The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 "long" (M1) and "short" (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Singlemolecule step photobleaching and intensity analysis of GFPlabeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37°C indicated that OAPs diffuse slowly (D ϳ 10 ؊12 cm 2 /s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramertetramer interactions. Aquaporin-4 (AQP4)2 water channels are expressed in glial cells throughout the central nervous system and in skeletal muscle, lung, kidney, stomach, and exocrine glands. In brain, AQP4 is involved in water balance, neuroexcitation, and glial cell migration (reviewed in Ref. 1). AQP4 deletion in mice improves clinical outcome in models of ischemic stroke (2), bacterial meningitis (3), and compression spinal cord injury (4) and alters seizure dynamics (5). AQP4 autoantibodies are found in the sera of most patients with the multiple sclerosis variant neuromyelitis optica (6), in which AQP4 autoantibodies are involved in the pathogenesis of central nervous system neuroinflammation (7).At the molecular level, AQP4 is expressed as two major isoforms, a short isoform (M23) with a translation initiation site at Met-23 and a longer isoform (M1) that initiates translation at Met-1. Additional AQP4 isoform(s) may be present at low levels, at least in rat brain (8). Like other aquaporins, AQP4 forms tetramers in membranes (9). AQP4 tetramers can form supramolecular assemblies called orthogonal arrays of particles (OAPs), which are regular square arrays of intramembrane particles seen by freeze-fracture electron microscopy in brain, skeletal muscle, and kidney (10). Our laboratory discovered that AQP4 is the OAP-forming protein by demonstrating OAPs in M23-transfected cells (11) and the absence of OAPs in brain and other tissues from AQP4 knock-out mice (12). Labelfracture electron microscopy subsequently confirmed AQP4 in OAPs (13). We found by quantum dot/single-particle tracking that M1, when expressed alone, diffuses f...
Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.
Aquaporins (AQPs) are membrane water channels that are involved in a diverse set of functions in mammalian physiology including epithelial fluid transport, brain water balance, cell migration, cell proliferation, neuroexcitation, fat metabolism, epidermal hydration, and others. Phenotype analysis of knockout mice has demonstrated an important role for AQPs in transepithelial fluid transport in kidney tubules, salivary and airway submucosal glands, choroid plexus and ciliary epithelium. The physiological functions of these epithelia, such as absorption of glomerular filtrate by proximal tubule and secretion of saliva by salivary gland, involve rapid transcellular water transport across epithelial cell barriers. Studies in knockout mice have also provided evidence that AQPs are not physiologically important in some epithelia where they are expressed, including lacrimal gland, sweat gland, gallbladder, alveoli and airways. Rates of transepithelial fluid transport per unit membrane surface area in these epithelia are substantially lower than transepithelial fluid transport rates in proximal tubule and salivary gland. Pharmacological inhibition of AQP water permeability in epithelia, with consequent reduced fluid transport, offers potential therapy for human diseases involving water imbalance such as congestive heart failure, hypertension and glaucoma.
Orthogonal arrays of particles (OAPs) have been visualized for many years by freeze-fracture electron microscopy. Our laboratory discovered that aquaporin-4 (AQP4) is the protein responsible for OAP formation by demonstrating OAPs in AQP4-transfected cells and absence of OAPs in AQP4 knockout mice. We recently developed live-cell, single-molecule imaging methods to study AQP4 diffusion and interactions in OAPs. The methods include single particle tracking of quantum-dot labeled AQP4, and total internal reflection fluorescence microscopy of green fluorescent protein (GFP) and small fluorophore-labeled AQP4. The full-length (M1) form of AQP4 diffuses freely in membranes and does not form OAPs, whereas the shorter (M23) form of AQP4 forms OAPs and is nearly immobile. Analysis of a series of AQP4 truncations, point mutants and chimeras revealed that OAP formation by AQP4-M23 is stabilized by hydrophobic tetramer-tetramer interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from blocking of this interaction by residues just upstream from Met23. These biophysical methods are being extended to identify the cellular site of AQP4 assembly, AQP4 isoform interactions, OAP size and dynamics, and the determinants of regulated OAP assembly.
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