Mechanistic And Functional Insights Into Ankyrin-G-Mediated Protein Complex In Neuronal Polarity Maintenance And Signal Transmission

Neurons are highly polarized cells with distinct compartments responsible for vital physiological functions.The axon initial segment(AIS)is a specialized region connecting the soma and axon that is characterized by enrichment for diverse proteins including voltage-gated ion channels,cell adhesion molecules,cytoskeleton proteins,and a scaffold protein Ankyrin-G(AnkG).In addition to being responsible for the initial depolarization and generation of action potential,the AIS also serves as an axonal-somatodendritic filter,contributing to selective transport of specific molecules to somatodendrites or axons to maintain the neuronal polarity.At the AIS,AnkG is a master scaffold protein,which is recognized as the marker of the AIS in organizing and stabilizing diverse complexes that it forms with Nav channels,Kv channels,L1-family CAMs,and ??-spectrin.Loss of AnkG,either in vitro or in vivo,leads to axonal polarity disruption and causes axons to acquire dendritic properties.Meanwhile,the AIS is the site of action potential(AP)initiation in most neurons and is thus a critical site in the regulation of neuronal excitability.GABAA receptor is also enriched at the AIS.These AIS GABAergic synapses are essential for normal inhibitory neurotransmission which is thought to contribute to the tight control of AP.AnkG could stabilize GABAergic synapses through opposing endocytosis of GABAA receptors,but the underlying meachnism is still elusive.Given the essential physiological roles of AnkG in maintaining the axonal polarity and neuronal signal transmission,it would be interesting and meaningful to investigate how AnkG-related protein complexs control cargo selective transport and regulate action potential initiation at the AIS.Inspired by a previous study which demonstrated that AnkG-dependent localization of the dynein regulator Ndell/Ndel at the AIS is responsible for efficient local cargo reversal,we first biochemically and structurally characterized the interaction between AnkG and Ndell/Ndel in detail,and found that an 1897-1966 aa section of the AnkG giant insertion region strongly binds to the C-terminal coiled-coil domains of Ndell/Ndel,and does so with a molar ratio of 1:2.We found that the 440kDa giant AnkB(gAnkB),which is specifically expressed in neurons,also binds to Ndell/Ndel in a similar manner.We then solved the high-resolution crystal structure of AnkB in complex with Ndell CT-CC and revealed that AnkB binds with Ndell by forming a stable,five-helix bundle dominated by hydrophobic interactions spread across six distinct interaction layers.Moreover,we demonstrated that AnkG is essential for Ndell accumulation at the AIS.Finally,and supporting a gatekeeper function of AnkG at the AIS,We show that the sorting of three distinct somatodendritic cargoes,TfR,GluR1,and CAR,is disrupted in cells by blocking the AnkG/Nde1(Ndell)complex formation using a peptide designed based on their structural data.Collectively,the atomic structure of the AnkB/Ndell complex together with studies of cargo sorting through the AIS establish the mechanistic basis for AnkG/Ndel 1 complex formation and for the maintenance of the axonal polarity.Our study will also be valuable for future studies of the interaction between AnkB and Ndell perhaps at distal axonal cargo transport.Previous studies have reported that GABARAP can bind to the TM3-TM4 intracellular loop of GABAA receptor ?2 subunit with low affinities and GABARAP supports the stability of GABAA receptors on cell membranes,although the underlying molecular mechanisms are still not well understood.Here,we show that GABARAP directly binds to a previously unappreciated intracellular loop of the y2 subunit of the GABAA receptor.Our solved GABARAPL1-GABAA receptor crystal structure reveals the detailed interaction mechanisms underlying LIR-dependent complex formation.Moreover,we demonstrate that overexpression of GABARAP in cells leads to an increase in GABAA receptor-mediated responses,whereas mutations in either GABARAP or GABAA receptor which block complex formation eliminate these effects.GABARAP functions to stabilize GABA receptors via a trafficking pathway rather than by blocking endocytosis.Finally,we demonstrate that mIPSC amplitudes are decreased upon by blockage of the GABARAP/GABAA receptor complex using a peptide from giant ankyrins which indicate that the delivery of the ankyrin peptides into neurons specifically weakens the functions of postsynaptic GABAA receptor most likely through disrupting the synaptic GABARAP/GABAA receptor interaction.AnkG could compete for binding to GABARAP and then to release GABAA receptor form the GABARAP/GABAA receptor complex,which is thought to maintain the dynamic balance in the cell surface level of GABAA receptors.In summary,we have dissected the detailed interactions of two important protein complexes(AnkG/Ndell,GABARAP/GABAA receptor)involved in neuronal polarity maintenance and neuronal signal transmission,respectively,through combination of multiple biological methods.These findings would provide valuable insights and new molecular targets for the study and treatment of these protein complex-related nervous system diseases.