TETRAMERIC ASSEMBLY OF CHIP28 WATER CHANNELS IN LIPOSOMES AND CELL-MEMBRANES - A FREEZE-FRACTURE STUDY

Channel forming integral protein of 28 kD (CHIP28) functions as a wate r 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 recon stituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (P(f) 0.04 cm/s) that was inhibited by HgCl 2. Freeze-fracture replicas showed a fairly uniform set of intramembra ne particles (IMPs); no IMPs were observed in liposomes without incorp orated 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 sim ilar size and appearance were seen on the P-face of plasma membranes f rom CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight t ubules. A distinctive network of complementary IMP imprints was observ ed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by nonlinear regr ession, were (in IMPs/mum2): 2,494 in CHO cells, 5,785 in TDL, and 1,9 28 in proximal straight tubules; predicted P(f), based on the CHIP28 s ingle channel water permeability of 3.6 x 10(-14) cm3/s (10-degrees-C) , was in good agreement with measured P(f) 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 arrange ment 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 prov ide a morphological signature for CHIP28 water channels and evidence f or a tetrameric assembly of CHIP28 monomers in reconstituted proteolip osomes and cell membranes.