{'fraction_non_alphanumeric': 0.025460331893612183, 'fraction_numerical': 0.012275517162991589, 'mean_word_length': 5.358381502890174, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Calcium‐dependent neurotransmitter release: Synaptotagmin to the rescue

One of the longest standing problems in neuroscience is to understand how calcium ions (Ca) act to trigger neurotransmitter release from presynaptic terminals. A variety of evidence has implicated the synaptic vesicle protein, synaptotagmin, in this process (Jahn and Südhof, 1999; Lin and Scheller, 2000). Among the most important evidence is the large body of work establishing that disruption of synaptotagmin dramatically affects neurotransmitter release (Augustine et al., 1999). For example, loss of a single synaptotagmin gene (there are at least 13 synaptotagmin genes in mammals) causes dramatic reductions in synaptic transmission in Drosophila, Caenorhabditis elegans, and mice (Littleton and Bellen, 1995). Although the specific causes of such defects are still being debated, evidence has been presented that this protein may be important not only for the activation of synaptic vesicle fusion by Ca but also may be involved in other vesicle trafficking reactions, such as the docking of vesicles at the plasma membrane or the budding of vesicles from the plasma membrane during endocytosis. In addition, the loss of synaptotagmin enhances spontaneous transmitter release in Drosophila. Given all of these actions, the synaptotagmin molecule must be capable of doing many things. With this in mind, current efforts are directed at defining the functional architecture of synaptotagmin. The crystal structures of various parts of various synaptotagmin isoforms have been solved, giving us a high-resolution picture of the structure of synaptotagmin. Now, it is necessary to map functions onto this structural framework. From in vitro biochemical experiments, we know that one part of synaptotagmin, the C2A domain, contains Ca -binding sites that control binding to phospholipids and to SNARE proteins, such as syntaxin and SNAP-25, that are important for neurotransmitter release. Another part of synaptotagmin, the C2B domain, also is capable of binding Ca . The C2B domain includes a region that is characterized by multiple lysine residues. This polylysine region is responsible for binding to several other molecules that are important for synaptic vesicle trafficking, including inositol polyphosphates, the clathrin adaptor protein AP-2, and the Ca channels that allow Ca to enter the presynaptic terminal. And, as if this was not enough, the polylysine motif of C2B also allows synaptotagmin molecules to bind to each other and oligomerize (Fukuda et al., 1995; Chapman et al., 1998). These intriguing properties suggest that the C2B domain, especially its polylysine motif, may mediate some of the important biological actions of synaptotagmin. However, there have been relatively few attempts to determine what happens to neurotransmitter release when the C2B domain is impaired. One of the first studies of synaptotagmin function—by Bommert et al. (1993)—reported that microinjecting a peptide representing the C2B polylysine motif completely abolishes neurotransmitter release. This suggests that binding of one or more proteins to the motif is important for neurotransmitter release, although why this is the case is not yet known. The excellent paper by Mackler and Reist in this issue of the Journal of Comparative Neurology (p. 4–16) goes some way toward clarifying the importance of the C2B domain in neurotransmitter release (see Fig. 1). The general strategy of their experiments was to use specific site-directed mutations in amino acids of the C2B domain to look at the relationship between the structure and function of synaptotagmin. Their specific goal was to take Drosophila that are missing a synaptotagmin gene and use transgenic methods to add back synaptotagmin, either wild type or mutant forms. Such gene replacement experiments, also called genetic rescue, are extremely powerful tools for addressing the function of protein-protein interactions in vivo. Genetic rescue experiments have been attempted before in Drosophila; for example, DiAntonio and Schwarz (1994) have shown that it is possible to restore synaptotagmin in synaptotagmin-deficient (null) flies. However, in their experiments, only a very low level of synaptotagmin was produced due to the promoter (elav) that was used to stimulate synaptotagmin expression. Mackler and Reist (2001) have avoided this limitation by