Freeze-fracture and immunogold analysis of aquaporin-4 (AQP4) square arrays, with models of AQP4 lattice assembly

Abstract

Each day, approximately 0.5-0.9 l of water diffuses through (primarily) aquaporin-1 (AQP1) channels in the human choroid plexus, into the cerebrospinal fluid of the brain ventricles and spinal cord central canal, through the ependymal cell lining, and into the parenchyma of the CNS. Additional water is also derived from metabolism of glucose within the CNS parenchyma. To maintain osmotic homeostasis, an equivalent amount of water exits the CNS parenchyma by diffusion into interstitial capillaries and into the subarachnoid space that surrounds the brain and spinal cord. Most of that efflux is through AQP4 water channels concentrated in astrocyte endfeet that surround capillaries and form the glia limitans. This report extends the ultrastructural and immunocytochemical characterizations of the crystalline aggregates of intramembrane proteins that comprise the AQP4 "square arrays" of astrocyte and ependymocyte plasma membranes. We elaborate on recent demonstrations in Chinese hamster ovary cells of the effects on AQP4 array assembly resulting from separate vs. combined expression of M1 and M23 AQP4, which are two alternatively spliced variants of the AQP4 gene. Using improved shadowing methods, we demonstrate sub-molecular crossbridges that link the constituent intramembrane particles (IMPs) into regular square lattices of AQP4 arrays. We show that the AQP4 core particle is 4.5 nm in diameter, which appears to be too small to accommodate four monomeric proteins in a tetrameric IMP. Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4. Keywordscross-bridges; furrows; FRIL; plastic deformation; water homeostasis Precise control of water homeostasis is critical in the brain and spinal cord because even minor changes in water metabolism may result in brain edema and rapid development of potentially fatal compressive forces within the rigid encasement of the skull (King and Agre, 1996). Moderate hyponatremia may result in failure of synaptic transmission or in altered neuronal excitability (Kandel et al., 1992;Jefferys, 1995;Nielsen et al., 1997) and is a contributing factor in the pathogenesis of epilepsy (Dudek et al., 1990;Roper et al., 1992), whereas severe hyponatremia contributes to medical complications that arise from brain and spinal cord NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript trauma, brain tumors, neonatal hydrocephalus, acute water intoxication, and drowning (Johnson and Loewy, 1990;King and Agre, 1996;Nielsen et al., 1997).Rates of secretion vs. resorption of cerebrospinal fluid (CSF) is a major controlling factor in osmoregulation of the CNS. The aqueous component of CSF passes primarily through aquaporin-1 (AQP1) in the choroid plexus epithelium , with the remainder arising from metabolism of glucose, from simple transmembrane diffusion through cell membrane lipid bilayers [reviewed by Agre et al. (2...

Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex

“…Electron microscopy data of 2D-AQP4 crystals indicates that AQP4, like all known water channels, is organized in the membrane in tetramers (Hiroaki et al 2006) which correspond to IMPs when visualized by freeze-fracture electron microscopy. Assuming that in astrocytes the number of IMPs in each OAP is 25 (Rash et al 2004), the predicted mass of an OAP is 3 MDa, a value which is close to the size of the largest AQP4 pool found in our BN/SDS-PAGE analysis. Furthermore, this large molecular weight pool appeared highly enriched with the M23 but not the M1 isoform of AQP4.…”

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

“…Astrocytes and their processes were identified by distinctive bundles of 10-nm "glial fibrillary acidic protein" (GFAP) filaments in their cytoplasms (but see cautionary note; Bushong et al, 2002), high density of intramembrane particles (IMPs) in both P-faces and E-faces, and by the presence of AQP4 "square arrays" in their plasma membrane P-faces and topographically "complementary" (Steere and Moseley, 1969;Challcroft and Bullivant, 1970) square arrays of pits in their plasma membrane E-faces (Landis and Reese, 1974;Rash et al, 1998bRash et al, , 2004aFurman et al, 2003). This report uses the internationally-recognized terminology for describing membrane faces in freeze-fracture replicas (Branton et al, 1975), wherein newly-cleaved and replicated "protoplasmic" fracture faces are designated by the mnemonic "P-face" and the newly-created "extraplasmic" leaflets (extracellular, extranuclear, extramitochondrial) are designated "E-faces".…”

“…Astrocyte processes were identified by the presence of GFAP filaments in their cytoplasms (Fig. 7C, asterisk) and by the presence of AQP4 square arrays (not shown) in their plasma membranes (Rash et al, 1998b(Rash et al, , 2004aFurman et al, 2003). Oligodendrocyte plasma membranes were characterized by the presence of one or more gap junctions that were surrounded by discontinuous rows of E-face IMPs (Fig.…”

Rôles des aquaporines dans le cerveau

“…Des expériences récentes de cocultures ont montré que la présence de cellules endothéliales était nécessaire pour induire cette polarisation [11]. L'AQP4 périvasculaire est localisée dans des figures géométriques observables en microscopie électronique après cryo-fracture, les OAP (orthogonal arranged particles) [12]. Les OAP sont localisées d'une part dans les pieds astrocytaires en contact avec des vaisseaux, et d'autre part, dans les pieds astrocytaires de la glie limitante en contact avec le LCR [12].…”

“…L'AQP4 périvasculaire est localisée dans des figures géométriques observables en microscopie électronique après cryo-fracture, les OAP (orthogonal arranged particles) [12]. Les OAP sont localisées d'une part dans les pieds astrocytaires en contact avec des vaisseaux, et d'autre part, dans les pieds astrocytaires de la glie limitante en contact avec le LCR [12]. La polarisation de l'AQP4 sur les pieds astrocytaires ( Figure 3C) se fait au moyen d'un ancrage de cette protéine par un domaine PDZ sur l'α-syntrophine (α-Syn) ou la dystrophine [13].…”