二价金属离子转运蛋白1——一个新发现的重要铁转运蛋白

二价金属离子转运蛋白1(divalent metal transporter 1,DMT1)的发现是近年铁代谢研究领域最重大的一项突破.DMT1是哺乳类跨膜铁转运蛋白.这种蛋白质广泛分布于人体各组织.DMT1 mRNA有两种形式,一种含有IRE(iron response element),而另一种则不含此结构.DMT1的功能主要是介导小肠上皮细胞的铁吸收以及参与铁从内吞小体移位到胞浆的过程.DMT1介导的铁转运是一个主动的和H⁺依赖的过程.DMT1也参与其他二价金属如Zn²⁺、Mn²⁺、Co²⁺、Cd²⁺、Cn²⁺、Ni²⁺和Pb²⁺的转运.小肠DMT1的表达受饮食或组织铁控制.第四跨膜区是DMT1的重要功能区.此区基因发生点突变（G185R）是导致不可逆性缺铁性贫血的原因.在帕金森氏病人的黑质发现DMT1表达异常增加,因而DMT1可能也与某些神经退行性疾病的形成有关.     

鸡二价金属转运蛋白1 cDNA的克隆与序列分析

鸡二价金属转运蛋白1（divalent metal transporter 1, DMT1）在动物胃肠道锰吸收过程中起重要作用.根据哺乳动物Dmt1同源蛋白氨基酸序列的保守性设计引物,应用3′RACE(rapid amplification of cDNA ends)技术,扩增并克隆获得鸡小肠Dmt1 cDNA 3′端1 289bp和1 092bp的2种片段,发现其3′端翻译区和非翻译区存在差异. 根据鸡Dmt1 cDNA 3′端片段的测序结果设计引物,扩增获得1个与3′端片段部分重叠的鸡Dmt1 cDNA 5′端907 bp片段,并对其进行了克隆测序. 根据鸡小肠Dmt1 3′RACE片段和5′RACE片段序列信息进行拼接,从而获得鸡小肠Dmt1 cDNA全序列信息.结果表明,鸡小肠Dmt1 cDNA有2种形式,1种全长为1 972个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 695个核苷酸,3′非翻译区为173个核苷酸,编码1个含564个氨基酸残基的蛋白质;另1种形式为1 775个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 593个核苷酸,3′非翻译区为78个核苷酸,编码1个含530个氨基酸残基的蛋白质.据鸡Dmt1 cDNA推测出的2种形式蛋白质的氨基酸序列与人、大鼠和小鼠的Dmt1蛋白具有高度同源性,它们的同源性分别为82%、82%、80%,和 84%、84%、83%. 对推测氨基酸序列进行疏水性和跨膜区分析表明,Dmt1蛋白为1种跨膜整合蛋白,具有膜转运蛋白糖基化位点和底物结合位点的保守序列.     

Iron,Copper, and Zinc Transport: Inhibition of Divalent Metal Transporter 1 (DMT1) and Human Copper Transporter 1 (hCTR1) by shRNA

Iron (Fe), copper (Cu), and zinc (Zn) fulfill various essential biological functions and are vital for all living organisms. They play important roles in oxygen transport, cell growth and differentiation, neurotransmitter synthesis, myelination, and synaptic transmission. Because of their role in many critical functions, they are commonly used in food fortification and supplementation strategies globally. To determine the involvement of divalent metal transporter 1 (DMT1) and human copper transporter 1 (hCTR1) on Fe, Cu, and Zn uptake, Caco-2 cells were transfected with four different shRNA plasmids to selectively inhibit DMT1 or hCTR1 transporter expression. Fe and Cu uptake and total Zn content measurements were performed in shRNA-DMT1 and shRNA-hCTR1 cells. Both shRNA-DMT1 and shRNA-hCTR1 cells had lower apical Fe uptake (a decrease of 51% and 41%, respectively), Cu uptake (a decrease of 25.8% and 38.5%, respectively), and Zn content (a decrease of 23.1% and 22.7%, respectively) compared to control cells. These results confirm that DMT1 is involved in active transport of Fe, Cu, and Zn although Zn showed a different relative capacity. These results also show that hCTR1 is able to transport Fe and Zn.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Divalent Metal Ions with Zn2+-Glycerophosphocholine Cholinephosphodiesterase from Ox Brain

