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.     

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 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.     

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.     

Potassium channels along the nephron

The K+ channels that are present in three different nephron segments, the Necturus proximal, Amphiuma early distal (diluting segment), and rabbit collecting tubule have been examined. Ca2+-sensitive K+ channels were present in the apical membranes of the cells lining all these segments. The channels were all voltage-sensitive and their open probability increased with membrane depolarization. Because of the ubiquitous distribution, it is suggested that this channel is responsible for K+ secretion by the nephron and that the same intracellular regulators act throughout the various segments. Basolateral K+ channels have been examined only in Necturus proximal tubules. This channel is apparently insensitive to Ca2+; the voltage dependence is exactly opposite to that of the apical K+ channels; that is, hyperpolarizing potentials caused an increase in open probability. These differences in regulatory factors permit the independent regulation of apical and basolateral membrane K+ permeabilities that must occur in renal cells.     

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane- spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.     

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.     

Neuronal sodium channels in ventricular heart cells are localized near T-tubules openings

Cardiac voltage-dependent sodium channels (VDSC) are known to be tetrodotoxin (TTX)-resistant. However, recent immunochemical studies suggest the presence of TTX-sensitive neuronal-type VDSC in the heart. Scanning ion conductance microscopy (SICM) coupled to electrophysiology was used to obtain more direct functional evidence. TTX sensitivities of whole-cell sodium currents (I(Na)) in control and detubulated cells were compared. Addition of 200 nM TTX decreased I(Na) of control cells by 20%, whereas detubulated cells were hardly effected. The remaining current peaked slightly earlier and inactivation decay was faster (as in neuronal VDSC) than in detubulated cells. Single-channel activity was first assayed at random on the plasmalemma, and after topography had been revealed by SICM, at patched T-tubules openings. In the latter case, a single-channel conductance of 11-12pS was observed with a higher rate of success. This study provides independent evidence for neuronal VDSC in cardiomyocytes where they could rapidly and synchronously couple T-tubule and cell surface depolarizations.     

Effects of low-potassium diet on N-ethylmaleimide-sensitive ATPase in the distal nephron segments.

The present study was undertaken to investigate whether or not potassium deficiency influences N-ethylmaleimide (NEM)-sensitive ATPase in the distal nephron segments of the rat. One group of animals was fed a low-K diet, whereas the normal K-group was given the same diet after supplementation with KCl. The nephron segments examined were: the medullary and cortical thick ascending limbs, the distal convoluted tubule, and the cortical, outer and inner medullary collecting ducts. NEM-sensitive ATPase activity in microdissected segments was measured by a fluorometric microassay. The plasma K+ concentration in the low-K group was 3.1 +/- 0.3 mEq/l compared with 4.2 +/- 0.1 mEq/l in the normal-K group. NEM-sensitive ATPase activity in the outer medullary collecting duct of low-K diet animals was significantly greater than in normal-K animals. There was no significant difference in NEM-sensitive ATPase activity between the two groups of animals in the other nephron segments examined. It is suggested that NEM-sensitive H-ATPase activity in the outer medullary collecting duct is modulated by the potassium status of the animal.     

Cytochemical observations on the transport of horseradish peroxidase in different segments of the nephron

Synopsis Kidney slices from rats injected with horseradish peroxidase (HRP) 5–10 min before sacrifice were fixed with formaldehyde vapour for 4 hr at 37°C and compared with tissue fixed by perfusion or by immersion. Much more of the injected protein was retained in extracellular and vascular spaces of the nephron in vapour-fixed than in perfusion-fixed or immersion-fixed tissue. The extracellular localization of HRP in the lateral intercellular spaces and in the infoldings of the basal cell membranes showed characteristic differences in different segments of the nephron. The high concentration of HRP in the lateral intercellular spaces of the collecting tubules, as well as the early location of small phagosomes containing HRP in the apical, lateral, and basal cell regions suggested that HRP was reabsorbed through the cytoplasm into the intercellular spaces or excreted in the opposite direction. The intercellular spaces in the terminal segments of the proximal tubules also showed high concentrations of HRP which suggests participation of these spaces in protein transport between the lumen and the peritubular capillaries. The extracellular concentration of HRP early after injection was found, by colorimetric assays of homogenates, to be several times higher in the papilla than in the cortex.     

