DataSheet1_Ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic membrane scaffolding of voltage-gated sodium channel NaV1.2 in neurons.pdf

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B’s inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites.

神经元锚蛋白-B（neuronal ankyrin-B）是一类细胞内支架蛋白，在轴突中发挥多种功能。与之形成对比的是，尽管锚蛋白-B也定位于成熟神经元的树突中，目前对其在树突中的功能仍知之甚少。此前我们的研究显示，在新皮层锥体神经元的树突中，锚蛋白-B可作为电压门控钠通道NaV1.2的支架蛋白。然而，锚蛋白-B自身如何被靶向至树突膜的机制仍未明确。本研究表明，锚蛋白-B可通过S-棕榈酰化（S-palmitoylation）进行脂质修饰，从而促进NaV1.2的树突定位。我们鉴定出棕榈酰酰基转移酶zDHHC17是介导异源细胞与神经元中锚蛋白-B棕榈酰化的关键因子。此外，我们发现zDHHC17可通过其锚蛋白重复结构域（ANK repeat domain）与锚蛋白-B的zDHHC锚蛋白重复结合基序之间的保守结合机制，在不依赖其S-酰化功能的前提下调控锚蛋白-B的蛋白水平。后续我们在锚蛋白-B的N端锚蛋白重复结构域中鉴定出5个半胱氨酸残基，它们是锚蛋白-B棕榈酰化所必需的。将这5个半胱氨酸残基突变为丙氨酸残基后，不仅完全消除了锚蛋白-B的棕榈酰化修饰，还因锚蛋白-B无法正常定位于树突，使其无法在神经元树突膜处支架NaV1.2。因此，我们证实棕榈酰化修饰对于锚蛋白-B在树突中的定位与功能至关重要。值得注意的是，锚蛋白-B棕榈酰化的缺失并不会影响锚蛋白-B介导的神经元内突触囊泡突触结合蛋白-1的轴突货物运输。本研究首次证实，S-棕榈酰化是锚蛋白-B实现其树突定位，并在树突处支架NaV1.2的核心机制。