Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1^K189-192A and Syt1^K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain–membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain–membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

Synaptotagmin-1 (Syt1) 是同步神经递质释放所需的囊泡钙传感器，由与两个 C2 结构域（C2A 和 C2B）连接的单程跨膜结构域组成，C2 结构域结合钙、酸性脂质和 SNARE 蛋白，驱动神经递质融合突触小泡与质膜。尽管 Syt1 具有重要作用，但人们对 Syt1 如何将钙进入与同步释放结合起来仍知之甚少。钙与 C2B 的结合对于同步释放至关重要，并且 C2B 还与 SNARE 复合物结合。 Syt1 功能也需要 C2A 域，但尚不清楚原因。在这里，我们询问 C2A 的哪些关键特征可能对其功能作用负责，并将其与 C2B 中的类似特征进行比较。我们重点关注位于 C2A (K189-192) 和 C2B (K324-327) 两侧的高度保守的聚赖氨酸斑块。我们并排测试了任一区域（Syt1 ^K189-192A和 Syt1 ^K326-327A ）中电荷中和突变的影响，以确定它们对来自任一性别小鼠的培养皮层神经元中的 Syt1 功能的相对贡献以及单分子实验。结合电生理记录和光镊测量来探测动态单 C2 结构域-膜相互作用，我们发现 C2A 和 C2B 多元斑块都有助于膜结合，并且两者都是诱发释放所必需的。易于释放的囊泡池的大小和自发释放的速率不受影响，因此这两个贴片可能专门用于同步释放。我们认为这些补丁有助于协同膜结合，增加 Syt1 对带负电膜的整体亲和力并促进诱发释放。

意义声明Synaptotagmin-1 是同步神经递质释放所需的囊泡钙传感器。其串联胞质 C2 结构域（C2A 和 C2B）结合钙、酸性脂质和 SNARE 蛋白，驱动突触小泡与质膜的融合。钙与 Synaptotagmin-1 结合如何导致释放以及 C2 结构域的相对贡献尚不清楚。结合培养神经元的电生理记录和单个 C2 结构域-膜相互作用的光镊测量，我们发现两个结构域中的保守多碱基区域有助于膜协同结合，并且两者都是诱发释放所必需的，可能是通过增加突触结合蛋白的整体亲和力来实现的。 1 用于酸性膜。