Modification of a carboxyl group that appears to cross the permeability barrier in the red blood cell anion transporter

A recently developed method for converting protein carboxyl groups to alcohols has been used to examine the functional role of carboxyl groups in the red blood cell inorganic anion-transport protein (band 3). A major goal of the work was to investigate the carboxyl group that is protonated during the proton-sulfate cotransport that takes place during net chloride-sulfate exchange. Three kinds of evidence indicate that the chemical modification (Woodward's reagent K followed by borohydride) converts this carboxyl to an alcohol. First, monovalent anion exchange is inhibited irreversibly. Second, the modification stimulates sulfate influx into chloride-loaded cells and nearly eliminates the extracellular pH dependence of the sulfate influx. (The stimulated sulfate influx in the modified cells is inhibitable by stilbenedisulfonate.) Third, the proton influx normally associated with chloride-sulfate exchange is inhibited by the modification. These results would all be expected if the titratable carboxyl group were converted into the untitratable, neutral alcohol. In addition to altering the extracellular pH dependence of sulfate influx, the chemical modification removes the intracellular pH dependence of sulfate efflux. The modification is performed under conditions in which the reagent does not cross the permeability barrier. The large effect on the intracellular pH dependence of sulfate transport suggests that a single carboxyl group can at different times be in contact with the aqueous medium on each side of the permeability barrier.     

Sulfanilation of lysozyme by carbodiimide reaction. Reactivities of individual carboxyl groups

The carboxyl groups of lysozyme were coupled with sulfanilic acid, a chromophoric nucleophile, using 1-ethyl-3-dimethylaminopropylcarbodiimide at pH 5. Other carbodiimides were less effective. Ninety percent of the carboxyl groups were sulfanilated through exhaustive reaction with 1.2 m nucleophile. Isolation and identification of the tryptic peptides from this material showed that all 10 of the carboxyls of lysozyme had reacted. In 0.05 m sulfanilic, Glu-35 and Asp-101 were most reactive while Glu-7, Asp-18, and Asp-66 were least. Change to high concentration of nucleophile (from 0.05 to 1.2 m sulfanilic) altered carboxyl reactivity. Addition of inhibitor reduced reactivity of Asp-101 and Glu-35. Side reactions were not important.     

Modification of cardiac sodium channels by carboxyl reagents. Trimethyloxonium and water-soluble carbodiimide

In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.     

Simple model can explain self-inhibition of red cell anion exchange.

Ion translocation in red cell anion exchange is assumed to occur by means of an alternating access mechanism, in which a critical binding site for the transported ion alternates between two conformational states, each accessible from only one side of the membrane. If this alternating site is located within the transport protein at some distance from one or both surfaces of the membrane, an access channel is required to connect the alternating site to the adjacent bulk solution. This automatically leads to inhibition of transport at high concentrations of the transported ion because release of the ion from the alternating site can occur only via unoccupied channel sites.     

Modification of essential carboxyl group in rabbit muscle phosphorylase by water-soluble carbodiimide

Water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) and glycine ethyl ester (GEE) as a nucleophile were used to modify the essential carboxyl group of phosphorylases. The inactive b form of the muscle phosphorylase was modified faster than the active a form and potato phosphorylases. Use of N,N,N',N'-tetramethyl-ethylenediamine (TEMED)-HCl buffer system (pH 6.2) resulted in a remarkable difference from the previous results obtained with phosphate and beta-glycerophosphate buffer systems. That is, the substrate glucose 1-phosphate gave the best protection of the three phosphorylase activities. Glucose and glycogen were also effective to retard the inactivation of muscle phosphorylases, though glycogen was not effective for the potato enzyme. The EDC-GEE-modified phosphorylase b retained the affinity for AMP-Sepharose, though partially modified enzyme completely lost the homotropic cooperativity. Phosphorylase b was subjected to differential labeling with [14C]GEE. A labeled peptide was obtained after CNBr cleavage and peptic digestions, and corresponded to the catalytic site sequence surrounding the GEE-substituted Asp 661 and Glu 664. Either or both of these EDC-modified carboxyl residues may have an important role in the catalytic reaction.     

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4′-dichloromercuric-2,2,2′,2′-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385–389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385-389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Relation between red cell anion exchange and water transport

A new distilbene compound, 4',4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid (DCMBT), has been synthesized for use in studies of anion and water transport in the human red cell. DCMBT combines features of both the specific stilbene anion transport inhibitor, DIDS, and the mercurial water transport inhibitor, pCMBS. This new compound inhibits anion transport almost completely with a Ki of 15 microM. DCMBT also inhibits water transport by about 15-20% with a Ki of about 8 microM. Treatment of red cells with DIDS inhibits the effect of DCMBT on water transport, suggesting that anion transport and water transport are mediated by the same protein.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Kinetic independence between red cell anion exchange and urea transport

Urea equilibrium exchange fluxes were measured in human red cells under conditions which recruit the anion transporter into an outward-facing or an inward-facing state (with respect to the anion transport site). Regardless of these conditions, urea transport always occurred at the same rate: 41 +/- 2 mol.(kg cell solids.min)-1 with 1.5 M urea at 0 degrees C. These data suggest that the pathway on the band-3 protein which mediates anion transport is kinetically uncoupled from urea transport and is probably not involved in the transport of urea across the red cell membrane.     

