One year after the Brose paper was published Littleton repor

One year after the Brose . paper was published, Littleton . reported the first measurements of evoked synaptic transmission in syt1 nulls, using the neuromuscular junction as a model system. These experiments revealed that syt1 was essential for rapid, robust evoked neurotransmitter release. Similar observations were subsequently made in other model systems and structure–function analyses ensued. These efforts were guided by structural biology pioneered by Sutton . , who solved the X-ray crystal structure of the first C2 domain of syt1 (C2A) providing key insights into how these domains bind Ca ions. Subsequent studies have suggested that syt1 C2A binds either two or three Ca ions while C2B binds two , consistent with the findings of Brose . . The next step was to determine whether the Ca-binding activity of syt1 was crucial for fast release. Ca binding is largely mediated by the negatively charged side chains of a conserved set of aspartic long queue residues and these residues have been targeted via mutagenesis (). However, it is difficult to predict the effect of disrupting individual Ca binding sites. For example, if Ca binds in an ordered and cooperative manner, disruption of the first Ca-binding site could impair binding to subsequent sites, but not vice versa. Alternatively, substitution of these residues could potentially mimic the Ca-bound state, resulting in a gain of function. These issues have been partially addressed via rescue experiments using mice and fruit flies lacking syt1. It is now clearly established that substitution of individual acidic Ca ligands in the second C2 domain (C2B) abolish the ability of syt1 to trigger fusion in response to Ca. These mutations also endowed syt1 with potent dominant-negative activity when expressed in wild-type neurons . The inability of these mutants to support release precludes the determination of a Hill slope. A consensus regarding the effects of neutralizing acidic Ca ligands in C2A has not yet been reached, with reports ranging from: (i) a gain of function and a concomitant decrease in Hill slope that appeared to partially mimic the Cabound state , (ii) no effect at all on single evoked responses , or (iii) a loss of function . While it remains unclear whether C2A must bind Ca to regulate release, the presence of this domain is essential for syt1 to function . Clearly, we are long way from ‘mapping’ individual Ca-binding sites of syt1 to the Hill coefficient of synaptic release. However, given current findings, it seems likely that the observed cooperativity may result from a more distributed action of Ca (e.g., across more than one copy of syt1) to govern release. In short, the evidence is now overwhelming that syt1 serves as a Ca sensor for rapid, synchronous SV release . But how does it trigger release? Initial insights into this question again stem from Brose . , who also showed that syt1 binds Ca poorly in the absence of acidic phospholipids, so interactions with phospholipid head groups appear to be essential for syt1 to sense relevant changes in [Ca]. Later, by scanning the surface of the C2 domains with fluorescent probes, it was discovered that the distal tips of the Ca-binding loops themselves are inserted into the target (plasma) membrane upon Ca binding (, ) , . These interactions are extremely rapid and reversible and may serve to transiently juxtapose the bilayers that are destined to fuse, bringing them into proximity to facilitate SNARE-catalyzed fusion . The membrane penetration by loops of Ca•syt1 might also directly drive structural changes in the target membrane, such as localized bending or buckling, to lower the energy barrier for fusion (). So, while structure–function experiments revealed that Ca•syt1–membrane interactions constitute an essential step in Ca-triggered SV exocytosis in living neurons, the precise effect of syt1 on bilayers – to accelerate fusion – remains somewhat elusive.