Aquaporin water channels: unanswered questions and unresolved controversies.

The long-standing biophysical question of how water crosses plasma membranes has been answered by the recent discovery of the aquaporins. Identification of this large family of membrane water-transport proteins has generated new questions about the physiological functions, tissue distributions, and regulatory mechanisms of individual aquaporins. The fast pace of developments in this field has also resulted in major discrepancies in published reports which warrant resolution.

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.

Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and C/EBP.

Both C/EBP and GCN4 are sequence-specific DNA binding proteins that control gene expression. Recent evidence implicates C/EBP as a transcriptional regulator of genes involved in lipid and carbohydrate metabolism. The C/EBP protein binds avidly to the dyad symmetric sequence 5'-ATTGCGCAAT-3'; GCN4 regulates the transcription of genes that control amino acid biosynthesis in yeast, and binds avidly to the dyad symmetric sequence 5'-ATGA(G/C)TCAT-3'. Both C/EBP and GCN4 bind DNA via the same structural motif. This motif has been predicted to be bipartite, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." Specificity of DNA binding has been predicted to be imparted by the basic region. As a test of this hypothesis, recombinant proteins were created wherein the basic regions and leucine zippers of GCN4 and C/EBP were reciprocally exchanged. In both of the recombinant polypeptides, DNA binding specificity is shown to track with the basic region.

Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein.

Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.

Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

Aquaporins: water channel proteins of plant and animal cells.

Certain biological membranes, such as the erythrocyte plasma membrane, have a high osmotic water permeability, and such membranes have long been suspected of harboring water channels. The molecular identity of these channels has now been established with the purification of water-channel proteins and the cloning of the genes encoding them. Homologous water-channel proteins, called 'aquaporins', are present in plants and animals. These channels are water selective and do not allow ions or metabolites to pass through them. Their discovery is providing new insights into how plant and animal cells facilitate and regulate the passage of water through their membranes.

Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis.

Whereas marked variations in the clinical manifestations of hereditary spherocytosis have long been recognized, we have only recently begun to define the molecular basis for this heterogeneity. An important unanswered question is whether decreased spectrin results in reduced membrane mechanical stability, and if this reduction in membrane mechanical stability can be related to in vivo surface area loss. Using the ektacytometer, we quantitated membrane surface area and stability in erythrocytes from 18 individuals with hereditary spherocytosis and deficiencies of spectrin (30-80% of normal spectrin level). Membrane mechanical stability was reduced and the magnitude of the reductions correlated with the spectrin content. Moreover, the reductions in mechanical stability correlated with in vivo loss of membrane surface area. These data indicate that decreased spectrin content results in reduced membrane mechanical stability and surface area loss in vivo. We conclude that partial deficiencies of spectrin, reductions in membrane mechanical stability, and loss of membrane surface area are directly related and are major features determining the heterogeneous clinical manifestations of hereditary spherocytosis.

Reductions of erythrocyte membrane viscoelastic coefficients reflect spectrin deficiencies in hereditary spherocytosis.

Hereditary spherocytosis is a common hemolytic anemia associated with deficiencies in spectrin, the principal structural protein of the erythrocyte membrane-skeleton. We have examined 20 different individuals from 10 spherocytosis kindreds and 2 elliptocytosis kindreds to determine the effects of different levels of spectrin deficiency on the viscoelastic properties of the erythrocyte membrane. Micropipettes were used to perform single-cell micromechanical measurements of approximately 1,000 individual cells to determine the membrane elastic shear modulus, the apparent membrane bending stiffness, and whole cell recovery time constant for the different cell populations. The membrane viscosity was calculated by the product of the shear modulus and the recovery time constant. Results show correlation between the fractional reduction in shear modulus and the fractional reduction in spectrin content (determined by spectrin radioimmunoassay) and spectrin density (determined by the ratios of spectrin to band 3 on electrophoresis gels) suggesting that membrane shear elasticity is directly proportional to the surface density of spectrin on the membrane (P less than 0.001). The apparent membrane bending stiffness is also reduced in proportion to the density of spectrin (P less than 0.001). The membrane viscosity is reduced relative to control (P less than 0.001), but the nature of the relationship between spectrin density and membrane viscosity is less clearly defined. These studies document striking relationships between partial deficiencies of erythrocyte spectrin and specific viscoelastic properties of the mutant membranes.

Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes.

Two new vascular smooth muscle relaxants, bepridil and cetiedil, were found to possess specific CaM-inhibitory properties which resembled those of trifluoperazine. Trifluoperazine, bepridil, and cetiedil inhibited Ca2+-dependent 125I-CaM binding to erythrocyte membranes and CaM activation of membrane Ca2+-ATPase with IC50 values of approximately 12, approximately 17, and approximately 40 microM, respectively. This does not appear to be the result of a nonspecific hydrophobic interaction since inhibition was not observed with micromolar concentrations of many other hydrophobic agents. The predominant inhibition of binding and Ca2+-ATPase activation was competitive with respect to CaM. Bepridil and cetiedil bind directly to CaM since these drugs displaced [3H]trifluoperazine from sites on CaM. Inhibition of Ca2+-ATPase and binding by the drugs was not due to interference with the catalytic activity of this enzyme since: (a) neither inhibition of CaM-independent basal Ca2+-ATPase activity nor inhibition of proteolytically-activated Ca2+-ATPase activities were produced by these agents, and (b) no drug-induced inhibition of CaM binding was detected when membranes were preincubated with these agents but washed prior to addition of 125I-CaM. Thus, bepridil and cetiedil competitively inhibit Ca2+-dependent interactions of CaM with erythrocyte membranes, most likely by a direct interaction between these drugs and CaM. The principal clinical actions of these drugs may be explained by their interactions with CaM or CaM-related proteins leading to reduced activation of Ca2+-regulated enzymes in certain other tissues, such as myosin light chain kinase in vascular smooth muscle.

Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat.

At birth water is rapidly reabsorbed from the distal lung in preparation for alveolar gas exchange. To investigate a potential role for the AQP1 water channel in development, lung membranes from fetal and perinatal rats were analyzed by immunoblot. First expression of AQP1 was noted in fetal rat lung at E19 (19th day of the 21-day gestation). The level of AQP1 increased fivefold from the last gestational day to the first postnatal day and persisted at high levels into adulthood. Maternal corticosteroids increased expression of AQP1 in fetal lung, an effect also seen in adult rats. AQP1 mRNA increased in rat pups treated with corticosteroids, suggesting at least partial regulation at the level of transcription. Immunohistochemical analyses with anti-AQP1 demonstrated the protein in peribronchial vessels and visceral pleura at E21 with increased postnatal expression. AQP1 was not expressed in airway epithelium, and only occasional alveolar pneumocytes were labeled. Immunoelectron microscopy revealed AQP1 on both apical and basolateral membranes of endothelial cells. The ontogeny and corticosteroid induction of AQP1 in rat lung coincide with major physiological alterations in lung development; however, the distribution of AQP1 predicts the existence of other water channels in the alveolar epithelium.

Isolation of proteins related to the Rh polypeptides from nonhuman erythrocytes.

It is thought that the Rh antigens may be important in maintaining normal erythrocyte membrane integrity. Despite their name, Rh antigens are serologically present only on human erythrocytes. Rh structural polymorphisms are known to reside within a family of nonglycosylated Mr 32,000 integral membrane proteins that can be purified by hydroxylapatite chromatography. Mr 32,000 integral membrane proteins were purified similarly from erythrocyte membrane vesicles prepared from rhesus monkeys, cows, cats, and rats, but could not be purified from human Rhmod erythrocytes, a rare syndrome lacking Rh antigens. The purified Mr 32,000 polypeptides were labeled with 125I, digested with chymotrypsin, and found to be 30-60% identical to human Rh polypeptides when compared by two-dimensional iodopeptide mapping. The physiologic function of the Rh polypeptides remains to be identified; however, the existence of related proteins in nonhuman erythrocytes supports the concept that the Rh polypeptides are erythrocyte membrane components of fundamental significance.

