The inhibitor effect of probenecid and structural analogues on organic anions and chloride permeabilities in ox erythrocytes

Probenecid inhibits anion movements (organic anions and chloride) in ox erythrocytes. The I₅₀ is 4 · 10^?5 M. Structural analogues such as carinamide, $\text{p-}\text{carboxybenzene}$ sulfonamide and $\text{p-}\text{carboxy}$$\text{N,N}$ diethyl benzene sulfonamide, which are drugs of the sulfonamide class, were also found to inhibit anion transport. These results reinforce the previously discussed view based on structural considerations, that sulfonamides act on the red cell membrane as competitors of anion transport. It is possible that probenecid and carinamide act in a similar way in the kidney.     

Dihydropteroate synthase from Streptococcus pneumoniae: structure, ligand recognition and mechanism of sulfonamide resistance

DHPS (dihydropteroate synthase) catalyses an essential step in the biosynthesis of folic acid and is the target for the sulfonamide group of antimicrobial drugs. In the present paper we report two crystal structures of DHPS from the respiratory pathogen Streptococcus pneumoniae: the apoenzyme at 1.8 A (1 A=0.1 nm) resolution and a complex with DHPP (6-hydroxymethyl-7,8-dihydropterin monophosphate) at 2.4 A resolution. The enzyme forms a alpha/beta barrel structure, with a highly conserved binding pocket for recognition of the pterin substrate, DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). There is a fixed order of substrate binding: DHPPP binds first, followed by the second substrate, pABA (p-aminobenzoic acid). Binding of PP(i) also allows the enzyme to recognize pABA or sulfonamide drugs, which act as pABA analogues. Using equilibrium and pre-steady state kinetic fluorescence measurements, we show that the on-rate for DHPPP binding to the enzyme is relatively low (2.6x10(5) M(-1) x s(-1)) and propose that binding of this substrate induces a large scale movement of the second loop in the enzyme structure to participate in the formation of the pABA-binding site. Two mutations which confer resistance to sulfonamide drugs do not affect DHPPP binding, but have a substantial effect on pABA and sulfonamide recognition. The results show that binding of DHPPP and pABA are separate distinguishable events in the reaction cycle, and that mutations which confer resistance to sulfonamide drugs act exclusively on the second step in the binding process.     

Effects of anion and cation inhibitors and carbonic anhydrase inhibitors upon the activity of the gypsy moth (Lepidoptera: Lymantriidae) nucleo-polyhedrovirus

Twenty chemicals that were reported to act as anion transport inhibitors, cation transport inhibitors, and inhibitors of carbonic anhydrase activity were tested at a 1% concentration (wt:wt) for their effects upon the biological activity of the gypsy moth, Lymantria dispar (L.), nucleopolyhedrovirus (LdMNPV). Among the five anion transport inhibitors tested, flufenamic acid acted as a viral enhancer. None of the seven inhibitors of K+ enhanced viral activity and three (4-aminopyridine, diacetyl, and procaine) significantly reduced the activity of LdMNPV. All four Na+ transport inhibitors (abietic acid, amiloride, dicyclohexylcarbodiimide, triamterene) acted as viral enhancers. Triamterene was the most active enhancer, as the LC50 was reduced by approximately 1,750-fold. Five carbonic anhydrase inhibitors were tested and four (acetazolamide, hydrochlorothiazide, methazolamide, sulfanilamide) enhanced the activity of LdMNPV. Acetazolamide (a carbonic anhydrase inhibitor), amiloride (a Na+ transport inhibitor), and flufenamic acid (an anion transport inhibitor) were tested singly and in different combinations. Every combination tested (acetazolamide/amiloride, acetazolamide/flufenamic acid, amiloride/flufenamic acid, acetazolamide/amiloride/flufenamic acid) significantly decreased the LC50 from 7.79 PIB/mm2 to a value as low as 0.008 PIB/mm2 (amiloride/flufenamic acid).     

Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells

Characterization of the phosphate transport system across the basolateral membrane of renal proximal tubule has been attempted using isolated proximal tubule cells prepared from chicks. The Pi efflux system is independent of Na+ ions and is not influenced by the nature of the chief anion present in the bathing medium. Pi efflux is not sensitive to DIDS and it is concluded that a generalized anion transporter of band III type is not the chief agent for facilitating Pi exit from the cell across the basolateral membrane. Inhibition of efflux by vanadate is evidence for a specific carrier protein in the membrane. The carrier probably possesses thiol group(s) that are essential for activity. The carrier may effect electroneutral transport of Pi possibly in exchange for OH- ions. The activity of the transport process is not stimulated by depleting the cells of phosphate or inhibited by rearing the chicks on a vitamin D-deficient diet. The system is unlikely to be of great importance for the expression of various regulatory mechanisms that act on the kidney to control the excretion of Pi. The activity declines as the chicks mature however.     

