Alternative Splicing Leads to Two Cholinergic Proteins in Caenorhabditis elegans

Alfonso, Aixa; Grundahl, Kiely; McManus, John; Asbury, Jeffrey M.; Rand, James B.

doi:10.1006/jmbi.1994.1538

Cited by 108 publications

(67 citation statements)

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“…By contrast, 5-HT and fluoxetine did not inhibit sensitivity to levamisole, a specific agonist of the nicotinic ACh receptor UNC-29 in the body-wall muscles (data not shown) (Nurrish et al 1999), suggesting that 5-HT regulates presynaptic ACh neurotransmission. We next examined a GFP reporter under the control of a common promoter element shared by the vesicular ACh transporter gene unc-17 and the choline acetyltransferase gene cha-1 (AChTgfp) (Alfonso et al 1994). 5-HT and fluoxetine did not cause any discernible changes in the morphology of cholinergic neurons, but they both reduced AChTgfp expression.…”

Section: Resultsmentioning

confidence: 99%

A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans

et al. 2010

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Fluoxetine is one of the most commonly prescribed medications for many behavioral and neurological disorders. Fluoxetine acts primarily as an inhibitor of the serotonin reuptake transporter (SERT) to block the removal of serotonin from the synaptic cleft, thereby enhancing serotonin signals. While the effects of fluoxetine on behavior are firmly established, debate is ongoing whether inhibition of serotonin reuptake is a sufficient explanation for its therapeutic action. Here, we provide evidence of two additional aspects of fluoxetine action through genetic analyses in Caenorhabditis elegans. We show that fluoxetine treatment and null mutation in the sole SERT gene mod-5 eliminate serotonin in specific neurons. These neurons do not synthesize serotonin but import extracellular serotonin via MOD-5/SERT. Furthermore, we show that fluoxetine acts independently of MOD-5/SERT to regulate discrete properties of acetylcholine (Ach), gamma-aminobutyric acid (GABA), and glutamate neurotransmission in the locomotory circuit. We identified that two G-protein-coupled 5-HT receptors, SER-7 and SER-5, antagonistically regulate the effects of fluoxetine and that fluoxetine binds to SER-7. Epistatic analyses suggest that SER-7 and SER-5 act upstream of AMPA receptor GLR-1 signaling. Our work provides genetic evidence that fluoxetine may influence neuronal functions and behavior by directly targeting serotonin receptors.

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Section: Resultsmentioning

confidence: 99%

A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans

et al. 2010

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“…We found that overexpression of the G347R protein increased the amount of UNC-17 immunoreactivity, but did not improve the behavioral deficits of the unc-17(e245) animals ( Figure 6). Next, we examined the behavior of unc-17(md1447) mutants; the md1447 mutation is a deletion in the 39-UTR of the unc-17 gene that results in a severe reduction in UNC-17 immunostaining (Alfonso et al 1994a). Although the decrease in UNC-17 immunoreactivity in this mutant is more :NLS-CFP-LacZ (green) and Punc-17::NLS-YFP (magenta) transgenes were imaged with a confocal microscope.…”

Section: Effects Of Sup-1 Mutations On Unc-17 Abundance and Traffickingmentioning

confidence: 99%

“…The unc-17 promoter (Punc-17) included 3180 bp upstream of the SL1 trans-splice site (Alfonso et al 1994a); it drives expression exclusively in cholinergic neurons (Rand et al 2000). A derivative of this promoter, designated Punc-17b, drives expression in the AS, DA, DB, VA, and VB cholinergic motor neurons of the ventral nerve cord (Charlie et al 2006).…”

Section: Transgene Production and Propertiesmentioning

confidence: 99%

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

et al. 2012

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The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-offunction mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-toarginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/ VAChT and synaptobrevin. C OORDINATED muscle contraction in both vertebrates and nematodes requires release of the neurotransmitter acetylcholine (ACh) from motor neurons. ACh is synthesized in motor neurons by the enzyme choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular ACh transporter (VAChT). Following transmitter release, cholinergic signaling is terminated primarily through the action of acetylcholinesterase (AChE), which hydrolyses the released ACh into choline and acetyl-CoA. In the nematode Caenorhabditis elegans, the ChAT and VAChT proteins are encoded by the cha-1 and unc-17 genes, respectively (Alfonso et al. , 1994b. Mutations that functionally eliminate either protein are lethal , while reduction-of-function mutations result in animals that are small, slow growing, uncoordinated, and resistant to AChE inhibitors such as aldicarb .The unc-17 missense mutation e245 replaces a glycine with an arginine (G347R) in the ninth transmembrane domain (TMD9) of the VAChT protein . Genetic screens designed to identify mutations that improve the locomotion of e245 homozygotes have identified several loci that suppress the deleterious effects of the VAChT G347R mutation. Sandoval et al. (2006) found that the sup-8 locus corresponds to the previously characterized snb-1 gene, which encodes the v-SNARE synaptobrevin. The single sup-8 allele is associated with a missense mutation that results in a charge alteration (I97D) in the TMD of synaptobrevin. T...

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“…Sequencing was performed either using an ABI Cycle Sequencing kit (Perkin Elmer-Cetus, Norwalk, CT) or a Sequenase II kit (United States Biochemical, Cleveland, OH). Genomic DNA and RNA were isolated from nematodes as previously described (Alfonso et al, 1994). Poly-A ϩ -enriched RNA was isolated from mixed or L1-enriched cultures as described previously (Nonet and Meyer, 1991).…”

Section: Molecular Analysis Of Unc-11mentioning

confidence: 99%

UNC-11, aCaenorhabditis elegansAP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

Nonet

Holgado

Brewer

et al. 1999

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The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second,unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

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Alternative Splicing Leads to Two Cholinergic Proteins in Caenorhabditis elegans

Cited by 108 publications

References 0 publications

A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans

A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

UNC-11, aCaenorhabditis elegansAP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

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