title: Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study.
abstract: Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.
authors.affiliation: Renal Unit, Massachusetts General Hospital.
authors.forename: J M
authors.initials: JM
authors.lastname: Verbavatz
authors.forename: D
authors.initials: D
authors.lastname: Brown
authors.forename: I
authors.initials: I
authors.lastname: Sabolić
authors.forename: G
authors.initials: G
authors.lastname: Valenti
authors.forename: D A
authors.initials: DA
authors.lastname: Ausiello
authors.forename: A N
authors.initials: AN
authors.lastname: Van Hoek
authors.forename: T
authors.initials: T
authors.lastname: Ma
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D006224:Cricetinae; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005614:Freeze Fracturing; D007473:Ion Channels / Q000378:metabolism / Q000648:ultrastructure; D007672:Kidney Cortex / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism / Q000648:ultrastructure; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification / Q000648:ultrastructure; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D008961:Models, Structural; D011510:Proteolipids / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D014162:Transfection
chemical_list: C496022:Aqp1 protein, rat; D020346:Aquaporins; D007473:Ion Channels; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D051398:Aquaporin 1