Aquaporin-4 (AQP4) can assemble into supramolecular aggregates called orthogonal arrays of particles (OAPs). differentially tagged AQP4 isoforms and in main glial cell ethnicities in which native AQP4 was labeled having a monoclonal recombinant Idasanutlin (RG7388) neuromyelitis optica autoantibody. OAP assembly was assessed individually by Blue Native gel electrophoresis. We found that OAPs in native glial cells could be reproduced in transfected cells expressing equivalent amounts of AQP4-M1 and -M23. Mutants of M23 that do not themselves form OAPs including M23-F26Q and M23-G28P were able to fully co-associate with Idasanutlin (RG7388) native M23 to form large immobile OAPs. Analysis of a palmitoylation-null M1 mutant (C13A/C17A) indicated palmitoylation-dependent OAP Idasanutlin (RG7388) assembly only in the presence of M23 with increased M1 palmitoylation causing progressive OAP disruption. Differential rules of OAP assembly by palmitoylation calcium elevation and protein kinase C activation was found in main glial cell ethnicities. We conclude that M1 and M23 co-assemble in AQP4 OAPs and that specific signaling events can regulate OAP assembly in glial cells. Intro Aquaporin-4 (AQP4)2 is the predominant water channel in the mammalian central nervous system where it is localized primarily in endfoot processes of pericapillary glial cells and in ependymal cells lining the ventricles (1 2 AQP4 provides the basic principle pathway for water entry into the mind and spinal cord through an undamaged blood-brain barrier in cytotoxic edema (3 -5) and the pathway for removal of excessive mind water in vasogenic edema and hydrocephalus (6 7 In addition to its central part in keeping in mind water balance AQP4 is also involved in neuroexcitation (8 9 and glial cell migration (10 11 AQP4 also Idasanutlin (RG7388) functions as a receptor for autoantibodies found in serum from individuals with the severe demyelinating autoimmune disease neuromyelitis optica (NMO) (12 13 Freeze-fracture electron microscopy of glial cell endfoot membranes offers revealed densely packed square arrays of intramembrane proteins known as orthogonal arrays of particles (OAPs) (14 15 Our laboratory first proposed that AQP4 forms OAPs based on its manifestation in the specific cell types where OAPs are seen (16). The involvement of AQP4 in OAP formation was verified by showing OAPs in AQP4-transfected cells (17) the absence of OAPs in Idasanutlin (RG7388) cells from AQP4 knock-out mice (18) and OAP labeling by AQP4 antibodies (2). The biological relevance of AQP4 assembly into OAPs is at present unknown. It has been speculated that OAPs might enhance AQP4 water permeability (19 -21) stabilize AQP4 polarization in glial cell foot-processes (22) and enhance glial cell-cell adhesion (23). The presence of OAPs has been correlated with neurological and neuromuscular diseases (24 25 and a recent Txn1 study suggested that OAPs contribute to the pathogenesis of NMO (26). The AQP4 transcript consists of at least two alternate translation initiation sites (27) yielding a “very long” (M1) isoform of ～34 kDa and a “short” (M23) isoform of ～31 kDa (28 -30) (Fig. 1(21 33 Number 1. Labeling strategy for two-color solitary particle tracking. (38). Briefly weighty- and light-chain constructs were co-transfected into HEK293 cells and the supernatant was harvested centrifuged to remove any cells and debris and then incubated immediately with protein A-Sepharose (Sigma) at 4 °C. The rAb was eluted in 0.1 m glycine 1 m NaCl (pH 3.0) and immediately adjusted to pH 7.5 by the addition of 0.1 m Tris-HCl (pH 8.0). Recombinant IgG was consequently exchanged and concentrated Idasanutlin (RG7388) in storage buffer (PBS plus 0.1% IgG/protease-free bovine serum albumin) using Ultracel YM-30 microconcentrators (Millipore Billerica MA). Antibody integrity was confirmed by gel electrophoresis and IgG concentration was determined by a human being IgG capture enzyme-linked immunosorbent assay. Purified rAb-53 showed specific binding to human being and mouse AQP4 in multiple self-employed assays (38). Immunostaining COS-7 cells transfected with Myc- and/or HA-tagged AQP4 isoforms were fixed with 4% paraformaldehyde and incubated for 20 min in obstructing buffer (PBS comprising 6 mm glucose 1 mm pyruvate 1 bovine serum albumin and 2% goat serum) and then for 20 min in 70 μg/ml mouse anti-Myc (Covance Emeryville CA) and rabbit anti-HA (Zymed Laboratories Inc. South San Francisco CA) antibodies in obstructing buffer. Cells were then rinsed and incubated another 20 min with 10 μg/ml goat anti-mouse.