The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron

We studied the distribution of the alpha 1-alpha 6 subunits of beta 1 integrins in developing and adult human kidney using a panel of mAbs in indirect immunofluorescence microscopy. Uninduced mesenchyme displayed a diffuse immunoreactivity for only the alpha 1 integrin subunit. At the S-shaped body stage of nephron development, several of the alpha subunits were characteristically expressed in distinct fetal nephron segments, and the pattern was retained also in the adult nephron. Thus, the alpha 1 subunit was characteristically expressed in mesangial and endothelial cells, the alpha 2 in glomerular endothelium and distal tubules, the alpha 3 in podocytes, Bowman's capsule, and distal tubules, and the alpha 6 subunit basally in all tubules, and only transiently in podocytes during development. Unlike the alpha 3 and alpha 6 subunits, the alpha 2 subunit displayed an overall cell surface distribution in distal tubules. It was also distinctly expressed in glomerular endothelia during glomerulogenesis. The beta 4 subunit was expressed only in fetal collecting ducts, and hence the alpha 6 subunit seems to be complexed with the beta 1 rather than beta 4 subunit in human kidney. Of the two fibronectin receptor alpha subunits, alpha 4 and alpha 5, only the latter was expressed, confined to endothelia of developing and adult blood vessels, suggesting that these receptor complexes play a minor role during nephrogenesis. The present results suggest that distinct integrins play a role during differentiation of specific nephron segments. They also indicate that alpha 3 beta 1 and alpha 6 beta 1 integrin complexes may function as basement membrane receptors in podocytes and tubular epithelial cells.     

Suppression of ouabain-insensitive K-ATPase activity in rabbit nephron segments during chronic hyperkalemia

Recently, we demonstrated that an ATPase stimulated by K (and not inhibited by ouabain, Na-K-ATPase inhibitor) is present in the connecting tubule (CNT) and collecting duct segments of the rabbit. In this study, we determined the effects of high- and low-K diet on K-ATPase activity in the CNT and collecting duct segments of rabbit. One group of animals was given a low-K diet (34 mEq/kg diet) and the other group was given a high-K diet (700 mEq/kg diet) for 1 week. K-ATPase activity was measured by a microfluorometric assay in which ATP hydrolysis is coupled to oxidation of NADH. Low-K animals had plasma K = 3.1 +/- 0.2 as compared with 5.5 +/- 0.5 mEq/l in high-K animals. Low-K animals had significant K-ATPase activity in CNT, CCD (cortical collecting duct) and MCD (medullary collecting duct). On the other hand, K-ATPase activity in all 3 segments from high-K animals was not significantly different from zero. These results support a hypothesis that chronic K loading suppresses the ouabain-insensitive K-ATPase in the distal nephron.     

Neurotoxic unc-8 mutants encode constitutively active DEG/ENaC channels that are blocked by divalent cations

Ion channels of the DEG/ENaC family can induce neurodegeneration under conditions in which they become hyperactivated. The Caenorhabditis elegans DEG/ENaC channel MEC-4(d) encodes a mutant channel with a substitution in the pore domain that causes swelling and death of the six touch neurons in which it is expressed. Dominant mutations in the C. elegans DEG/ENaC channel subunit UNC-8 result in uncoordinated movement. Here we show that this unc-8 movement defect is correlated with the selective death of cholinergic motor neurons in the ventral nerve cord. Experiments in Xenopus laevis ooctyes confirm that these mutant proteins, UNC-8(G387E) and UNC-8(A586T), encode hyperactivated channels that are strongly inhibited by extracellular calcium and magnesium. Reduction of extracellular divalent cations exacerbates UNC-8(G387E) toxicity in oocytes. We suggest that inhibition by extracellular divalent cations limits UNC-8 toxicity and may contribute to the selective death of neurons that express UNC-8 in vivo.     

Tumor antigens are constitutively presented in the draining lymph nodes

Tumor growth is rarely associated with a strong specific CTL response, suggesting that the immune system is ignorant of the presence of tumor because the Ags are not readily available to or are sequestered from potential effector cells. We studied the in vivo activation of naive TCR transgenic hemagglutinin (HA)-specific CD8+ T cells adoptively transferred into mice bearing HA-expressing tumor using 5,6-carboxy-succinimidyl-fluorescein-ester labeling, which allows the identification of proliferating HA-specific T cells. We demonstrate that tumor Ags are constitutively presented in the lymph nodes draining tumors and are powerfully mitogenic for responding T cells despite the absence of anti-tumor CTL responses. Importantly, this proliferative signal occurs throughout tumor growth and is still detectable 6 mo after tumor inoculation when tumor is not palpable. These results provide the first evidence that there is constitutive presentation of tumor Ags in draining lymph nodes.     

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.