Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications

Aquaporin water channels in the nervous system.

The aquaporins (AQPs) are plasma membrane water-transporting proteins. AQP4 is the principal member of this protein family in the CNS, where it is expressed in astrocytes and is involved in water movement, cell migration and neuroexcitation. AQP1 is expressed in the choroid plexus, where it facilitates cerebrospinal fluid secretion, and in dorsal root ganglion neurons, where it tunes pain perception. The AQPs are potential drug targets for several neurological conditions. Astrocytoma cells strongly express AQP4, which may facilitate their infiltration into the brain, and the neuroinflammatory disease neuromyelitis optica is caused by AQP4-specific autoantibodies that produce complement-mediated astrocytic damage

Wide Scale Range Structure in Polyelectrolyte-protein Dense Complexes : Where Sans Meets Freeze-fracture Microscopy

International audienc

Localization and functional analysis of CHIP28k water channels in stably transfected Chinese hamster ovary cells.

CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proximal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced approximately 8 x 10(6) copies of CHIP28k protein/cell. Plasma membrane osmotic water permeability (Pf), measured by stopped-flow light scattering, was 0.004 cm/s in control (vector-transfected) cells (10 degrees C) and 0.014 cm/s in the CHIP28k-transfected cells. Pf in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgCl2. CHIP28k expression did not affect the transport of protons and the small polar non-electrolytes urea and formamide. CHIP28k immunoreactivity and function was then determined in subcellular fractions. Pf in 6-carboxyfluorescein-labeled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002 cm/s (control cells) and 0.011 cm/s (CHIP28k-transfected cells); Pf in transfected cells was inhibited by HgCl2. Immunoblotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (approximately 5000 monomers/microns 2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies establish a stably transfected somatic cell line that strongly expresses functional CHIP28k water channels. As in the original proximal tubule cells, the expressed CHIP28k protein is a selective water channel that is functional in endocytic vesicles and the cell plasma membrane

Expression of aquaporin-4 in fast-twitch fibers of mammalian skeletal muscle

In this study we analyzed the expression of aquaporin-4 (AQP4) in mammalian skeletal muscle. Immunohistochemical experiments revealed that affinity-purified AQP4 antibodies stained selectively the sarcolemma of fast-twitch fibers. By immunogold electron microscopy, little or no intracellular labeling was detected. Western blot analysis showed the presence of two immunopositive bands with apparent molecular masses of 30 and 32 kD specifically present in membrane fraction of a fast-twitch rat skeletal muscle (extensor digitorum longus, EDL) and not revealed in a slow-twitch muscle (soleus). PCR Southern blot experiments resulted in a selective amplification in EDL of a 960-bp cDNA fragment encoding for the full-length rat form of AQP4. Functional experiments carried out on isolated skeletal muscle bundle fibers demonstrated that the osmotic response is faster in EDL than in soleus fibers isolated from the same rat. These results provide for the first time evidence for the expression of an aquaporin in skeletal muscle correlated to a specific fiber-type metabolism. Furthermore, we have analyzed AQP4 expression in skeletal muscle of mdx mice in which a decreased density of orthogonal arrays of particles, a typical morphological feature of AQP4, has been reported. Immunofluorescence experiments showed a marked reduction of AQP4 expression suggesting a critical role in the membrane alteration of Duchenne muscular dystrophy

Effects of salt acclimation on water and urea permeabilities across the frog bladder: relationship with intramembrane particle aggregates.

In salt-acclimated frogs, water and urea bladder permeabilities are markedly higher than in tap water-acclimated animals. 2. Intra-membrane particle aggregates (IMPA) cover an unusually large surface area of the salt-acclimated frog bladder apical plasma membrane. 3. In saline-adapted animals, proteins extracted from the apical plasma membrane contain additional species of 19, 26, 31 and 53-61 kDa. These proteins might be related to the water channels contained by IMPA

Localization of the CHIP28 water channel in reabsorptive segments of the rat male reproductive tract

Abstract: The water channel protein CHIP28 is responsible for the high constitutive plasma membrane permeability to water of erythrocytes, renal proximal tubule, and thin descending limb of Henle. The male reproductive tract is embryologically related to kidney and some segments, particularly the efferent ducts, exhibit a high rate of solute flux-dependent reabsorption of luminal fluid. To determine whether this could occur through water channels in the plasma membrane of reproductive tract epithelial cells, we used anti-CHIP28 antibodies to localize this protein by Western blotting and immunocytochemistry. Western blotting of proteins from efferent duct homogenate indicated the presence of CHIP28 in the efferent duct cells. By indirect immunofluorescence and protein A-gold immunolabeling, CHIP28 was localized to the brush-border and basolateral membranes of nonciliated cells. Ciliated cells in the same epithelium showed no plasma membrane staining for CHIP28. In accord with immunocytochemical findings, freeze-fracture of nonciliated efferent duct cells revealed a plasma membrane organization resembling that of renal proximal tubule cells that are rich in CHIP28. The anti-CHIP28 antibodies also stained plasma membranes of epithelial cells in the ampulla of the vas deferens, seminal vesicles, and prostate, but not the cells in seminiferous tubules, epididymis, and proximal parts of the vas deferens. Therefore, CHIP28 may be a principal mediator of the transmembrane water transport in absorptive epithelial cells of efferent ducts, as well as in epithelia of several other segments of the male reproductive tract that show both secretory and reabsorptive functions