The effect of divalent metal ions on the activity of glycerophosphocholine cholinephosphodiesterse from ox brain was examined. Zn²⁺- and Co²⁺-glycerophosphocholine cholinephosphodiesterases were prepared from the exposure of apoenzyme to Zn²⁺ and Co²⁺, respectively, and the properties of two metallo-phosphodiesterases were compared to those of native phosphodiesterase. Although two metallo-enzymes were similar in expressing Km value, optimum pH or sensitivity to Cu²⁺, they differed in the susceptibility to the inhibition by thiocholine or tellurite; while Co²⁺-phosphodiesterase was more sensitive to tellurites, Zn²⁺-phosphodiesterase was more susceptible to inhibition by thiocholine. In addition, Zn²⁺-phosphodiesterase was more thermo-stable than Co²⁺ enzyme. Separately, when properties of native phosphodiesterase were compared to those of each metallo-phosphodiesterase, native phosphodiesterase was found to be quite similar to Zn²⁺-phosphodiesterase in many respects. Even in thermo-stability, native enzyme resembled Zn²⁺-phosphodiesterase rather than Co²⁺-enzyme. Consistent with this, the stability of native phosphodiesterase was maintained in the presence of Zn²⁺, but not Co²⁺. Mn²⁺ was also as effective as Zn²⁺ in the stabilization of the enzyme. Noteworthy, the native enzyme was found to be inhibited competitively by Cu²⁺ with a Ki value of 20 M, and its inhibitory action was antagonized effectively by Zn²⁺ or Co²⁺. Also, choline, another competitive inhibitor of the enzyme, appeared to antagonize the inhibitory action of Cu²⁺. Taken together, it is suggested that there may be multiple binding sites for divalent metal ions in the molecule of glycerophosphocholine cholinephosphodiesterase.     

The Divalent Metal Transporter Homologues SMF-1/2 Mediate Dopamine Neuron Sensitivity in Caenorhabditis elegans Models of Manganism and Parkinson Disease

Parkinson disease (PD) and manganism are characterized by motor deficits and a loss of dopamine (DA) neurons in the substantia nigra pars compacta. Epidemiological studies indicate significant correlations between manganese exposure and the propensity to develop PD. The vertebrate divalent metal transporter-1 (DMT-1) contributes to maintaining cellular Mn²⁺ homeostasis and has recently been implicated in Fe²⁺-mediated neurodegeneration in PD. In this study we describe a novel model for manganism that incorporates the genetically tractable nematode Caenorhabditis elegans. We show that a brief exposure to Mn²⁺ increases reactive oxygen species and glutathione production, decreases oxygen consumption and head mitochondria membrane potential, and confers DA neuronal death. DA neurodegeneration is partially dependent on a putative homologue to DMT-1, SMF-1, as genetic knockdown or deletion partially inhibits the neuronal death. Mn²⁺ also amplifies the DA neurotoxicity of the PD-associated protein α-synuclein. Furthermore, both SMF-1 and SMF-2 are expressed in DA neurons and contribute to PD-associated neurotoxicant-induced DA neuron death. These studies describe a C. elegans model for manganism and show that DMT-1 homologues contribute to Mn²⁺- and PD-associated DA neuron vulnerability.     

Lead and Cadmium Synergistically Enhance the Expression of Divalent Metal Transporter 1 Protein in Central Nervous System of Developing Rats

Divalent metal transporter 1 (DMT1) can transport a large range of ions, including toxic lead (Pb) and cadmium (Cd), across membranes. In this study, a total of 24 rats were divided into four groups for intragastrical perfusion treatment: control, Pb alone, Cd alone, and Pb + Cd. Pb and Cd contents in blood were detected, and the mRNA and protein levels of DMT1 were analyzed in the cerebellum, cortex, and hippocampus. Both Pb and Cd levels were elevated in all groups perfused with Pb and/or Cd, except for Pb level in the Cd-alone group (P < 0.05). The mRNA level of DMT1 did not differ among the four groups (P > 0.05). However, the DMT1 protein expression was significantly increased by 0.9-, 1.0-, and 1.1-fold in cerebellum, cortex, and hippocampus of the Pb + Cd group than in controls, respectively. Pb and Cd exposure can synergistically induce DMT1 protein synthesis and has implications for transportation of toxic ions in the developing rat’s brain. Chengwu Gu and Songjian Chen contributed equally to this work, they are joint first authors.     

FGT-1 Is a Mammalian GLUT2-Like Facilitative Glucose Transporter in Caenorhabditis elegans Whose Malfunction Induces Fat Accumulation in Intestinal Cells

Caenorhabditis elegans (C. elegans) is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS), that are conserved in mammalian cells. To explore C. elegans as a model system to study the regulation of the facilitative glucose transporter (GLUT), we have characterized the GLUT gene homologues in C. elegans: fgt-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1 and Y37A1A.3. The exogenous expression of these gene products in Xenopus oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (K _m) of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1-mediated 2-deoxy-D-glucose uptake (P < 0.01), indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in C. elegans, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of fgt-1 resulted in increased fat staining in both wild-type and daf-2 (mammalian insulin receptor homologue) mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by fgt-1 knockdown in wild-type or daf-2 mutants. Our results indicated that in C. elegans, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism.     

