Michael L. Jennings

Oligomeric structure and the anion transport function of human erythrocyte band 3 protein.

ConclusionsEvidence from many laboratories using several different techniques strongly suggests that, in the intact red cell, band 3 exists as dimers which can associate with other dimers to form tetramers. The kinetics of anion transport inhibition by stilbenedisulfonates indicate that irreversible inhibition of one subunit does not detectably affect anion transport by the other subunit. This does not imply that monomeric band 3 could necessarily transport anions; the native conformation of each subunit may require stabilizing interactions with another subunit, as indicated by the recent work of Boodhoo and Reithmeier [10]. A more detailed understanding of the structure of the band 3 dimer/tetramer will require information on which specific segments of the primary structure are involved in subunit-subunit contact. The combination of chemical cross-linking with proteolysis [136] is a promising approach to this problem.

Proton Fluxes Associated with Erythrocyte Membrane Anion Exchange

SummaryTransient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942.Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloridesulfate exchange. The pH rise can be reversed by the addition of HCO3− (4 μm) or 2,4-dinitrophenol (90 μm). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloríde exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972.In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Roth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.

Characteristics of CO2-independent pH equilibration in human red blood cells.

SummaryThe rate of dissipation of a pH gradient across the red cell membrane has been measured at pH 5.7–7.5 in a medium free of CO2 and other penetrating acids or bases. The measured rates and extents of pH movements are influenced only slightly by valinomycin-induced changes in the membrane potential. This indicates that the primary process involved is electrically silent OH−/Cl− exchange or H+/Cl− cotransport. This electrically silent pH equilibration has several characteristics which suggest the involvement of the red cell anion exchange protein.1.It is strongly inhibited by phloretin and DIDS (4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid).2.The rates of pH equilibration depend on the halide present in the medium, the relative rates being 100, 18, and 2 in NaCl, NaBr, and NaI media, respectively.3.The pH equilibration has apparent activation energies of 27 kcal/mol atT<13°C and 16 kcal/mol atT>13°C. The pH dependence of the equilibration rate, however, is much more consistent with H+/Cl− cotransport than with OH−/Cl− exchange; the rate increases steeply with the H+, rather than the OH− concentration. It is suggested therefore that the transport event is H+/Cl− cotransport, but that this transport is mediated by the membrane protein that catalyzes anion exchange.

Chloride Homeostasis in Saccharomyces cerevisiae: High Affinity Influx, V-ATPase-dependent Sequestration, and Identification of a Candidate Cl− Sensor

Chloride homeostasis in Saccharomyces cerevisiae has been characterized with the goal of identifying new Cl− transport and regulatory pathways. Steady-state cellular Cl− contents (∼0.2 mEq/liter cell water) differ by less than threefold in yeast grown in media containing 0.003–5 mM Cl−. Therefore, yeast have a potent mechanism for maintaining constant cellular Cl− over a wide range of extracellular Cl−. The cell water:medium [Cl−] ratio is >20 in media containing 0.01 mM Cl− and results in part from sequestration of Cl− in organelles, as shown by the effect of deleting genes involved in vacuolar acidification. Organellar sequestration cannot account entirely for the Cl− accumulation, however, because the cell water:medium [Cl−] ratio in low Cl− medium is ∼10 at extracellular pH 4.0 even in vma1 yeast, which lack the vacuolar H+-ATPase. Cellular Cl− accumulation is ATP dependent in both wild type and vma1 strains. The initial 36Cl− influx is a saturable function of extracellular [36Cl−] with K1/2 of 0.02 mM at pH 4.0 and >0.2 mM at pH 7, indicating the presence of a high affinity Cl− transporter in the plasma membrane. The transporter can exchange 36Cl− for either Cl− or Br− far more rapidly than SO4 =, phosphate, formate, HCO3 −, or NO3 −. High affinity Cl− influx is not affected by deletion of any of several genes for possible Cl− transporters. The high affinity Cl− transporter is activated over a period of ∼45 min after shifting cells from high-Cl− to low-Cl− media. Deletion of ORF YHL008c (formate-nitrite transporter family) strongly reduces the rate of activation of the flux. Therefore, Yhl008cp may be part of a Cl−-sensing mechanism that activates the high affinity transporter in a low Cl− medium. This is the first example of a biological system that can regulate cellular Cl− at concentrations far below 1 mM.

