中枢神经系统内神经递质释放研究进展
an SNAP has the same name as the independently identified and molecularly unrelated SNAP-25 is one of the great coincidences in modern biology), and VAMP/synaptobrevin. Rothman immediately proposed that VAMP is what he called a v-SNARE (SNARE stands for SNAP receptor) and syntaxin and SNAP-25 are t-SNAREs. The Rothman SNARE hypothesis holds that a v (vesicular)-SNARE associates with t (target)-SNAREs to form the molecular complex responsible for membrane fusion. Rothman noted, in support of his identification, that metaloproteases specific for VAMP, tetanus and botulinum B toxins, were both known to completely block neurotransmitter release. And because synaptotagmin (already proposed as the calcium sensor for neurotransmitter release [Brose et al., 1992]) interacts with syntaxin, he pointed out that this molecule could provide the required regulation for the constitutive fusion machinery used in membrane trafficking.
In the original SNARE hypothesis, the idea was that ATP hydrolysis by NSF probably was responsible for the membrane fusion event, but it later turned out that this action is instead used for dissociating the SNARE complex after fusion. The 1993 Rothman paper also identified some of the yeast genes that appear to be homologs of the mammalian fusion proteins, an idea developed more completely later that year in a review article (Bennett and Scheller, 1993). In the decade since 1993, evidence for the SNARE hypothesis has accumulated, and this is currently the conceptual framework for investigations of exocytosis. That said, it should be noted that the precise role of the SNAREs in exocytosis remains unclear (Duman and Forte, 2003). Exactly what forms the fusion pore is still uncertain (Peters et al., 2001), and this, together with how vesicle docking is controlled, are two of the most important unsettled questions.
The Calcium Hypothesis
One of Katz's large contributions was a beautiful series of experiments establishing his calcium hypothesis (Smith and Augustine, 1988). Since the work of Dodge and Rahamimoff (1967), neurobiologists had talked about the calcium sensor for release, but this hypothetical construct had no molecular reality. With the cloning of P65 (Perin et al., 1990), it became clear that this molecule, soon renamed synaptotagmin, was a good candidate because it consisted mainly of two C2 domains, motifs that are present to bind calcium ions in many different proteins. Because of its properties, synaptotagmin was proposed as the major calcium sensor controlling synaptic vesicle fusion (Brose et al., 1992), an idea that gained strong support from the analysis of mutant mice in which synaptotagmin I was deleted: these mice had no synchronous neurotransmitter release, but asynchronous release (and its calcium sensitivity) was completely preserved (Geppert et al., 1994); furthermore, the number of docked vesicles was normal (Geppert et al., 1997) and they could be released normally by a calcium-independent mechanism. Although many studies of synaptotagmin carried out in Drosophila have added greatly to our understanding of this molecule (Kidokoro, 2003; Tokuoka and Goda, 2003), certain differences between these results and those on mammalian synapses (Tokuoka and Goda, 2003) suggest that the mammalian central synapse is not always a very good model for the fly neuromuscular junction.
A recent analysis of the calcium binding pocket of the first C2 domain of synaptotagmin (C2A) (Stevens and Sullivan, 2003), carried out by rescue of hippocampal neurons from synaptotagmin knockout mice by overexpression of modified synaptotagmins, finds that neutralization of a negative charge at a specific aspartate residue is the same (with respect to changes in the Dodge-Rahamimoff equation) as binding two calcium ions. The authors interpret this to mean that two synaptotagmins are required to initiate fusion and that there are four binding sites for calcium ions (two per synaptotagmin), one of which is in the C2A domain and the other presumably is in C2B. If this interpretation is confirmed by further studies, Katz's calcium hypotheses and the quantitative model originally proposed by Dodge and Rahamimoff will have been an amazing insight.
Synapses at the Time of Neuron's 25th Birthday
Given the rapid progress of the past decade, I would predict that, by Neuron's 25th birthday, we will have cleared up some of the persisting uncertainties (what is a release site?) and will know what constitutes the fusion pore. We also should understand the molecular basis for historic effect of use on release probability (facilitation, augmentation, etc.), something that is still quite mysterious. Furthermore, we may understand how the synapse structure is maintained and how vesicles are moved around in the synapse and may, perhaps, even have a crystal structure of a fusion pore. But most likely, the really interesting advances will be the ones that cannot now be anticipated.
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