Immunocytochemical localization of Na+-HCO3- cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells

In corneal endothelium, there is evidence for basolateral entry of HCO(3)(-) into corneal endothelial cells via Na(+)-HCO(3)(-) cotransporter (NBC) proteins and for net HCO(3)(-) flux from the basolateral to the apical side. However, how HCO(3)(-) exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO(3)(-) similarly to fresh tissue. In addition, Cl(-) channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl(-) channels, CO(2) diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.     

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO ₃ ^– /Cl^– exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO₂ to HCO ₃ ^– . CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.We should like to express our thanks to Dr. I.M. Wiener for kindly supplying us with the impermeable sulfonamide, ZBI, which we used in preliminary experiments and to Dr. T.H. Maren for analysis of a sample of BCA II.     

A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

In contrast to animal cells, plants use nitrate as a major source of nitrogen. Following the uptake of nitrate, this major macronutrient is fed into the vasculature for long-distance transport. The Arabidopsis thaliana shoot expresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1 HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chloride ions. By contrast, we identified SLAH2 as a nitrate-specific channel that is impermeable for chloride. To understand the molecular basis for nitrate selection in the SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, a distantly related bacterial member. Structure-guided site-directed mutations converted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Our findings indicate that two pore-occluding phenylalanines constrict the pore. The selectivity filter of SLAC/SLAH anion channels is determined by the polarity of pore-lining residues located on alpha helix 3. Changing the polar character of a single amino acid side chain (Ser-228) to a nonpolar residue turned the nitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, the molecular basis of the anion specificity of SLAC/SLAH anion channels seems to be determined by the presence and constellation of polar side chains that act in concert with the two pore-occluding phenylalanines.     

Docking Substrates to Metalloenzymes

Abstract

Carbonic Anhydrase (CA) is a metalloenzyme that reversibly catalyzes the interconversion between carbon dioxide and bicarbonate anion. A class of sulfa drugs, sulfonamides, are known to inhibit CA. One approach to identifying important binding and specificity interactions between sulfonamides and CA is to analyze the results from docking studies. Previous docking studies have mainly focused on the encounters of substrates with non-metalloenzymes. Here we report the application of MOE-Dock to the CA II – sulfonamide system. After developing a standard docking protocol for the CA II – sulfonamide system we then used the protocol to determine other CA II – sulfonamide complexes.     

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.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Segmental differences in short-chain fatty acid transport in rabbit colon: Effect of pH and Na

Short-chain fatty acids (SCFAs) are the predominant luminal anion in the mammalian colon. Although they are rapidly absorbed in vivo, little is known about the mechanisms of transepithelial transport in vitro. Previous studies have suggested that SCFA transport may be linked to Na absorption or an anion exchange mechanism. We compared the transport of propionate under short-circuit conditions in rabbit proximal and distal colon to determine whether there were segmental differences, how SCFAs may be linked to either Na absorption or anion transport, and whether SCFAs, as weak electrolytes, may be affected by transepithelial pH gradients. In distal colon, propionate transport was not significantly altered by stimulation of electrogenic Na absorption, epinephrine or Cl removal. However, a modest transepithelial pH gradient (luminal 6.8/serosal 7.4) stimulated propionate absorption. In proximal colon, propionate transport was significantly altered by manuevers that either stimulated (lowered [Na] in the bathing media) or inhibited (theophylline) apical Na−H exchange. Neither Cl removal, nor the anion exchange inhibitor DIDS, nor a transepithelial bicarbonate gradient, altered propionate transport. A transepithelial pH gradient inhibited propionate secretion, but not in a manner entirely consistent with the effect of pH on the distribution of a weak electrolyte. These results suggest that there is significant segmental heterogeneity in colonic SCFA transport; that transepithelial propionate fluxes are altered by changes in pH or electroneutral Na absorption (Na−H exchange), but not by chloride removal, bicarbonate gradients or electrogenic Na absorption. Regulation of SCFA transport may be an important factor in the physiology of colonic fluid balance.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Carbonic anhydrase inhibitors. Inhibition studies with anions and sulfonamides of a new cytosolic enzyme from the scleractinian coral Stylophora pistillata

The catalytic activity and the inhibition of a new coral carbonic anhydrase (CA, EC 4.2.1.1), from the scleractinian coral Stylophora pistillata, STPCA-2, has been investigated. STPCA-2 has high catalytic activity for the physiological reaction being less sensitive to anion and sulfonamide inhibitors compared to STPCA, a coral enzyme previously described. The best STPCA-2 anion inhibitors were sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (K_Is of 5.7-67.2 μM) whereas the best sulfonamide inhibitors were acetazolamide and dichlorophenamide (K_Is of 74-79 nM). Because this discriminatory effect between these two coral CAs, sulfonamides may be useful to better understand the physiological role of STPCA and STPCA-2 in corals and biomineralization processes.     

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.     