Transport of H2S and HS− across the human red blood cell membrane: rapid H2S diffusion and AE1-mediated Cl−/HS− exchange

The rates of H2S and HS(-) transport across the human erythrocyte membrane were estimated by measuring rates of dissipation of pH gradients in media containing 250 μM H2S/HS(-). Net acid efflux is caused by H2S/HS(-) acting analogously to CO2/HCO3(-) in the Jacobs-Stewart cycle. The steps are as follows: 1) H2S efflux through the lipid bilayer and/or a gas channel, 2) extracellular H2S deprotonation, 3) HS(-) influx in exchange for Cl(-), catalyzed by the anion exchange protein AE1, and 4) intracellular HS(-) protonation. Net acid transport by the Cl(-)/HS(-)/H2S cycle is more efficient than by the Cl(-)/HCO3(-)/CO2 cycle because of the rapid H2S-HS(-) interconversion in cells and medium. The rates of acid transport were analyzed by solving the mass flow equations for the cycle to produce estimates of the HS(-) and H2S transport rates. The data indicate that HS(-) is a very good substrate for AE1; the Cl(-)/HS(-) exchange rate is about one-third as rapid as Cl(-)/HCO3(-) exchange. The H2S permeability coefficient must also be high (>10(-2) cm/s, half time <0.003 s) to account for the pH equilibration data. The results imply that H2S and HS(-) enter erythrocytes very rapidly in the microcirculation of H2S-producing tissues, thereby acting as a sink for H2S and lowering the local extracellular concentration, and the fact that HS(-) is a substrate for a Cl(-)/HCO3(-) exchanger indicates that some effects of exogenous H2S/HS(-) may not result from a regulatory role of H2S but, rather, from net acid flux by H2S and HS(-) transport in a Jacobs-Stewart cycle.

Culture of embryonic renal collecting duct epithelia in a gradient container.

Abstract. During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.

Mouse Ae1 E699Q mediates SO42−i/aniono exchange with [SO42−]i-dependent reversal of wild-type pHo sensitivity

The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.

Mouse Slc4a11 expressed in Xenopus oocytes is an ideally selective H+/OH− conductance pathway that is stimulated by rises in intracellular and extracellular pH

The SLC4A11 gene encodes the bicarbonate-transporter-related protein BTR1, which is mutated in syndromes characterized by vision and hearing loss. Signs of these diseases [congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome] are evident in mouse models of Slc4a11 disruption. However, the intrinsic activity of Slc4a11 remains controversial, complicating assignment of its (patho)physiological role. Most studies concur that Slc4a11 transports H+ (or the thermodynamically equivalent species OH-) rather than HCO3-, but disparities have arisen as to whether the transport is coupled to another species such as Na+ or NH3/NH4+ Here for the first time, we examine the action of mouse Slc4a11 in Xenopus oocytes. We simultaneously monitor changes in intracellular pH, membrane potential, and conductance as we alter extracellular pH, revealing the electrical and chemical driving forces that underlie the observed ion fluxes. We find that mSlc4a11 is an ideally selective H+/OH- conductive pathway, the action of which is uncoupled from the cotransport of any other ion. We also find that the activity of mSlc4a11 is independently enhanced by both extracellular and intracellular alkalinization, suggesting OH- as the most likely substrate and providing a novel explanation for the apparent NH3-dependence of Slc4a11-mediated currents reported by others. We suggest that the unique properties of Slc4a11 action underlie its value as a pH regulator in corneal endothelial cells.

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct &agr;-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation.

Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo

Osteoporosis, osteopenia, and pathological bone fractures are frequent complications of iron-overload conditions such as hereditary hemochromatosis, thalassemia, and sickle cell disease. Moreover, animal models of iron overload have revealed increased bone resorption and decreased bone formation. Although systemic iron overload affects multiple organs and tissues, leading to significant changes on bone modeling and remodeling, the cell autonomous effects of excessive iron on bone cells remain unknown. Here, to elucidate the role of cellular iron homeostasis in osteoclasts, we generated two mouse strains in which solute carrier family 40 member 1 (Slc40a1), a gene encoding ferroportin (FPN), the sole iron exporter in mammalian cells, was specifically deleted in myeloid osteoclast precursors or mature cells. The FPN deletion mildly increased iron levels in both precursor and mature osteoclasts, and its loss in precursors, but not in mature cells, increased osteoclastogenesis and decreased bone mass in vivo. Of note, these phenotypes were more pronounced in female than in male mice. In vitro studies revealed that the elevated intracellular iron promoted macrophage proliferation and amplified expression of nuclear factor of activated T cells 1 (Nfatc1) and PPARG coactivator 1β (Pgc-1β), two transcription factors critical for osteoclast differentiation. However, the iron excess did not affect osteoclast survival. While increased iron stimulated global mitochondrial metabolism in osteoclast precursors, it had little influence on mitochondrial mass and reactive oxygen species production. These results indicate that FPN-regulated intracellular iron levels are critical for mitochondrial metabolism, osteoclastogenesis, and skeletal homeostasis in mice.