title: Neuromyelitis optica IgG does not alter aquaporin-4 water permeability, plasma membrane M1/M23 isoform content, or supramolecular assembly.
abstract: Neuromyelitis optica (NMO) is thought to be caused by immunoglobulin G autoantibodies (NMO-IgG) against astrocyte water channel aquaporin-4 (AQP4). A recent study (Hinson et al. (2012) Proc Natl Acad Sci USA 109:1245-1250) reported that NMO-IgG inhibits AQP4 water permeability directly and causes rapid cellular internalization of the M1 but not M23 isoform of AQP4, resulting in AQP4 clustering, enhanced complement-dependent cytotoxicity, and tissue swelling. Here, we report evidence challenging this proposed mechanism of NMO-IgG-mediated pathology. We measured osmotic water permeability by stopped-flow light scattering on plasma membrane vesicles isolated from AQP4-expressing CHO cells, an approach that can detect changes in water permeability as small as 5% and is not confounded by internalization effects. We found similar single-molecule water permeability for M1-AQP4 tetramers and M23-AQP4 clusters (orthogonal arrays of particles, OAPs). Exposure of AQP4 to high concentrations of NMO-IgG from six seropositive NMO patients, and to high-affinity recombinant monoclonal NMO antibodies, did not reduce AQP4 water permeability. Also, NMO-IgG did not reduce water permeability in AQP4-reconstituted proteoliposomes. In transfected cells expressing M1- or M23-AQP4 individually, NMO-IgG caused more rapid internalization of M23- than M1-AQP4. In cells coexpressing both isoforms, M1- and M23-AQP4 comingled in OAPs that were internalized together in response to NMO-IgG. Super-resolution imaging and native gel electrophoresis showed that the size of AQP4 OAPs was not altered by NMO sera or recombinant NMO antibodies. We conclude that NMO-IgG does not: (i) inhibit AQP4 water permeability, (ii) cause preferential internalization of M1-AQP4, or (iii) cause intramembrane AQP4 clustering.
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rossi
authors.forename: Julien
authors.initials: J
authors.lastname: Ratelade
authors.forename: Marios C
authors.initials: MC
authors.lastname: Papadopoulos
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Bennett
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D051401:Aquaporin 4 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001323:Autoantibodies / Q000502:physiology*; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism* / Q000473:pathology; D002463:Cell Membrane Permeability / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000502:physiology*; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D009471:Neuromyelitis Optica / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000378:metabolism
chemical_list: C496030:AQP4 protein, human; D000911:Antibodies, Monoclonal; D051401:Aquaporin 4; D001323:Autoantibodies; D007074:Immunoglobulin G; D046912:Multiprotein Complexes; D020033:Protein Isoforms; D014867:Water