Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system.

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.

Synaptic exocytosis and nervous system development impaired in Caenorhabditis elegans unc-13 mutants.

C. elegans mutants defective in unc-13 exhibited severe behavioral abnormalities including paralyzed locomotion and slow pharyngeal pumping and irregular defecation cycle. Consistent with the phenotypes, the mutants accumulated abnormally high levels of the neurotransmitter acetylcholine and were resistant to acetylcholinesterase inhibitors. The unc-13 gene was expressed in most, if not all, neurons when analyzed by using chimeric constructs consisting of the unc-13 promoter and green fluorescence protein or beta-galactosidase reporter gene. While Ca(2+)-regulated acetylcholine release is lacking, the mutants were still able to release acetylcholine in vivo and in vitro at similar levels to that mediated by the regulated mechanism. Double mutants defective in both unc-13 and other genes involved in synaptic transmission showed the Unc-13 phenotype, rather than other mutant phenotypes, in terms of locomotion as well as of acetylcholine accumulation. Furthermore, electron microscopic reconstruction of the mutant nervous system uncovered that a majority of neurons developed and connected as those in the wild type except for subtle abnormalities including inappropriate connections through gap junctions and morphological alterations of neurons. These results demonstrate that the unc-13 gene product plays an essential role at a late stage in Ca(2+)-regulated synaptic exocytosis. Neurotransmitters released through the Ca(2+)-regulated mechanism are required for, but do not play major roles in the nervous system development. The large amount of Ca(2+)-independent neurotransmitter release observed in the unc-13 mutants suggests that there may be a distinct mechanism from evoked or spontaneous release in neurotransmission.

Multiple stores of calcium are released in the sea urchin egg during fertilization.

Fertilization initiates a transient increase in intracellular Ca2+ principally by Ca2+ release from intracellular stores. Possible multiple Ca2+ stores and multiple receptor regulation of the same store have been reported. Here we report the presence of at least two independent intracellular Ca2+ stores in the sea urchin egg, which are released during fertilization. Ca2+ release from one store is mediated by inositol 1,4,5-trisphosphate (IP3) and is sensitive to low molecular weight heparin. The other store is heparin insensitive and independent of IP3 regulation, but the regulatory factor remains unidentified. A transient increase in Ca2+ in heparin-loaded eggs is observed during fertilization, which suggests that IP3-independent Ca2+ release mediates the production of IP3 and release of the IP3-dependent store. Experiments presented here do not support the idea of sperm receptor coupling to inositol phospholipid hydrolysis through a GTP-binding protein mediating the fertilization response.

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.

Synergistic calcium release in the sea urchin egg by ryanodine and cyclic ADP ribose.

A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to release from an intracellular store. Two intracellular receptor Ca2+ channels for inositol 1,4,5-trisphosphate (IP3) and ryanodine have been identified by physiological and immunological techniques. While IP3 is the endogenous messenger for the IP3 receptor, a corresponding physiological messenger for the ryanodine receptor is unknown. A variety of recent experimental evidences suggest that cyclic ADP ribose (cADPR) may be a possible candidate. In this study using both egg homogenates and intact eggs, we show that subthreshold concentrations of cADPR and ryanodine can act synergistically to potentiate Ca2+ release. Addition of 10-20 nM cADPR, which causes little net increase in Ca2+, generally enhances the action of subthreshold concentrations of ryanodine. Similarly the addition of 60-80 microM ryanodine causes a slight transient increase but potentiates maximal Ca2+ increase by a subsequent subthreshold addition of cADPR. While the target of Ca2+ release by ryanodine and cADPR may be the ryanodine receptor, their actions appear to be different and more complex than simply opening the release mechanism. There are significant differences in the kinetics of release by the two agonists. In addition we used a poorly metabolized analog of IP3 and an inhibitor of endoplasmic reticulum Ca2+ ATPase activity, to show that the unfertilized egg contains a rapidly filled Ca2+ store, which is commonly released by both IP3-mediated and ryanodine-mediated release mechanisms.