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Sunday. Gapjunctions I (525-530) 90a Membrane Domains and Polarity (520-522). Sunday 520 521 CHOLESTEROL VARIATION IN COCHLEAR OUTER HAIR CELL TRANSMEMBRANE LIPID ASYMMETRY AND FLIP-FLOP IN MEMBRANE DOMAINS DIABETIC ERYTHROCYTES ((Michael J. Wilson, Eric B. Sputh, Jason Cholesterol is an important membrane constituent that influences B. Butler, Delaina Eash, David L. Daleke.)) Medical Sciences Program, membrane fluidity and the activity of membrane-bound enzymes. Indiana University, Bloomington, IN 47405 Cholesterol is not uniformly distributed within the cochlear outer hair cell Exposure of non-diabetic erythrocytes to hyperglycemic conditions in vitro membrane. Functionally, this cell can be divided into three domains: the alters transmembrane phospholipid asymmetry, induces the expression of apex plays a role in mechanico-electrical transduction; the base is involved phosphatidylserine (PS) on the cell surface, and converts the cells to a pro- with synaptic transmission; and the lateral wall exerts electromotility, or coagulant state. Loss of asymmetry is accompanied by an increase in lipid rapid length changes in response to electrical energy. Using filipin, a oxidation and depletion of cellular o-tocopherol and can be suppressed with polyene fluorescent antibiotic that binds to cholesterol, we find that the a combination of lipophilic and hydrophilic antioxidants. Additional in vitro lateral wall contains less cholesterol than the apical and basal membranes. experiments have demonstrated that the likely mechanism is an increase in This difference in filipin fluorescence between the lateral walls and the ends passive lipid flip-flop; bidirectional movements of short chain analogs of each diminishes when cells are incubated in water-soluble cholesterol prior to class of endogenous erythrocyte phospholipid are increased 2-20 fold in cells staining, suggesting that exogenous cholesterol preferentially enters the treated with high concentrations of glucose. These results suggest that hyper- confocal we the lateral wall. Under microscopy, study incorporation pattern glycemia induces an oxidant-mediated increase in passive lipid flip-flop which a of fluorescent cholesterol analogue, NBD-cholesterol. The confocal results in an apparent loss of asymmetry. In the present work, we examine findings are consistent with the filipin results in that NBD-cholesterol does the oxidative status of, and transmembrane lipid movements in, erythrocytes while it labels the lateral wall. The not stain the apical membrane intensely isolated from diabetic individuals and rats. Lipid oxidation by-products were micropipette aspiration technique is used to assess the effect of cholesterol increased in diabetic erythrocytes but the activities of catalase and glutathione on lateral wall stiffness. The stiffness cholesterol-treated = of cells (n 23) is peroxidase were unchanged. Passive lipid flip-flop of di[C10:0]PC and [1-"C]- than that of controls = = 0.76 0.24 significantly higher (n 27): S + (mean 1-palmitoyl-lyso PC was increased in erythrocytes from diabetic animals and versus S = 0.46 + Student's = 5 E-6. In SD) 0.10, t-test, p conclusion, poorly-controlled diabetic humans, but not in well-controlled diabetics or an- cholesterol has different distributions among the three membrane domains, imals treated with insulin to normalize plasma glucose content. Furthermore, and when an additional amount is incorporated, cholesterol will decrease the erythrocytes from poorly controlled diabetic individuals displayed increased elasticity of the outer hair cell, which may affect electromotility. This project is supported by the American Academy of prothrombin converting activity than cells from well-controlled individuals. These results support a model in which glucose induces an oxidant-mediated Otolaryngology -- Head and Neck Surgery Foundation Resident Research Grant and the NIDCD/DC-00354 Grant. increase in transbilayer lipid movements that converts cells to a procoagulant state. These perturbations may contribute to the microvascular complications of the disease. 522 DETERGENT-RESISTANT, GLYCOSPHINGOLIPID-ENRICHED, MEMBRANOUS MICRODOMAINS CONTAIN BOTH THE CELLULAR AND SCRAPIE PRION PROTEINS. Martin Vey', Susanne Pilkuhnl, Holger Willel, Richard G. W. Anderson2, and Stanley B. Prusinerl. 