The further concentration of AMIGO-1 in high density clusters, which we estimate comprise < 20% of the PM area of a neocortical cell body, would further contribute to the robust AMIGO-1 immunolabeling signal from PM clustered AMIGO-1 as present in sections from WT mice relative to signal from predominantly intracellular AMIGO-1 present in dKO mouse sections. The relative impact of eliminating expression of Kv2.1 and Kv2.2 in the single and double KOs around the expression levels of AMIGO-1 may also provide valuable insights into the relative expression levels of these Kv2 subunits, information not available from Kv2.1- and Kv2.2-specific immunolabeling. fluorescence. The label depicts presence/absence of PK. Image2.TIF (1.2M) GUID:?6429F529-D96D-48C5-BAAD-C538B6494D36 Physique S3: Initial immunoblot used as the source for the representative immunoblot shown in Physique 11A. Representative immunoblot of crude whole brain homogenates from WT, Kv2.1 KO, Kv2.2 KO, and Kv2 double KO mice. Immunoblots were probed with mAbs against Kv2.1 (K89/34 mAb, green), Kv2.2 (N372B/60 mAb, red), AMIGO-1 (AMIGO-1 pAb, red), and Grp75 (N52A/42 mAb, green) as a loading control. The leftmost lane is usually prestained molecular excess weight standards, only some of which show up in fluorescence. Image3.TIF (3.4M) GUID:?854F1DEF-70B3-430D-A2B4-76CD65A8DB13 Abstract Voltage-gated K+ (Kv) channels play important functions in regulating neuronal excitability. Kv channels comprise four principal PDGFC subunits, and transmembrane and/or cytoplasmic auxiliary subunits that change diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Phenethyl alcohol Kv2.1-containing Kv channels. We show that AMIGO-1 is usually extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent populace of AMIGO-1 outside of that colocalized with these Kv2 subunits. AMIGO-1 is usually coclustered with Kv2 subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This unique PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is usually exclusively associated with Kv2 subunits, and that Kv2 subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering. and auxiliary subunit of Kv2.1-containing channels. However, the full extent of AMIGO-1 association with the Kv2.1 and Kv2.2 subunits in brain, and the role of Kv2 subunits in determining the expression and localization of AMIGO-1, has not been investigated. Here, we use newly developed and KO-validated anti-AMIGO-1 antibodies (Abs) to define the expression and colocalization of AMIGO-1 with Kv2.1 and Kv2.2 in adult brain. We also analyze the impact of the Kv2 subunits on expression and localization of AMIGO-1 in studies employing single and double Kv2.1 and Kv2.2 KO mice, and heterologous cells expressing WT and mutant Kv2 subunits. These studies reveal an important role for Kv2 channels Phenethyl alcohol in supporting the expression and localization Phenethyl alcohol of AMIGO-1 in adult brain neurons. Materials and methods Unless normally stated, all chemicals were from Sigma-Aldrich. Antibodies Antibodies used here are outlined in Table ?Table11. Table 1 Antibodies used in this study. counterstained with uranyl acetate, dehydrated and smooth embedded in Durcupan resin (ACM Fluka, Sigma-Aldrich). Ultrathin sections (70 nm) were collected Phenethyl alcohol on formvar coated single-slot copper grids, counterstained briefly with freshly prepared 1% lead citrate and analyzed using a Philips transmission electron microscope (EM208S) equipped with a MegaView III CCD video camera (Olympus-SIS). Heterologous cell culture and transfection HEK293 cells were managed in Dulbecco’s altered Eagle’s medium supplemented with 10% Fetal Clone III (HyClone), 1% penicillin/streptomycin, and 1X GlutaMAX (ThermoFisher). HEK293 cells were split to 15% confluence then transiently transfected 24 h later with the respective plasmids. These included plasmids encoding rat Kv2.1 (Frech et al., 1989; Shi et al., 1994) or the non-clustering rat Kv2.1 mutant S586A (Lim et al., 2000), and/or rat Kv2.2 (Kihira et al., 2010), or the non-clustering rat Kv2.2 mutant S605A (Bishop et al., 2015), all in the mammalian expression vector pRBG4 (Lee et al., 1991) and/or mouse AMIGO-1 in the mammalian expression vector PC DNA6 V5 His Version A (Peltola et al., 2011). Transfections were performed using LipofectAMINE 2000 (Invitrogen/ThermoFisher) transfection reagent following the manufacturer’s protocol. HEK293 cells were transfected in DMEM without supplements, then returned to regular growth media 4 h after transfection. For live cell imaging experiments, HEK293 cells were transiently transfected with the general ER marker SEC61-BFP, and DsRed-Kv2.1 and/or YFP-AMIGO-1 using the same approach. YFP-AMIGO-1 for.