Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.

Water rapidly crosses the plasma membranes of red blood cells (RBCs) and renal tubules through highly specialized channels. CHIP28 is an abundant integral membrane protein in RBCs and renal tubules, and Xenopus laevis oocytes injected with CHIP28 RNA exhibit high osmotic water permeability, Pf [Preston et al. (1992) Science 256, 385-387]. Purified CHIP28 from human RBCs was reconstituted into proteoliposomes in order to establish if CHIP28 is itself the functional unit of water channels and to characterize its physiological behavior. CHIP28 proteoliposomes exhibit Pf which is up to 50-fold above that of control liposomes, but permeability to urea and protons is not increased. Like intact RBC, the Pf of CHIP28 proteoliposomes is reversibly inhibited by mercurial sulfhydryl reagents and exhibits a low Arrhenius activation energy. The magnitude of CHIP28-mediated water flux (11.7 x 10(-14) cm3/s per CHIP28) corresponds to the known Pf of intact RBCs. These results demonstrate that CHIP28 protein functions as a molecular water channel and also indicate that CHIP28 is responsible for most transmembrane water movement in RBCs.     

Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28

The localization and transporting properties of a kidney protein homologous to human erythrocyte protein CHIP28 was evaluated. The cDNA encoding rat kidney protein CHIP28k was isolated from a rat renal cortex cDNA library. A 2.8-kb cDNA was identified which contained an 807 bp open reading frame encoding a 28.8 kD protein with 94% amino acid identity to CHIP28. in vitro translation of CHIP28k cDNA in rabbit reticulocyte lysate generated a 28-kD protein; addition of ER-derived microsomes gave a 32-kD transmembrane glycoprotein. Translation of truncated RNA demonstrated glycosylation of residue Asn42 which is predicted to lie between the first and second transmembrane domains. Expression of in vitro transcribed mRNA encoding CHIP28k in Xenopus oocytes increased oocyte osmotic water permeability (Pf) from (4 +/- 1) x 10(-4) to (33 +/- 4) x 10(-4) cm/s at 10 degrees C; the increase in oocyte Pf was weakly temperature dependent and inhibited by HgCl2. Two- electrode voltage clamp measurements indicated that CHIP28k was not permeable to ions. Oocyte Pf also increased with expression of total mRNA from kidney cortex and papilla; the increase in Pf with mRNA from cortex, but not kidney papilla, was blocked by coinjection with excess antisense CHIP28k cRNA. In situ hybridization of a 150 base cRNA antisense probe to tissue sections from rat kidney showed selective CHIP28k localization to epithelial cells in proximal tubule and thin descending limb of Henle. Pf in purified apical membrane vesicles from rat and human proximal tubule, and in proteoliposomes reconstituted with purified protein, was very high and inhibited by HgCl2; stripping of apical vesicles with N-lauroylsarcosine enriched a 28-kD protein by 25-fold and yielded a vesicle population with high water, but low urea and proton permeabilities. CHIP28k identity was confirmed by NH2- terminus sequence analysis. These results indicate that CHIP28k is a major and highly selective water transporting protein in the kidney proximal tubule and thin descending limb of Henle, but not collecting duct.     

Functional reconstitution of the isolated erythrocyte water channel CHIP28.

Measurements of water permeability indicate the existence of a facilitated water transporting pathway in erythrocytes, kidney tubules and amphibian urinary bladder. Two lines of evidence suggest that one type of water channel is an approximately 30-kDa protein: the approximately 30-kDa target size determined by radiation inactivation (van Hoek, A. N., Hom, M. L., Luthjens, L. H., de Jong, M. D., Dempster, J. A., and van Os, C. H. (1991) J. Biol. Chem. 266, 16633-16635) and the increased water permeability in oocytes that express mRNA encoding a 28-kDa erythrocyte protein (CHIP28, Preston, B. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387). We report direct evidence that CHIP28 is the erythrocyte water channel. Osmotic water permeability (Pf) remained high (0.029 cm/s, 37 degrees C) when erythrocyte membranes were stripped of nearly all proteins except for CHIP28. N-terminal sequence analysis confirmed that the 28-kDa protein was CHIP28. Pf in proteoliposomes reconstituted with solubilized CHIP28 was high (Pf = 0.03 cm/s, 37 degrees C), the activation energy was low (2.2 kcal/mol), and Pf was decreased by greater than 50-fold by mercurial sulfhydryl reagents and Me2SO. The single-channel water permeability was approximately 10(-13) cm3/s, slightly higher than that of the gramicidin A channel. The water channel excluded the small solute urea. These data establish a procedure to reconstitute functional water channels into liposomes and demonstrate that CHIP28 is the erythrocyte water channel.     