1University of California, San Francisco, CA 94143; 2University of Texas Southwestern Medical Center, Dallas, TX 75235. GPI-anchored proteins cluster in detergent-resistant membranous microdomains called DIGs (detergent-insoluble, glycosphingolipid- enriched complexes) which are enriched for both cholesterol and glycosphingolipids. To determine if prion protein (PrP) also associates with these membrane domains, we used two independent methods for isolation of DIGs from scrapie-infected and uninfected mouse neuroblastoma cells. In the detergent-based method, cells were lysed in cold Triton-X100 and the DIGs were separated from the lysates by flotation in sucrose gradients. Alternatively, postnuclear supernatants were prepared from cell homogenates in the absence of detergent and fractionated on Percoll gradients. Isolated plasma membrane fractions were further disrupted by sonication and the DIGs separated from membranous debris by flotation in OptiPrep gradients. Analysis of DIGs showed that the cellular prion protein (PrPc)was concentrated in DIGs. Prion infection did not interfere with formation of DIGs and even after conversion, the pathologic, scrapie isoform PrPsc was still associated with them. The PrP 27-30 core of PrPSc was detected after limited digestion with proteinase K. Furthermore, a 17 kDa degradation product of PrPC could be identified. These results raise the possibility that prion propagation occurs in microdomains of cellular membranes which are enriched for cholesterol, glycosphingolipids and GPI-anchored proteins. GapJunctions I (523-524). 523 524 FREEZE-FRACTURE ANALYSIS OF GAP JUNCTIONS AND SQUARE ARRAYS AT DOCKING SPECIFICITY BETWEEN CONNEXINS IS MODIFIED BY THIE SUB-NANOMETER RESOLUTION. ((J. Rash and T. Yasumura)), Department of TERTIARY STRUCTURE OF THE EXTRACELLULAR LOOPS, INDEPENDENT Anatomy and Neurobiology, Colorado State University, Ft. Collins, CO 80523 OF PRIMARY SEQUENCE. ((Brnce J. Nicholson, Xing Zhu, Hui Zhu and Cynthia I. Foote)). Dept Biological Sciences, SUNY at Buffalo, Buffalo, NY 14260 A new method for deposition of continuous, subnanometer-thick platinum/carbon ((Jurgen films on freeze-fractured membranes from rat liver and spinal cord reveals structural Schwarz and Klaus Willecke)). Inst. for Genetik, U. Bonn., Germany. details smaller than 1 nm. Stereoscopic Images of gap junction P-faces reveal connexons as conical tubes 6-7 nm In diameter, each containing a central 2 nm The specificity of docking between liemichannels composed of different connexins dimple' at Its apex. The corresponding E-face pits are 7-8 nm In diameter, with to form an intact heterotypic gap junction is dictated by the two extracellular loops Intervening lipidic septa forming a continuous honeycomb of 2 nm-thick ridges, all of (El & E2). To achieve a better understanding of the docking process, we recently which fracture at the same height as the extrajunctbonal E-face lipids. Within each undertook systesadic movements ofthe 6 conseved cysteines within the extcellular E-face pit, a central 'peg' corresponding to the aqueous matrix of the connexon Ion loops (3 in ea) in order to map the pattern of disulfide bridges (all disulfides are channel (J. Coll Blol. 115: 190a, 1991) Is resolved. At 600 local shadowing angle, known to form within a single connexin). All single movements predictably produced each peg exhibits a 1.3 nm cap of platinum, whereas, at 90° shadowing angie, the non-functional channels whenpairedwidi wtCx32. Th pattenofpairedmiant diat pegs are resolved as cones with a basal diameter of 3 nm and projecting from a pit rescue coupling demonstrated that all 3 disulfides form between the loops that appear whose bottom has been reduced to 4 nm In diameter. Slmilar-quality replicas from to be in P-sheet confirmation based on the periodicity ofmovements. Among the non- spinal cord astrocytes reveal new details In E-face Images of square arrays". Unlike functional mutants, two revealed a surprising result (one single mutant and one In gap lipic Images square arrays those junctions, the septa within E-face of fracture inappropriately paired mutant). While these failed to pair with wtCx32, they now 1.5-2 nm below the surrounding fracture face. The E-face of square arrays are pits formed hoterotypic channels with XenCx38, a pairing which does not occur in the 5 nm In diameter, with each pit containing a smame, conical peg. At 600 local case of wtCx32. Thus, a change in the folding patten of these regions with minimal shadowing angle, the tip of each peg has a 0.7 nm diameter cap of platinum, which change the primary sequence causes a marked suppression of docking characteristics is one-half the diameter of the pegs in adjacent gap junctions. Stereoscopic Imaging and a concommitant modification ofvoltage gating. In independent experiments, we reveals that the two previousiy-identified morphologies of square arrays result examined docking specificity using a chimeric construct of Cx40 with El and E2 primarily from two primary patterns of deposition of platinum on diagonally- vs contributed by Cx43. wtCx40 does not form heterotypic junctions with either Cx43 perpendicularly-oriented rows of pits in the orthogonal lattices of 'square arrays". or As These data show that very thin
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