文章基本信息

标题：Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A domain in asynchronous neurotransmitter release
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作者：Rashmi Voleti ; Diana R. Tomchick ; Thomas C. Südhof 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：40
页码：E8518-E8527
DOI：10.1073/pnas.1710708114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Synaptotagmins (Syts) act as Ca²⁺ sensors in neurotransmitter release by virtue of Ca²⁺-binding to their two C₂ domains, but their mechanisms of action remain unclear. Puzzlingly, Ca²⁺-binding to the C₂B domain appears to dominate Syt1 function in synchronous release, whereas Ca²⁺-binding to the C₂A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C₂A domain and C₂AB region, and analyses of intrinsic Ca²⁺-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca²⁺ saturating conditions, we show that the Syt7 C₂A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of l -α-phosphatidylinositol 4,5-diphosphate (PIP₂). For Syt1, the two Ca²⁺-saturated C₂ domains have similar affinities for membranes lacking PIP₂, but the C₂B domain dominates binding to PIP₂-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C₂A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C₂ domains in neurotransmitter release.
关键词：synaptotagmin-7 ; synaptotagmin-1 ; membrane binding ; X-ray crystallography ; neurotransmitter release