Differential control of band 3 lateral and rotational mobility in intact red cells.

Measurements of integral membrane protein lateral mobility and rotational mobility have been separately used to investigate dynamic protein--protein and protein-lipid interactions that underlie plasma membrane structure and function. In model bilayer membranes, the mobilities of reconstituted proteins depend on the size of the diffusing molecule and the viscosity of the lipid bilayer. There are no direct tests, however, of the relationship between mechanisms that control protein lateral mobility and rotational mobility in intact biological membranes. We have measured the lateral and rotational mobility of band 3 in spectrin-deficient red blood cells from patients with hereditary spherocytosis and hereditary pyropoikilocytosis. Our data suggest that band 3 lateral mobility is regulated by the spectrin content of the red cell membrane. In contrast, band 3 rotational mobility is unaffected by changes in spectrin content. Band 3 lateral mobility and rotational mobility must therefore be controlled by different molecular mechanisms.

Quantification of Aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.

Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, the actual abundance of these molecules has not been established. To accomplish this goal, we have developed a fluorescence-based ELISA method and have used it to quantitate Aquaporin-CHIP (AQP-CHIP) water channel protein in rat kidney tubules. Microdissected tubules (2 mm/sample, permeabilized with 0.5% Triton X-100) or purified AQP-CHIP standards (0-200 fmol) were utilized in a fluorescence ELISA protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit of detection was 2.4 fmol of AQP-CHIP. Preabsorption with excess purified AQP-CHIP or use of nonimmune serum eliminated the signal. In proximal segments, the measured AQP-CHIP was linearly related to tubule length (1-10 mm). The measured AQP-CHIP was (mean +/- SE, fmol/mm): S-1 proximal, 10.8 +/- 2.1; S-2, 10.0 +/- 2.3; S-3, 21.3 +/- 3.1; type 1 thin descending limb (DTL), 12.9 +/- 4.6; type 2 DTL, 86.5 +/- 19.5; type 3 DTL, 43.0 +/- 11.2. In thin ascending limbs, thick ascending limbs, distal convoluted tubules, connecting tubules, and collecting ducts, the AQP-CHIP signal was indistinguishable from zero. Based on the unit water conductance of single CHIP molecules, our calculations show that the content of AQP-CHIP is sufficient to explain water permeability measured in isolated proximal tubules and DTL segments.

A molecular defect in two families with hemolytic poikilocytic anemia: reduction of high affinity membrane binding sites for ankyrin.

Patients from two families with chronic hemolytic anemia have been studied. The erythrocytes are very fragile and appear microcytic with a great variety of shapes. Clinical evaluation failed to identify traditionally recognized causes of hemolysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed no significant abnormality of the major polypeptide bands. Erythrocytes spectrin-ankyrin and ankyrin-membrane interactions were analyzed with 125I-labeled spectrin, 125I-labeled ankyrin, and inside-out vesicles. Patients' vesicles bound 125I-spectrin normally. Likewise, patients' spectrin and ankyrin competed normally for the binding sites on control membranes. None of the individual components appeared to have abnormal thermal sensitivity. Ankyrin-stripped, inside-out vesicles prepared from the patients bound less 125I-ankyrin than did vesicles prepared from normals (P less than 0.05 for all corresponding points in the high-affinity region). Scatchard analysis showed the most significant abnormality to be a 50% reduction in the high affinity ankyrin binding sites. Similar experiments were performed with blood from patients with spherocytosis and splenectomized controls, but no abnormalities were detected. The water soluble 43,000-dalton fragments of band 3 (the high-affinity ankyrin binding sites) were prepared from one of the patients and competed normally for 125I-ankyrin binding in solution. This suggests that the primary structural defect is a reduction in the number of high affinity membrane binding sites for ankyrin, and is consistent with an abnormal organization of band 3 in the membrane.

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. II. Determination of molecular genetic origins of rearrangements.