The relationship between anion exchange and net anion flow across the human red blood cell membrane

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.     

Effects of pH and temperature on the interaction of an impermeant probe with surface proteins of the human red blood cell.

The conformation of the outer surface of the human red cell membrane has been studied under various conditions using the impermeant probe [125I]diazodiiodosulfanilic acid. At least seven polypeptides were labeled by the reagent, including the three extractable glycoproteins separable by the electrophoretic method employed. The Mr = 43,000 protein band was shown to contain two labeled species, one a glycoprotein, in addition to its major constituent, red cell actin. The extent and pattern of labeling were very sensitive to changes in pH and temperature. Total labeling increased with increasing pH and was greater at 4 degrees C than 37 degrees C. Binding of the probe to the Mr = 90,000 polypeptide and the major glycoprotein were relatively increased with increasing pH and temperature while opposite effects were observed for the Mr = 43,000 peptide(s). The pH effects on external membrane labeling were rapidly reversible. Results were similar in cells of different densities, suggesting that the pH and temperature effects were not related to cell age. The data presented emphasize the lability of membrane conformation and reactivity and thus the necessity to consider carefully the conditions of labeling in interpretation of studies using impermeant probes.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Asymmetry in the mechanism for anion exchange in human red blood cell membranes. Evidence for reciprocating sites that react with one transported anion at a time

The kinetics of chloride and bromide transport were examined in intact human red blood cells and resealed ghosts. Because the influx and efflux of halide ions are almost equal (less than 0.01% difference), the stimulation of the exchange flux by external halides could be determined by measuring 36Cl or 82Br efflux. When the external halide concentration was increased by replacement of isoionic, isotonic solutions of sucrose and the nontransported anion citrate, the stimulation of the exchange flux was hyperbolic and was maximum at 20 mM halide externally. The K 1/2-out, the external concentration of chloride or bromide which stimulated the efflux to half of its maximum value, was 3 and 1 mM respectively, 15-fold smaller than K 1/2-in which we found to be about equal to the K 1/2 of halide self-exchange with nearly equal internal and external concentrations. Thus, the transport mechanism behaves asymmetrically with respect to these transported halides. Bromide flux was two-fold greater in bromide-chloride heteroexchange than in bromide-bromide self-exchange but it was still much smaller than the chloride self-exchange flux. The maximum influx and efflux of bromide in exchange for chloride were roughly eqal. Thus, since the maximum transport rates in the two directions are nearly equal, the kinetics of bromide equilibrium exchange with equal concentrations on the two sides are controlled on the inside where K 1/2 is greatest. The K 1/2-out Cl was a hyperbolic function of internal chloride concentration and was proportional to the maximum flux at each internal chloride concentration. These results are evaluated in terms of two broad categories of models. We conclude that, in contrast to other ion transport systems which have been shown to have kinetics of a sequential mechanism, anion exchange is compatible with a ping-pong mechanism in which a single site reciprocates between inside- and outside-facing orientations with asymmetric K 1/2 values.     

Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with a water-soluble carbodiimide: identification of carboxyl groups protected by MgATP and inhibitor peptides

The catalytic subunit of cAMP-dependent protein kinase typically phosphorylates protein substrates containing basic amino acids preceding the phosphorylation site. To identify amino acids in the catalytic subunit that might interact with these basic residues in the protein substrate, the enzyme was treated with a water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), in the presence of [14C]glycine ethyl ester. Modification of the catalytic subunit in the absence of substrates led to the irreversible, first-order inhibition of activity. Neither MgATP nor a 6-residue inhibitor peptide alone was sufficient to protect the catalytic subunit against inactivation by the carbodiimide. However, the inhibitor peptide and MgATP together completely blocked the inhibitory effects of EDC. Several carboxyl groups in the free catalytic subunit were radiolabeled after the catalytic subunit was modified with EDC and [14C]glycine ethyl ester. After purification and sequencing, these carboxyl groups were identified as Glu 107, Glu 170, Asp 241, Asp 328, Asp 329, Glu 331, Glu 332, and Glu 333. Three of these amino acids, Glu 331, Glu 107, and Asp 241, were labeled regardless of the presence of substrates, while Glu 333 and Asp 329 were modified to a slight extent only in the free catalytic subunit. Glu 170, Asp 328, and Glu 332 were all very reactive in the apoenzyme but fully protected from modification by EDC in the presence of MgATP and an inhibitor peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the murine anion exchange protein

A full-length clone encoding the mouse erythrocyte anion exchange protein, band 3, has been isolated from a cDNA library using an antibody against the mature erythrocyte protein. The complete nucleotide sequence has been determined. Substantial homology is evident between the deduced murine amino acid sequence and published sequences of fragments of human band 3 protein. The amino-terminal 420 and the carboxy-terminal 32 residues constitute polar, soluble domains, while the intervening 475 amino acids are likely to be intimately associated with the lipid bilayer. Hydrophobic analysis of this sequence, together with structural studies on the human protein, suggests the possibility of at least 12 membrane spans, predicting that both the amino- and carboxy-termini are intracellular.     

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.