Phospholipase A2-activating protein induces mitophagy through anti-apoptotic MCL1-mediated NLRX1 oligomerization

Mitochondrial protein homeostasis is fine-tuned by diverse physiological processes such as mitochondria-associated degradation (MAD), which is regulated by valosin-containing protein (VCP) and its cofactors. As a cofactor of VCP, the mutation of phospholipase A-2-activating protein (PLAA) is the genetic cause of PLAA-associated neurodevelopmental disorder (PLAAND). However, the physiological and pathological roles of PLAA in mitochondria remain unclear. Here, we demonstrate that PLAA partially associates with mitochondria. Deficiency in PLAA increases mitochondrial reactive oxygen species (ROS) production, reduces mitochondrial membrane potential, inhibits mitochondrial respiratory activity and causes excessive mitophagy. Mechanically, PLAA interacts with myeloid cell leukemia-1 (MCL1) and facilitates its retro-translocation and proteasome-dependent degradation. The upregulation of MCL1 promotes the oligomerization of NLR family member X1 (NLRX1) and activation of mitophagy. Whereas downregulating NLRX1 abolishes MCL1 induced mitophagy. In summary, our data identify PLAA as a novel mediator of mitophagy by regulating MCL1-NLRX1 axis. We propose mitophagy as a target for therapeutic intervention in PLAAND.     

三磷酸腺苷结合盒转运体A1在脑疾病发生发展中的作用

三磷酸腺苷结合盒转运体A1(ATP binding cassette transporter A1,ABCA1)在脑组织中广泛表达,它将脑细胞内胆固醇转运给载脂蛋白E(apolipoprotein E,apoE)及载脂蛋白A1(apolipoprotein A1,apoA-Ⅰ)形成高密度脂蛋白(high density lipoprotein,HDL),从而调控脑内胆固醇平衡.研究表明,ABCA1与胆固醇代谢相关脑疾病存在密切联系,包括阿尔茨海默病(Alzheimer's disease,AD)、创伤性脑损伤(traumatic brain injury,TBI)及脑梗死.虽然近来在ABCA1与相关脑疾病的研究取得了一些进展,但仍存在许多问题尚未阐明.本文对ABCA1在各种相关脑疾病发生发展中的作用做一综述,期望为相关脑疾病的治疗寻找新的靶点和方法.     

Partitioning of Photosynthetically Fixed 14CO2 Into Oil and Curcumin Accumulation in Curcuma Longa Grown Under Iron Deficiency

Changes in leaf growth, photosynthetic efficiency, and incorporation pattern of photosynthetically fixed ¹⁴CO₂ in leaves 1 and 2 from plant apex, in roots, and rhizome induced in Curcuma by growing in a solution culture at Fe concentration of 0 and 5.6 g m^–3 were studied. ¹⁴C was incorporated into primary metabolites (sugars, amino acids, and organic acids) and secondary metabolites (essential oil and curcumin). Fe deficiency resulted in a decrease in leaf area, its fresh and dry mass, chlorophyll (Chl) content, and CO₂ exchange rate at all leaf positions. The rate of ¹⁴CO₂ fixation declined with leaf position, maximum being in the youngest leaf. Fe deficiency resulted in higher accumulation of sugars, amino acids, and organic acids in leaves at both positions. This is due to poor translocation of metabolites. Roots and rhizomes of Fe-deficient plants had lower concentrations of total photosynthate, sugars, and amino acids whereas organic acid concentration was higher in rhizomes. ¹⁴CO₂ incorporation in essential oil was lower in the youngest leaf, as well as incorporation in curcumin content in rhizome. Fe deficiency influenced leaf area, its fresh and dry masses, CO₂ exchange rate, and oil and curcumin accumulation by affecting translocation of assimilated photosynthates.     

Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.     

