Walter F. Boron

Cloning and characterization of a mammalian proton-coupled metal-ion transporter

Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. Despite their pivotal physiological roles, however, there is no molecular information on how metal ions are actively absorbed by mammalian cells. We have now identified a new metal-ion transporter in the rat, DCT1, which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency, and may represent a key mediator of intestinal iron absorption. DCT1 is a member of the ‘natural-resistance-associated macrophage protein’ (Nramp) family and thus its properties provide insight into how these proteins confer resistance to pathogens.

The SLC4 family of HCO3 − transporters

The SLC4 family consists of ten genes. All appear to encode integral membrane proteins with very similar hydropathy plots—consistent with the presence of 10–14 transmembrane segments. At least eight SLC4 members encode proteins that transport HCO3− (or a related species, such as CO32−) across the plasma membrane. Functionally, these eight proteins fall into two major groups: three Cl-HCO3 exchangers (AE1–3) and five Na+-coupled HCO3− transporters (NBCe1, NBCe2, NBCn1, NDCBE, NCBE). Two of the Na+-coupled HCO3− transporters (NBCe1, NBCe2) are electrogenic; the other three Na+-coupled HCO3− transporters and all three AEs are electroneutral. At least NDCBE transports Cl− in addition to Na+ and HCO3−. Whether NCBE transports Cl–—in addition to Na+ and HCO3−—is unsettled. In addition, two other SLC4 members (AE4 and BTR1) do not yet have a firmly established function; on the basis of homology, they fall between the two major groups. A characteristic of many, though not all, SLC4 members is inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS). SLC4 gene products play important roles in the carriage of CO2 by erythrocytes, the absorption or secretion of H+ or HCO3− by several epithelia, as well as the regulation of cell volume and intracellular pH.

Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells

Trypanosoma cruzi invades most nucleated cells by a mechanism distinct from classical phagocytosis. Although parasites enter at the lysosome-poor peripheral cell margins, lysosomal markers are immediately incorporated into the parasitophorous vacuole. No accumulation of polymerized actin was detected around recently internalized parasites, and disruption of microfilaments significantly facilitated invasion. Lysosomes were observed to aggregate at the sites of trypanosome attachment and to fuse with the vacuole at early stages of its formation. Experimentally induced, microtubule-dependent movement of lysosomes from the perinuclear area to the cell periphery enhanced entry. Conditions that deplete cells of peripheral lysosomes or interfere with lysosomal fusion capacity inhibited invasion. These observations reveal a novel mechanism for cell invasion:recruitment of lysosomes for fusion at the site of parasite internalization.

Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes

It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM[Formula: see text] to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 × 10-4 pH units/s in control oocytes and 13.3 × 10-4 pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 × 10-4 pH units/s). Ethoxzolamide (ETX; 10 μM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 × 10-4 pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were ∼40% faster than in the water/CA oocytes (75 × 10-4 pH units/s), and ETX reduced the rates substantially (14.7 × 10-4 pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux, it would remain to be determined whether this CO2movement is quantitatively important.

An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel

Two electroneutral, Na+-driven HCO-3 transporters, the Na+-driven Cl-/HCO-3 exchanger and the electroneutral Na+/ HCO-3 cotransporter, have crucial roles in regulating intracellular pH in a variety of cells, including cardiac myocytes, vascular smooth-muscle, neurons and fibroblasts; however, it is difficult to distinguish their Cl- dependence in mammalian cells. Here we report the cloning of three variants of an electroneutral Na+/HCO-3 cotransporter, NBCn1, from rat smooth muscle. They are 89–92% identical to a human skeletal muscle clone, 55–57% identical to the electrogenic NBCs and 33–43% identical to the anion exchangers. When expressed in Xenopus oocytes, NBCn1-B (which encodes 1,218 amino acids) is electroneutral, Na+-dependent and HCO-3-dependent, but not Cl--dependent. Oocytes injected with low levels of NBCn1-B complementary RNA exhibit a Na+ conductance that 4,4-diisothiocyanatostilbene-2,2′-disulphonate stimulates slowly and irreversibly.

Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG

The water channel aquaporin 1 (AQP1) and certain Rh-family members are permeable to CO2 and NH3. Here, we use changes in surface pH (pHS) to assess relative CO2 vs. NH3 permeability of Xenopus oocytes expressing members of the AQP or Rh family. Exposed to CO2 or NH3, AQP1 oocytes exhibit a greater maximal magnitude of pHS change (ΔpHS) compared with day-matched controls injected with H2O or with RNA encoding SGLT1, NKCC2, or PepT1. With CO2, AQP1 oocytes also have faster time constants for pHS relaxation (τpHs). Thus, AQP1, but not the other proteins, conduct CO2 and NH3. Oocytes expressing rat AQP4, rat AQP5, human RhAG, or the bacterial Rh homolog AmtB also exhibit greater ΔpHS(CO2) and faster τpHs compared with controls. Oocytes expressing AmtB and RhAG, but not AQP4 or AQP5, exhibit greater ΔpHS(NH3) values. Only AQPs exhibited significant osmotic water permeability (Pf). We computed channel-dependent (*) ΔpHS or Pf by subtracting values for H2O oocytes from those of channel-expressing oocytes. For the ratio ΔpHS(CO2)*/Pf*, the sequence was AQP5 > AQP1 ≅ AQP4. For ΔpHS(CO2)*/ΔpHS(NH3)*, the sequence was AQP4 ≅ AQP5 > AQP1 > AmtB > RhAG. Thus, each channel exhibits a characteristic ratio for indices of CO2 vs. NH3 permeability, demonstrating that, like ion channels, gas channels can exhibit selectivity.

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane

We report here the application of a previously described method to directly determine the CO2 permeability (PCO2) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1‐expressing Xenopus laevis oocytes. This method measures the exchange of 18O between CO2, HCO3–, and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pHS) transients caused by the dominant effect of entry of CO2 vs. HCO3– into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces PCO2 from the normal value of 0.15 ± 0.08 (SD; n85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases PCO2 2‐fold and doubles the alkaline pHS gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces PCO2 of RBCs solely by action on AQP1 as it has no effect in AQP1‐deficient RBCs. 3) PCO2 determinations of RBCs and pHS measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS‐sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high PCO2 of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total PCO2.—Endeward, V., Musa‐Aziz, R., Cooper, G. J., Chen, L., Pelletier, M. F., Virkki, L. V., Supuran, C. T., King, L. S., Boron, W. F., Gros, G. Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J. 20, 1974–1981 (2006)

Cloning, characterization, and chromosomal mapping of a human electroneutral Na(+)-driven Cl-HCO3 exchanger.

The electroneutral Na+-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pH i ) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBankTM accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); ∼50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; ∼73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na+-driven anion exchanger (NDAE1) fromDrosophila. Northern blot analysis of NDCBE1 shows a robust ∼12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed inXenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pH i and [Na+] i (monitored with microelectrodes) that require HCO 3 − and are blocked by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The pH i increase also requires extracellular Na+. The Na+:HCO 3 − stoichiometry is 1:2. Forward-running NDCBE1 mediates a36Cl efflux that requires extracellular Na+ and HCO 3 − and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl−. Thus, NDCBE1 encodes a human, electroneutral Na+-driven Cl-HCO3 exchanger.

Immunolocalization of the electrogenic Na+-HCO3/- cotransporter in mammalian and amphibian kidney

Electrogenic cotransport of Na+ and[Formula: see text] is a crucial element of[Formula: see text] reabsorption in the renal proximal tubule (PT). An electrogenic Na+-[Formula: see text]cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928-1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a ∼130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl-/[Formula: see text]exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at ∼130 kDa in both rat and rabbit, as well as a single ∼160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-μm cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.

Acid-Base Transport by the Renal Proximal Tubule

One of the major tasks of the renal proximal tubule is to secrete acid into the tubule lumen, thereby reabsorbing approximately 80% of the filtered HCO3- as well as generating new HCO3- for regulating blood pH. This review summarizes the cellular and molecular events that underlie four major processes in HCO3- reabsorption. The first is CO2 entry across the apical membrane, which in large part occurs via a gas channel (aquaporin 1) and acidifies the cell. The second process is apical H+ secretion via Na-H exchange and H+ pumping, processes that can be studied using the NH4+ prepulse technique. The third process is the basolateral exit of HCO3- via the electrogenic Na/HCO3 co-transporter, which is the subject of at least 10 mutations that cause severe proximal renal tubule acidosis in humans. The final process is the regulation of overall HCO3- reabsorption by CO2 and HCO3- sensors at the basolateral membrane. Together, these processes ensure that the proximal tubule responds appropriately to acute acid-base disturbances and thereby contributes to the regulation of blood pH.