CHIP28 water channels are localized in constitutively water-permeable segments of the nephron

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.     

Freeze-Fracture Analysis of Plasma Membranes of CHO Cells Stably Expressing Aquaporins 1-5

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1–5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10–20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in ∼5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13.5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays. Received: 17 March 1998/Revised: 19 June 1998     

Isolation of a cDNA for rat CHIP28 water channel: high mRNA expression in kidney cortex and inner medulla.

The cDNA coding for the rat CHIP28 water channel was isolated from a kidney library. At the amino acid level, rat CHIP28 is 93% identical to the recently published human protein (1). Expression of rat CHIP28 mRNA was highest in the renal inner medulla, unchanged during antidiuresis and twice the level expressed in outer cortex, with lower expression levels also apparent in parotid gland, urinary bladder and prostate. The evidence suggests that CHIP28 water channels in the ADH-sensitive collecting tubules are identical to those of the ADH-insensitive proximal convoluted tubules and possibly other tissues specialised in fluid transport.     

Myelinating glia of earthworm giant axons: thermally induced intramembranous changes

The median and lateral giant axons in the ventral nerve cord of the earthworm Lumbricus terrestris are ensheathed by extensive spiral glial cell wrappings which resemble vertebrate myelin. The other, smaller, axons are encompassed by attenuated glial processes, as is typical of invertebrates. The fine structural details of the glial cells have been studied in thin sections and in replicas produced by freeze-fracturing where the intramembranous particle (IMP) populations within the lipid bilayer are visible. These consist of both low-profile IMPs as well as prominent ones 6-8 nm in diameter, scattered at random over the lipid interface in the myelinating glia. The larger IMPs on both P and E faces number about 80/mum2 at 16 degrees C in contrast to the IMP density of 400/mum2 in the other glial membranes. After acclimation to 5, 16 and 26 degrees C, the loose myelin glial membranes show variations in the density of their larger IMP population; in animals acclimated over 3 or more weeks to 5 degrees C, the number of these IMPs is significantly (P less than 0.001) less per unit area than in animals acclimated to 16 or 26 degrees C. The size of the particles at 5 degrees C is significantly (P less than 0.001) smaller than those at 16 or 26 degrees C. When animals are subjected to a sudden differential in ambient temperature, from 26 or 16 to 5 degrees C, or from 5 to 26 degrees C, and their giant axons with encompassing glia are fixed and frozen 30 min after this temperature change, the IMP population of the glial membranes remaining does not appear to alter. The differences in the IMP population of the myelinating glial membranes at different temperatures may reflect the extent to which they insulate and/or influence the velocity of impulse propagation.     

Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes

Intramembrane particles (IMPs) of the plasmalemma of mature, synapsing neurons are evenly distributed along the axon shaft. In contrast, IMPs of growing olfactory axons form density gradients: IMP density decreases with increasing distance from the perikarya, with a slope that depends upon IMP size (Small, R., and K. H. Pfenninger, 1984, J. Cell Biol., 98: 1422-1433). These IMP density gradients resemble Gaussian tails, but they are much more accurately described by the equations formulated for diffusion in a system with a moving boundary (a Stefan Problem), using constants that are dependent upon IMP size. The resulting model predicts a shallow, nearly linear IMP density profile at early stages of growth. Later, this profile becomes gradually transformed into a steep nonlinear gradient as axon elongation proceeds. This prediction is borne out by the experimental evidence. The diffusion coefficients calculated from this model range from 0.5 to 1.8 X 10(-7) cm2/s for IMPs between 14.8 and 3.6 nm, respectively. These diffusion coefficients are linearly dependent upon the inverse IMP diameter in accordance with the Stokes-Einstein relationship. The measured viscosity is approximately 7 centipoise. Our findings indicate (a) that most IMPs in growing axons reach distal locations by lateral diffusion in the plasma membrane, (b) that IMPs-- or complexes of integral membrane proteins--can diffuse at considerably higher rates than previously reported for iso-concentration systems, and (c) that the laws of diffusion determined for macroscopic systems are applicable to the submicroscopic membrane system.     