Characterization of anion permeability in AH-66 hepatoma ascites cells

The sulfate transport in AH-66 hepatoma ascites cells was examined under various controlled conditions using 35SO42- as a tracer. The sulfate efflux rate was dependent on temperature, pH and anion species of the cell suspending medium. The efflux rate became saturated as the concentration of extracellular anions was increased. The efflux of anion was inhibited by some chemical reagents specifically reactive with amino or sulfhydryl groups. The results obtained in this study suggest that sulfate anions were transported by a facilitated transport system(s), and that some membrane protein(s) is involved in the anion transport system(s) of AH-66 cells. Both amino and sulfhydryl groups are thought to play a determinant role at the sulfate transport site in AH-66 cells.     

Relationship of net chloride flow across the human erythrocyte membrane to the anion exchange mechanism

The parallel effects of the anion transport inhibitor DIDS (4,4'- diisothiocyanostilbene-2,2'-disulfonate) on net chloride flow and on chloride exchange suggest that a major portion of net chloride flow takes place through the anion exchange system. The "slippage" model postulates that the rate of net anion flow is determined by the movement of the unloaded anion transport site across the membrane. Both the halide selectivity of net anion flow and the dependence of net chloride flux on chloride concentration over the range of 75 to 300 mM are inconsistent with the slippage model. Models in which the divalent form of the anion exchange carrier or water pores mediate net anion flow are also inconsistent with the data. The observations that net chloride flux increases with chloride concentration and that the DIDS- sensitive component tends to saturate suggest a model in which net anion flow involves "transit" of anions through the diffusion barriers in series with the transport site, without any change in transport site conformation such as normally occurs during the anion exchange process. This model is successful in predicting that the anion exchange inhibitor NAP-taurine, which binds to the modifier site and inhibits the conformational change, has less effect on net chloride flow than on chloride exchange.     

Second transmembrane domain of human uncoupling protein 2 is essential for its anion channel formation

Uncoupling proteins (UCP) are known to transport anions, such as Cl-, in addition to H+ transport. Although H+ transport by UCP is clearly involved in thermogenesis, the mechanism of its anion transport is not clearly understood. In this study, we examined the anion channel characteristics of the six individual helical transmembrane (TM) domains of the human UCP2. The second TM domain peptide (TM2) forms multi-state channels by assemblies of conductive oligomers. Furthermore, the TM2 exhibited voltage-dependent anion channels with properties comparable to those of UCP1 chloride channel. However, the other five TM peptides did not form UCP1-like channels. Moreover, an analog of TM2 in which two Arg residues were substituted by Ala residues did not form stable channels, implying the significance of Arg residues for anion transport. These results suggest that the anion channel structure of UCP2 protein is oligomeric and the second TM domain is essential for the voltage-dependence of this anion channel.     

Hydrogen peroxide downregulates human organic anion transporters in the basolateral membrane of the proximal tubule

Hydrogen peroxide (H2O2) is known to be involved in drug-induced and ischemic proximal tubular damage. The purpose of this study was to elucidate the effects of hydrogen peroxide on organic anion transport mediated by human organic anion transporters 1 and 3 (hOAT1 and hOAT3), which are localized at the basolateral side of the proximal tubule. For this purpose, we established and utilized the second segment of the proximal tubule cells from mice stably expressing hOAT1 or hOAT3 (S2 hOAT1 or S2hOAT3, respectively). H2O2 induced a dose- and a time-dependent decrease in organic anion transport mediated by hOAT1 and hOAT3. Kinetic analysis revealed that H2O2 decreased the Vmax, but not Km of organic anion transport both in S2hOAT1 and S2hOAT3. The effects of gentamicin, known to induce proximal tubular damage via the production of H2O2, on the organic anion transporters were also examined. Gentamicin induced a significant decrease in organic anion transport in S2hOAT1 but not S2hOAT3. H2O2-induced decrease in organic anion transport was significantly inhibited by pretreatment with pyruvate as well as catalase, whereas the gentamicin-induced decrease was significantly inhibited by pretreatment with pyruvate but not with catalase. In conclusion, these results suggest that H2O2, which is produced during tubular injuries, downregulates organic anion transport mediated by both hOAT1 and hOAT3, leading to further modulation of pathophysiology.     

Probenecid, a gout remedy, inhibits pannexin 1 channels

Probenecid is a well-established drug for the treatment of gout and is thought to act on an organic anion transporter, thereby affecting uric acid excretion in the kidney by blocking urate reuptake. Probenecid also has been shown to affect ATP release, leading to the suggestion that ATP release involves an organic anion transporter. Other pharmacological evidence and the observation of dye uptake, however, suggest that the nonvesicular release of ATP is mediated by large membrane channels, with pannexin 1 being a prominent candidate. In the present study we show that probenecid inhibited currents mediated by pannexin 1 channels in the same concentration range as observed for inhibition of transport processes. Probenecid did not affect channels formed by connexins. Thus probenecid allows for discrimination between channels formed by connexins and pannexins.