Protein 4.1 is an approximately 80-kD structural protein in the membrane skeleton which underlies and supports the erythrocyte plasma membrane. The preceding companion paper presents a biochemical study of two abnormal protein 4.1 species from individuals with the red blood cell disorder, hereditary elliptocytosis. These variants, "protein 4.1(68/65)" and "protein 4.1(95)," have altered molecular weights due to internal deletions and duplications apparently localized around the spectrin-actin binding domain. Here we use polymerase chain reaction (PCR) techniques to clone and sequence the corresponding mutant reticulocyte mRNAs, and correlate the deletion/duplication end points with exon boundaries of the gene. Protein 4.1(68/65) mRNA lacks sequences encoding the functionally important spectrin-actin binding domain due to a 240 nucleotide (nt) deletion spanning the codons for Lys407-Gly486. Protein 4.1(95) mRNA encodes a protein with two spectrin-actin binding domains by virtue of a 369 nt duplication of codons for Lys407-Gln529. These deletions and duplications correspond to gene rearrangements involving three exons encoding 21, 59, and 43 amino acids, respectively. The duplicated 21 amino acid exon in the 4.1(95) gene retains its proper tissue-specific expression pattern, being spliced into reticulocyte 4.1 mRNA and out of lymphocyte 4.1 mRNA.

Mutant forms of spectrin alpha-subunits in hereditary elliptocytosis.

Two variant spectrins have been described in hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP). Both are characterized by increased susceptibility of the alpha I (N-terminal) 80-kD domain to mild tryptic digestion, yielding peptides of 46-50 or 65-68 kD (T50a and T68 in our terminology). In this report we add a third unstable spectrin alpha I domain found in three kindreds with HE; alpha IT80 in this type of spectrin is cleaved by mild tryptic digestion to a 50-kD peptide (T50b) distinguished from T50a by its more basic isoelectric point. All three spectrins show impaired self-association to form oligomers. Intermediate tryptic peptides of the three unstable alpha I domains from HE spectrins were characterized by monoclonal immunoblotting and I125 limit peptide mapping and affinity purified using polyclonal anti-alpha IT80. Partial amino acid sequences of alpha I domain peptides were obtained from two unrelated patients for each of the three variant spectrins. T50a results from cleavage at arginine 250 or lysine 252 of alpha IT80; a proline replaced the normal leucine or serine at residues 254 and 255, respectively. T50b and a 19-kD peptide result from cleavage at arginine 462 or arginine 464; a proline replaced the normal residue 465 (in T19b) in one of the two patients studied. T68 results from cleavage at arginine 131. In both 68-kD peptides examined, a leucine is inserted at residue 150. The relationship of the sequence changes to the new tryptic cleavages, to the current model of alpha I domain structure, and to defective spectrin self-association is discussed.

Concurrent expression of erythroid and renal aquaporin CHIP and appearance of water channel activity in perinatal rats.

Major phenotypic changes occur in red cell membranes during the perinatal period, but the underlying molecular explanations remain poorly defined. Aquaporin CHIP, the major erythroid and renal water channel, was studied in perinatal rats using affinity-purified anti-CHIP IgG for immunoblotting, flow cytometry, and immunofluorescence microscopy. CHIP was not detected in prenatal red cells but was first identified in circulating red cells on the third postnatal day. Most circulating red cells were positive for CHIP by the seventh postnatal day, and this proportion rose to nearly 100% by the 14th day. The ontogeny of red cell CHIP correlated directly with acquisition of osmotic water permeability and inversely with Arrhenius activation energy. Only minor alterations in the composition of red cell membrane lipids occurred at this time. Immunohistochemical analysis of perinatal kidneys demonstrated a major induction of CHIP in renal proximal tubules and descending thin limbs at birth, coincident with the development of renal concentration mechanisms. Therefore, water channels are unnecessary for oxygen delivery or survival in the prenatal circulation, however CHIP may confer red cells with the ability to rehydrate rapidly after traversing the renal medulla, which becomes hypertonic after birth.

Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens.

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane.