Gupta Julie P, Jenkins Paul M
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, United States.
Department of Psychiatry, University of Michigan Medical School, Ann Arbor, MI, United States.
Front Physiol. 2023 Mar 30;14:959660. doi: 10.3389/fphys.2023.959660. eCollection 2023.
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, Na1.2, 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 Na1.2. 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 Na1.2 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 Na1.2 at dendrites.
神经元锚蛋白B是一种细胞内支架蛋白，在轴突中发挥多种作用。相比之下，人们对锚蛋白B在树突中的功能了解较少，而在成熟神经元中，锚蛋白B也定位于树突。最近，我们发现锚蛋白B在新皮质锥体神经元的树突中作为电压门控钠通道Na1.2的支架。锚蛋白B自身如何靶向树突膜尚不清楚。在此，我们报告锚蛋白B通过S-棕榈酰化进行脂质修饰，以促进Na1.2在树突中的定位。我们确定棕榈酰酰基转移酶zDHHC17是异源细胞和神经元中锚蛋白B棕榈酰化的关键介质。此外，我们发现zDHHC17通过zDHHC17的ANK重复结构域与锚蛋白B的zDHHC锚蛋白重复结合基序之间的保守结合机制，独立于其S-酰化功能调节锚蛋白B的蛋白水平。我们随后在锚蛋白B的N端锚蛋白重复结构域中鉴定出五个半胱氨酸，它们是锚蛋白B棕榈酰化所必需的。将这五个半胱氨酸突变为丙氨酸不仅消除了锚蛋白B的棕榈酰化，还由于锚蛋白B无法正确定位于树突而阻止其在神经元树突膜上作为Na1.2的支架。因此，我们表明棕榈酰化对于锚蛋白B在树突中的定位和功能至关重要。令人惊讶的是，锚蛋白B棕榈酰化的缺失并不影响神经元中锚蛋白B介导的突触囊泡突触结合蛋白-1的轴突货物运输。这是首次证明锚蛋白B的S-棕榈酰化是锚蛋白B在树突中定位以及作为Na1.2支架发挥功能的潜在机制。
