Heini Murer

A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems.

We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.

A high yield preparation for rat kidney brush border membranes Different behaviour of lysosomal markers

Rat kidney cortex slices were homogenized with a polytron in a isoosmotic medium containing 5 mmol/l EGTA. By two precipitations with MgCl2 (12 mmol/l) and differential centrifugation, brush border membranes were purified. The brush border marker enzymes alkaline phosphatase and aminopeptidase M were found to be enriched 17.0 +/- 5.3-fold and 16.7 +/- 3.7-fold, respectively. By this method, a high yield of brush border membranes was obtained (48.3 +/- 7.9% for alkaline phosphatase; 47.0 +/- 9.5% for aminopeptidase M). The acid phosphatase was enriched 5-fold, whereas other lysosomal enzymes (glucosaminidase, glucuronidase, cathepsin D) were enriched only 0.2-fold. Acid phosphatase activity could not be washed out, but could be separated from alkaline phosphatase and leucine aminopeptidase by means of free flow electrophoresis and sucrose density gradient centrifugation. Vesicles prepared by the presently described Mg/EGTA-method show better transport properties, compared to vesicles prepared by the calcium method of Evers et al. (Evers, C., Haase, W., Murer, H. and Kinne, R. (1978) Membrane Biochem. 1, 203-219), whereas by SDS-polyacrylamide gel electrophoresis, no differences in the protein patterns were observed.

The sodium phosphate cotransporter family SLC34

This review summarizes the characteristics of the solute carrier family SLC34 that is represented by the type ll Na/Pi-cotransporters NaPi-lla (SLC34A1), NaPi-llb (SLC34A2) and NaPi-llc (SLC34A3). Other Na/Pi-cotransporters are described within the SLC17 and SLC20 families. Type ll Na/Pi-cotransporters are expressed in several tissues and play a major role in the homeostasis of inorganic phosphate. In kidney and small intestine, type ll Na/Pi-cotransporters are located at the apical sites of epithelial cells and represent the rate limiting steps for transepithelial movement of phosphate. Physiological and pathophysiological regulation of renal and small intestinal epithelial transport of phosphate occurs through alterations in the abundance of type ll Na/Pi-cotransporters.

The use of isolated membrane vesicles to study epithelial transport processes

SummaryEpithelia are multicompartment and multicomponent systems performing transcellular and paracellular transport in a very complex manner. One way to get a deeper understanding of the function of such a complex system is to dissect it into the single components and then, after having defined the components under well-controlled conditions, to try to describe the behavior of the whole system on the basis of the properties of the single components.This article deals with the analysis of isolated plasma membranes derived from the luminal and contraluminal face of epithelial cells, predominantly renal proximal tubular and small intestinal cells. It is aimed to give an overview of methods used to isolate and separate plasma membranes, to study their transport properties as membrane vesicles, and also to address the question of how information gained with the isolated membranes corresponds to observations made in the intact cell using other, notably electrophysiological, measurements. The review also critically evaluates the limitations of the approach and thereby tries to set the work on isolated membranes in the proper perspective within the field of transport physiology.

Interaction of the Type IIa Na/Pi Cotransporter with PDZ Proteins

The type IIa Na+-dependent inorganic phosphate (Na/Pi) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathioneS-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/Pi cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.

Expression Cloning of a Cdna From Rabbit Small-Intestine Related to Proton-Coupled Transport of Peptides, Beta-Lactam Antibiotics and Ace-Inhibitors

Injection of poly(A)+ RNA from rabbit small intestine intoXenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino ß-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H+-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in ooyctes clamped at a holding potential of-60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.

PDZ-domain interactions and apical expression of type IIa Na/Pi cotransporters

Type IIa Na/Pi cotransporters are expressed in renal proximal brush border and are the major determinants of inorganic phosphate (Pi) reabsorption. Their carboxyl-terminal tail contains information for apical expression, and interacts by means of its three terminal amino acids with several PSD95/DglA/ZO-1-like domain (PDZ)-containing proteins. Two of these proteins, NaPi-Cap1 and Na/H exchanger-regulatory factor 1 (NHERF1), colocalize with the cotransporter in the proximal brush border. We used opossum kidney cells to test the hypothesis of a potential role of PDZ-interactions on the apical expression of the cotransporter. We found that opossum kidney cells contain NaPi-Cap1 and NHERF1 mRNAs. For NHERF1, an apical location of the protein could be documented; this location probably reflects interaction with the cytoskeleton by means of the MERM-binding domain. Overexpression of PDZ domains involved in interaction with the cotransporter (PDZ-1/NHERF1 and PDZ-3/NaPi-Cap1) had a dominant–negative effect, disturbing the apical expression of the cotransporter without affecting the actin cytoskeleton or the basolateral expression of Na/K-ATPase. These data suggest an involvement of PDZ-interactions on the apical expression of type IIa cotransporters.

Regulation of small intestinal Na-Pi type IIb cotransporter by dietary phosphate intake

Dietary restriction of phosphate is a well-known stimulator (acting indirectly via vitamin D3) of small intestinal apical Na-Picotransport. In the present study, we document by Western blots and immunohistochemistry that, in mice, a low-Pi diet given for several days leads (in parallel to a stimulation of Na-Pi cotransport) to an increase of the abundance of the type IIb Na-Pi cotransporter in the brush-border membrane of mouse enterocytes. Similar results were also obtained by an injection of cholecalciferol. The abundance of the type IIb transcript was investigated by Northern blots. These results indicated that the amount of the type IIb transcript was not changed by either low-Pi diet or cholecalciferol. It is concluded that stimulation of intestinal Na-Pi cotransport by low-Pi diet and vitamin D3 can be explained by an increased amount of type IIb Na-Picotransporters in the brush-border membrane and that augmentation of type IIb Na-Pi cotransporters is not related to an increased rate of transcription of the type IIb gene.

Regulation of phosphate transport in proximal tubules

Homeostasis of inorganic phosphate (Pi) is primarily an affair of the kidneys. Reabsorption of the bulk of filtered Pi occurs along the renal proximal tubule and is initiated by apically localized Na+-dependent Pi cotransporters. Tubular Pi reabsorption and therefore renal excretion of Pi is controlled by a number of hormones, including phosphatonins, and metabolic factors. In most cases, regulation of Pi reabsorption is achieved by changing the apical abundance of Na+/Pi cotransporters. The regulatory mechanisms involve various signaling pathways and a number of proteins that interact with Na+/Pi cotransporters.

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

SummaryFor patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na+ and K+ as well as to Cl−. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.