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.     

The ultrastructural effects of β-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features.

Summary The fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of β- 1–40 (β-AP). PC12 cells treated with β-AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of β-AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and β-AP treated cells had two major P-face IMP populations, small-diameter (4–8 nm) IMPs, and large-diameter (≤ 9nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the β-AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the β-AP. These results demonstrate that β-AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.

The pre-steady-state kinetics of the vasopressin-induced increase in collecting tubule osmotic water permeability (Pf) has been measured by a new fluorescence technique. Isolated cortical collecting tubules (CCT) from rabbit kidney were perfused with physiological buffers containing the impermeant fluorophores fluorescein sulfonate (FS) and pyrenetetrasulfonic acid (PTSA). Tubules were subject to a 120 mOsm bath-to-lumen osmotic gradient in the presence and absence of 250 microU/ml vasopressin. The magnitude of transepithelial volume flow was determined from the self-quenching of FS, or from the ratio of PTSA/FS fluorescence, measured at 380 nm excitation and 420 +/- 10 nm (PTSA) and greater than 530 nm (FS) emission wavelengths. Pf was calculated from the magnitude of transepithelial volume flow, lumen and bath osmolarities, lumen perfusion rate, and tubule geometry. The instrument response time for a change in bath osmolality was less than 3 s. At 37 degrees C, CCT Pf was (in units of cm/s x 10(4] 13 +/- 2 (mean +/- SE, 16 tubules) before, and 227 +/- 10 after addition of vasopressin to the bath. CCT Pf began to increase in 23 +/- 3 s after vasopressin addition and was half-maximal after 186 +/- 20 s. At 23 degrees C, Pf was 9 +/- 1 (seven tubules) before, and 189 +/- 12 after vasopressin addition. Pf began to increase in 40 +/- 4 s and was half-maximal after 195 +/- 35 s. After vasopressin removal from the bath, Pf decreased to its baseline value with a half-time of 14 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell volume measured by total internal reflection microfluorimetry: application to water and solute transport in cells transfected with water channel homologs.

Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (approximately 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, Pf, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2-sensitive, threefold increase in Pf compared to nontransfected cells (Pf = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5) cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.     

Purification and Functional Reconstitution of the Human CHIP28 Water Channel Expressed inSaccharomyces cerevisiae

The yeastSaccharomyces cerevisiaewas used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus. Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.     

Gap junctions and rhombic particle arrays in the freeze-fractured mouse oocyte-follicle cell complex

Intact follicles as well as defolliculated oocytes of the mouse were studied by freeze-fracture electron microscopy. In intact follicles the oocyte plasma membrane shows two prominent types of intra-membrane particle array:gap junctions and yet undescribed rhombic particle arrays. The gap junctions vary in size (from 5 to 500 IMPs) and shape. Occasionally they are organized in so-called formation plaques. The rhombic particle arrays consist of 25 IMPs on an average, the IMP diameter is 10.5 nm, the mean IMP distance is 19.8 nm and the acute angle in the array is 81.3 degrees. After defolliculation the gap junctions disassemble and change transiently into linear IMP arrays. The rhombic particle arrays persist indicating that they are of a non-junctional nature. The possible function of the rhombic particle arrays is discussed in relation to similar membrane specializations in excitable cells.     

Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes

The existence and identity of protein water transporters in biological membranes has been uncertain. Osmotic water permeability (Pf) was measured in defolliculated Xenopus oocytes microinjected with water or mRNA from kidney cortex, kidney papilla, reticulocyte, brain, and muscle. Pf was measured by quantitative image analysis from the time course of oocyte swelling in response to an osmotic gradient. When assayed at 10 degrees C, Pf in water-injected oocytes increased from (3.6 +/- 0.9) x 10(-4) cm/s (S.D., n = 16) to 74 x 10(-4) cm/s with addition of amphotericin B, showing absence of unstirred layers. At 48-72 h after injection of 50 ng of unfractionated mRNA, Pf (in cm/s x 10(-4] was: 4.0 +/- 1.5 (rabbit brain, n = 15), 4.2 +/- 1.8 (rabbit muscle, n = 10), 18.4 +/- 6.3 (rabbit reticulocyte, n = 20), 16.1 +/- 5.6 (rat renal papilla, n = 24), 12.9 +/- 6.3 (rat renal cortex, n = 20), 14.4 +/- 6.1 (rabbit renal papilla, n = 15), and 11.8 +/- 3.4 (rabbit renal cortex, n = 8). In oocytes injected with mRNA from rat renal papilla, Pf was inhibited reversibly by 0.3 mM HgCl2 (4.1 +/- 1.6, n = 10); expressed water channels from kidney and red cell had activation energies of less than 4 kcal/mol. These results show functional oocyte expression of water channels from red cell, kidney proximal tubule (cortex), and the vasopressin-sensitive kidney collecting tubule (papilla), indicating that water channels are proteins, and providing an approach for the expression cloning of water channels.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional water channels are present in clathrin-coated vesicles from bovine kidney but not from brain

Targeting of water channels in renal epithelia may involve trafficking of clathrin-coated vesicles. We have isolated and measured the osmotic water permeability (Pf) of purified clathrin-coated vesicles from bovine kidney cortex and inner medulla, and bovine brain, a tissue not expected to contain "water channels." Brain-coated vesicles had a diameter of 80 nm in negatively stained preparations. Pf was measured by a stopped-flow light scattering technique. In brain-coated vesicles, water transport was functionally homogeneous with a low Pf of 0.0016 +/- 0.0001 cm/s (seven preparations, 23 degrees C). Pf was independent of osmotic gradient size (25-300 mOsm), not inhibited by mercurials, and not altered by removal of the clathrin coat. The activation energy (Ea) for Pf was high (11 +/- 1 kcal/mol less than 34 degrees C, 17 +/- 2 kcal/mol greater than 34 degrees C). Therefore, water channels are absent from brain-coated vesicles. In contrast, there were two functional populations of vesicles in coated vesicle preparations from both kidney cortex and medulla. One population of vesicles had low water permeability and no water channels, whereas a second population had high Pf (0.02 cm/s, 21 degrees C) that was inhibited by HgCl2, and low Ea (2-3 kcal/mol). The fraction of vesicles with high Pf was 52 +/- 3% (S.D., n = 3, cortical vesicles) and 26 +/- 3% (medullary vesicles). These results provide evidence that functional water channels are not present in clathrin-coated vesicles from the brain, whereas they are found in a population of coated vesicles from kidney cortex and medulla, tissues in which water channels are recycled between the plasma membrane, and an intracellular compartment.     

Freeze-fracture analysis of phloem structure in plant tissue cultures. II. The sieve element plasma membrane

Sieve element plasma membranes reveal a unique distribution of intramembrane particles (IMPs) in tissue cultures fixed and cyroprotected prior to freeze-fracturing. Sieve element IMPs are smaller than those found in the plasma membranes of callus parenchyma cells from these same cultures. The PF/EF ratio of plasma membrane IMPs is 9.6 for parenchyma cells and 1.21 for sieve elements. The increased binding of IMPs to the sieve element E face may be related to the role of membrane proteins in the loading of sucrose and other molecules by these cells. The enlargement of the cell wall at the site of sieve area pores creates complementary ridges and depressions in the E and P fracture faces of sieve element plasma membranes. No alteration of IMP density is seen at the sieve area pore site.