C. elegans as a model to study the role of SM proteins in neurotransmitter release in vivo

Communication between cells occurs via chemical messengers such as hormones and neurotransmitters. Many of these chemical messengers are normally stored in membrane-enclosed vesicles inside cells and can only be released by fusion of these vesicles with the cell membrane in the process known as exocytosis. The importance of this mechanism is illustrated by the fatal paralysis caused by botulism, which works by blocking the exocytosis of neurotransmitter from nerve cells. Exactly how exocytosis occurs was mysterious until the last decade, when it emerged that a set of similar proteins - the 'SNARE proteins' - controlled this process in all cells. The SNARE proteins are believed to directly drive membrane fusion during exocytosis, but other nerve cell proteins regulate the activity of the SNAREs, enabling sophisticated control of neurotransmitter release. A good candidate for one such regulatory protein in mammals is Munc18. Indeed, Munc18 binds tightly to the SNARE protein, syntaxin, and is essential for neurotransmitter release in vivo. Munc18 was named after a similar protein called unc-18 that was discovered years earlier in worms. Like the mammalian protein, unc-18 binds to syntaxin and is important for neurotransmission in vivo. It therefore seems likely that the critical function of unc-18/Munc18 in nerve cells involves binding to syntaxin. However, Munc18 also binds to different nerve cell proteins, so it is not clear which interactions are most important for exocytosis. Furthermore, Munc18 affects multiple stages of the exocytosis process and some of these do not appear to require syntaxin binding. We aim to shed light on this issue by engineering mutant versions of unc-18 that bind to some binding partners of unc-18, but not others. Analysis of worms containing these mutant proteins in nerve cells will identify which unc-18 interactions are important for exocytosis. As these mutants should also change the amount of neurotransmitter released from nerve cells, we will study how this contributes to behaviour in the living animal. As the proteins being studied are similar to those in our own nerve cells, the results of this study will illuminate the molecular basis of human brain function.

Neurotransmission occurs by the regulated exocytosis of synaptic vesicles. In recent years, it has emerged that this process is controlled by a number of evolutionarily conserved proteins. Central among these are the SNARE proteins, which are believed to directly drive membrane fusion. In addition, SM proteins serve as important regulators of exocytosis. Indeed, null mutants of the mammalian SM protein, Munc18-1, and its C. elegans orthologue, UNC-18, exhibit a profound inhibition of neurotransmitter release. Both proteins bind to the SNARE protein, syntaxin, suggesting that this interaction may underlie the critical function of UNC-18/Munc18 in nerve terminals. However, both proteins also bind to different neuronal proteins involved in exocytosis, so it is not clear which interactions are important physiologically. Furthermore, overexpression of different Munc18 mutants in cultured mammalian chromaffin cells changes either the overall frequency of exocytosis or the quantal size of individual release events. As both effects can be seen in mutants with no defect in syntaxin binding, this suggests that SM proteins perform multiple functions in exocytosis, some of which may be syntaxin-independent. We now wish to test if the modulation in quantal size induced by Munc18 mutants in chroamffin cells also occurs in synaptic exocytosis, where this would be predicted to result in alterations in synaptic transmission and hence behaviour. In addition, we aim to evaluate the contribution of the different protein interactions of SM proteins to their functions at the synapse. In order to adress the physiological roles of SM proteins in neurotransmitter release in vivo, we propose to generate and analyse a variety of unc-18 mutations. The basic approach will be to transform unc-18 null mutant worms with wild type and mutant unc-18 mutant constructs and assess the extent of rescue of defined phenotypes at the cellular and behavioural levels. In complementary experiments, wild type and mutant recombinant proteins will be analysed to determine the effect of the mutations on UNC-18 protein interactions. Taken together, these approaches will identify physiologically relevant mechanisms by which UNC-18 regulates neurotransmission and how this contributes to behaviour in the living animal. In view of the structural and functional conservation between the C. elegans and mammalian orthologues, it is expected that these findings will be relevant for neuronal function in humans also.