNS using immunohistochemistry with affinity purified anti-csp antibodies. Specific csp immunoreactivity having a punctate appearance is present throughout the neuraxis. Csp immunoreactivity is particularly abundant in synapse-rich regions including those of the retina, main olfactory bulb, hippocampal formation, and cerebellum. White matter tracts are devoid of csp immunoreactivity. Neuromuscular junctions show strong csp immunoreactivity. This localization of csp immunoreactivity is compatible with a role for csps in presynaptic events at a wide variety of synapses. Immunohistochemical analysis of a non-neuronal, secretory tissue, the adrenal gland, reveals prominent csp immunoreactivity in the chromaffin cells of the adrenal medulla. However, csp immunoreactivity is not detected in adrenal cortical regions. These findings are confirmed and extended by immunoblot and Northern analyses which identify a 35 kDa and a 5 kb product, respectively, in extracts of adrenal. The presence of csps in the adrenal medulla suggests that these proteins may also participate in secretion-related events in certain non-neuronal cells.

Adrenal Glands↗
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↗