Mammalian exchangers and co-transporters.

In the past year, novel mammalian exchanger and co-transporter isoforms have been characterized. Specialized subdomains within these oligomeric transporters have been shown to be involved in biosynthesis, targeting, transport and regulation. Progress on the structural front has been limited due to the lack of high-resolution structures, but transport mutants responsible for disease states continue to be identified.

Characterization and modeling of membrane proteins using sequence analysis.

The current libraries of amino acid sequences of membrane proteins are a valuable resource for the analysis of elements common to these proteins. Multiple-sequence alignment techniques and the identification of conserved features of transmembrane segments have improved the prediction of membrane protein topology. Molecular modeling in combination with structural studies or site-directed mutagenesis is proving to be a powerful link between theory and experiment. Unfortunately, the number of high-resolution structures of intrinsic membrane proteins, although increased recently, presents a restricted and perhaps biased view of membrane protein structure.

Inhibition of anion transport in human red blood cells by 5,5'-dithiobis(2-nitrobenzoic acid).

The uptake of [32P]phosphate into human red blood cells was inhibited (Ki = 0.6 mM) by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor (Ki = 7 mM). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[14C]-benzamido-4'-aminostilbene-2,2'-disulfonate, a potent inhibitor of anion transport (Ki = 1-2 microM), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.

The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide.

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.

Terbium-binding properties of calsequestrin from skeletal muscle sarcoplasmic reticulum.

Calsequestrin (Mr = 40,000) is a calcium-binding protein (Kd = 1 mM, 50 sites/molecule) located within the terminal cisternae of the sarcoplasmic reticulum of skeletal muscle cells. The interaction of terbium, a calcium analog, with rabbit skeletal muscle calsequestrin was studied by fluorescence and circular dichroism spectroscopy. Direct measurement of terbium binding using a fluorescence assay for terbium revealed that calsequestrin bound approx. 30 mol of terbium per mol of protein with an affinity of approx. 7 microM. The fluorescence of terbium measured at 545 nm was enhanced dramatically upon binding to calsequestrin, reaching a maximum value at a terbium to protein ratio of 28. The excitation spectrum of protein-bound terbium and chemical modification studies revealed that energy transfer occurred between aromatic residues, including tryptophan and bound terbium. Terbium bound to calsequestrin could be removed by EGTA, or displaced by Ca2+ or La3+. In the presence of Ca2+ or La3+ terbium bound to calsequestrin with a higher apparent affinity and lower capacity. 0.1 M KCl or 5 mM MgCl2 had little effect on terbium binding. Terbium increased the intrinsic fluorescence of calsequestrin 2-fold, and increased the alpha-helical content of calsequestrin from 16 to 33%. Terbium binding induces the same conformational changes in calsequestrin as does calcium, confirming that terbium is a useful calcium analog in this system.

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes.

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS; Ki = 10 microM)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4'-aminostilbene 2,2'-disulfonate (BADS; Ki = 2 microM), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4 degrees C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4 degrees C, the weakly bound form was slowly converted to the strongly bound form. At 37 degrees C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37 degrees C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37 degrees C.

Cross-linking of the proteins in the outer membrane of Escherichia coli.

1. The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A. Both forms of this protein, proteins A1 and A2, produced similar cross-linking products. No cross-linking of protein A to the peptidoglycan was detected. 2. The proteins of the isolated outer membrane varied in their ease of cross-linking. The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions. The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked. No linkage of protein A to protein B was detected. 3. Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. 4. These results suggest that specific protein-protein interactions occur in the outer membrane.

Inhibition of phosphate transport in human erythrocytes by water-soluble carbodiimides.

Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37 degrees C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37 degrees C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.

Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl).

Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) leads to inhibition of anion transport as measured by [32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37 degrees C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N-ethylmaleimide and p-chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or beta-mercaptoethanol. Prolonged incubation (60 min, 37 degrees C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or beta-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37 degrees C, pH 8.3 in sucrose-citrate medium) and p-nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37 degrees C, pH 8.1 in sucrose-citrate medium) but not by N-acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37 degrees C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter.

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes.

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.

Accessibility of the N-ethylmaleimide-unreactive sulfhydryl of human erythrocyte Band 3.

The human erythrocyte anion exchange protein, Band 3, was reacted with N-ethylmaleimide (NEM) in cells to a stoichiometry of 5.3 mol NEM per mol Band 3, indicating that all NEM-reactive cysteines in Band 3 were labeled. Quantitatively NEM-blocked Band 3 was still able to bind to and be eluted by reducing agents from a mercurial affinity resin, [p-(chloromercuribenzamido)ethylene]amino-Sepharose. Reaction of NEM-blocked Band 3 with p-chloromercuribenzoate (pCMB) did not prevent binding to the resin due to exchange of pCMB for the immobilized mercurial. pCMB has been reported to inhibit water and urea permeation across the red cell membrane, and this has been attributed to reaction with a NEM-reactive sulfhydryl in Band 3. The interaction of Band 3 with the immobilized ligand directly demonstrates the reaction of NEM-blocked Band 3 with a mercurial and indicates that the NEM-unreactive, pCMB-reactive sulfhydryl residue is accessible to within approximately equal to 12 A (the distance from the solid support to the Hg) of the surface of the solubilized Band 3 protein.

