Li LiYi, Shin Ok-Ho, Rhee Jeong-Seop, Araç Demet, Rah Jong-Cheol, Rizo Josep, Südhof Thomas, Rosenmund Christian
Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 2006 Jun 9;281(23):15845-52. doi: 10.1074/jbc.M600888200. Epub 2006 Apr 4.
Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.
钙离子依赖的磷脂与突触结合蛋白1的C2A和C2B结构域的结合被认为可触发快速神经递质释放，但只有钙离子与C2B结构域的结合对释放至关重要。为了研究其潜在机制，我们比较了碱性残基在钙离子/磷脂结合及释放过程中的作用。C2B结构域β折叠一侧的多碱性序列中的突变，或C2A结构域顶部钙离子结合环中的一个碱性残基（R233）的突变，会导致突触结合蛋白1的表观钙离子亲和力及释放的钙离子敏感性出现类似程度的降低，而C2B结构域中与Arg233同源的残基（Lys366）的突变则没有影响。磷脂酰肌醇多磷酸共同激活钙离子依赖和非依赖的磷脂与突触结合蛋白1的结合，但这些突变对释放的影响仅与其对钙离子依赖成分的影响相关。这些结果揭示了突触结合蛋白1 C2结构域在钙离子依赖的磷脂结合模式上的明显差异，这可能是其功能不对称的基础，并表明磷脂酰肌醇多磷酸可能在体内作为突触结合蛋白1钙离子亲和力的生理调节剂。