Self-association of Band 3, the human erythrocyte anion exchanger, in detergent solution.

Dimeric Band 3 purified in n-dodecyl octaethyleneglycol (C12E8) underwent an irreversible, temperature-dependent association, resulting in a complex with a Stokes radius slightly larger than a native tetramer, before forming a higher molecular weight aggregate. Self-association occurred with a half-time of about 1 h at 37 degrees C but did not occur at 0 degrees C after several days. No change in the secondary structure of Band 3, as observed by circular dichroism, occurred during the association process. However, self-association of Band 3 was accompanied by loss of the stilbene disulfonate inhibitor binding site. No association or loss of inhibitor binding occurred with the dimeric membrane domain under similar incubation conditions. The membrane domain dimer was also stable over a wide range of pH (5.5-9.5) and buffer conditions, while Band 3 aggregated below pH 6.5. Inhibitors of anion transport, which stabilize the membrane domain, slowed the association. Band 3, depleted of phospholipids by extensive washing of resin-bound protein with detergent or, incubated with excess detergent, was more prone to aggregation. The membrane domain also showed some aggregation when depleted of lipids. Preparations could be stabilized by adding dimyristoylphosphatidylcholine (DMPC) prior to the 37 degrees C incubation. The effect of inhibitors and DMPC was additive, with a combination of 1 mM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) and 1:1 (wt/wt) DMPC:Band 3 stabilizing 90% of the protein to a 24-h incubation at 37 degrees C. The results suggest that self-association of Band 3 dimers is promoted by the cytoplasmic domain but results in alterations to the membrane domain involving the loss of essential phospholipids. Addition of phospholipid or inhibitors to Band 3 results in a stable preparation of the intact protein that may be suitable for crystallization studies.

Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.

Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins.

The distribution of amino acids in the transmembrane segments and flanking regions of 115 human type I single span (amino terminus extracellular and carboxyl terminus cytosolic) plasma membrane proteins was found to be non-random. In this sample, Ile was preferentially localized to the amino-terminal region of the hydrophobic transmembrane segments, followed by Val, while Leu predominated in the carboxyl-terminal half of the segment. Although Gly residues were preferentially located in the transmembrane segment, this residue was excluded from the carboxyl-terminal and adjacent boundary regions. Aromatic residues (Tyr, Trp and Phe) occurred preferentially at the cytoplasmic boundary, with Trp also favored at the extracellular boundary. The extracellular flanking sequence amino-terminal to the transmembrane segment was enriched in residues predicted to initiate helix formation (Pro, Asn and Ser), while Arg and Lys were enriched in the cytoplasmic flank where they may function as topological determinants. The positional preferences of these particular amino acids within the transmembrane segment and flanking regions suggests that, in addition to lipid-protein interactions, these residues may participate in specific protein-protein interactions. A consensus sequence motif for type I membrane proteins is proposed and its role in the biosynthesis, folding, assembly and function of these segments is discussed.

Ion pathways in proteins of the sarcoplasmic reticulum.

In summary, we have begun to characterize three different ion pathways in the sarcoplasmic reticulum. Ca2+-ionophoric activity has been traced to a 13,000-dalton CNBr fragment localized at the amino terminus of the ATPase molecule... The pathway involved in Ca2+ release can be distinguished from the pathway involved in Ca2+ uptake by its insensitivity to quercetin. An anion pathway is sensitive to DIDS and appears to be localized in the ATPase molecule

Carbonic anhydrase: in the driver's seat for bicarbonate transport.

Carbonic anhydrases are a widely expressed family of enzymes that catalyze the reversible reaction: CO(2) + H(2)O <=> HCO(3)(-) + H(+). These enzymes therefore both produce HCO(3)(-) for transport across membranes and consume HCO(3)(-) that has been transported across membranes. Thus these enzymes could be expected to have a key role in driving the transport of HCO(3)(-) across cells and epithelial layers. Plasma membrane anion exchange proteins (AE) transport chloride and bicarbonate across most mammalian membranes in a one-for-one exchange reaction and act as a model for our understanding of HCO(3)(-) transport processes. Recently it was shown that AE1, found in erythrocytes and kidney, binds carbonic anhydrase II (CAII) via the cytosolic C-terminal tail of AE1. To examine the physiological consequences of the interaction between CAII and AE1, we characterized Cl(-)/HCO(3)(-) exchange activity in transfected HEK293 cells. Treatment of AE1-transfected cells with acetazolamide, a CAII inhibitor, almost fully inhibited anion exchange activity, indicating that endogenous CAII activity is essential for transport. Further experiments to examine the role of the AE1/CAII interaction will include measurements of the transport activity of AE1 following mutation of the CAII binding site. In a second approach a functionally inactive CA mutant, V143Y, will be co-expressed with AE1 in HEK293 cells. Since over expression of V143Y CAII would displace endogenous wild-type CAII from AE1, a loss of transport activity would be observed if binding to the AE1 C-terminus is required for transport.