Perinatal Exposure to Bisphenol-A Impairs Spatial Memory through Upregulation of Neurexin1 and Neuroligin3 Expression in Male Mouse Brain

Bisphenol-A (BPA), a well known endocrine disruptor, impairs learning and memory in rodents. However, the underlying molecular mechanism of BPA induced impairment in learning and memory is not well known. As synaptic plasticity is the cellular basis of memory, the present study investigated the effect of perinatal exposure to BPA on the expression of synaptic proteins neurexin1 (Nrxn1) and neuroligin3 (Nlgn3), dendritic spine density and spatial memory in postnatal male mice. The pregnant mice were orally administered BPA (50 µg/kgbw/d) from gestation day (GD) 7 to postnatal day (PND) 21 and sesame oil was used as a vehicle control. In Morris water maze (MWM) test, BPA extended the escape latency time to locate the hidden platform in 8 weeks male mice. RT-PCR and Immunoblotting results showed significant upregulation of Nrxn1 and Nlgn3 expression in both cerebral cortex and hippocampus of 3 and 8 weeks male mice. This was further substantiated by in-situ hybridization and immunofluorescence techniques. BPA also significantly increased the density of dendritic spines in both regions, as analyzed by rapid Golgi staining. Thus our data suggest that perinatal exposure to BPA impairs spatial memory through upregulation of expression of synaptic proteins Nrxn1 and Nlgn3 and increased dendritic spine density in cerebral cortex and hippocampus of postnatal male mice.     

A Possible Pathway of Phosphoinositide Metabolism Through EDTA-Insensitive Phospholipase A1 Followed by Lysophosphoinositide-Specific Phospholipase C in Rat Brain

Abstract— Incubation of [2-³H]glycerol-labeled phosphatidylinositol with a crude cytosol fraction of rat brain in the presence of EDTA yielded [³H]lysophosphatidylinositol predominantly without accumulation of labeled monoacylglycerol and diacylglycerol. The pH optimum of this Phospholipase A activity was 8.0. The activity for phosphatidylinositol was twofold higher than for phosphatidylethanolamine, whereas phosphatidylcholine, phosphatidylserine, and phosphatidic acid were not hydrolyzed significantly under the conditions used. The phospholipase A activity for phosphatidylethanolamine was resolved in part from that for phosphatidylinositol by ammonium sulfate fractionation of the cytosol, indicating the existence of at least two forms of EDTA-insensitive phospholipase A. The positional specificity of the phosphatidylinositol-hydrolyzing activity was found to be that of a phospholipase A₁, as radioactive lysophosphatidylinositol was produced from 1 -stearoyl-2-[1-¹⁴C]arachidonyl- sn -glycero-3-phosphoinositol without release of free arachidonate. A phospholipase C activity specific for lysophosphoinositides was found in a membrane fraction from rat brain, which was similar to that characterized in porcine platelets. The phospholipase C was demonstrated to hydrolyze the 2-acyl isomer as well as the 1-acyl isomer of lysophosphatidylinositol. Taken together, our results suggest a possible pathway through which phosphatidylinositol is selectively degraded to the 2-acyl isomer of lysophosphatidylinositol in a Ca²⁺-independeht manner, and subsequently converted to 2-monoacylglycerol in rat brain.     

Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.     

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis Epidermis Induces Cell Death and Suggests a Critical Role for Phospholipase A2 in This Process

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific β-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.     

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO₃ transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO₃ transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO₃⁻ currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO₃-dependent pH recovery from a CO₂-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO₂/HCO₃⁻-free solution that was abolished by Cl⁻ removal from the bath. In the presence of CO₂/HCO₃⁻, however, the outward current produced by D555E decreased only slightly after Cl⁻ removal. Starting from a Cl⁻-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO₂/HCO₃⁻. The substitution of Asp⁵⁵⁵ with an asparagine also produced a Cl⁻ current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO₃⁻. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp⁵⁵⁵ plays a role in HCO₃⁻ selectivity.The electrogenic Na/HCO₃ cotransporter NBCe1 (SLC4A4) is one of the SLC4A gene family members transporting HCO₃⁻ across the plasma membrane (1–3). NBCe1 plays a role in transepithelial HCO₃⁻ movement and pH_i regulation in many tissues (4–6). NBCe1 is responsible for HCO₃⁻ reabsorption in the proximal tubules of the kidney (7). The proximal tubule cells reclaim HCO₃⁻ from the lumen through a series of reactions involving titration of HCO₃⁻ by H⁺ secretion via the apical Na/H exchanger, production of CO₂, and regeneration of HCO₃⁻ and H⁺ in the tubule cells. HCO₃⁻ then moves to the interstitium via the basolateral NBCe1. The essential feature driving this basolateral Na⁺/HCO₃⁻ exit is the stoichiometry of 1:3 Na⁺:HCO₃⁻, which makes the equilibrium potential for NBCe1 more positive than the resting membrane potential of the proximal tubule cells (8). The stoichiometry of 1Na⁺:1HCO₃⁻ or 1Na⁺:2HCO₃⁻ causes both ions to move into cells in other tissues such as pancreas, brain, and cardiovascular tissues (9, 10).Despite the importance of NBCe1 for basolateral HCO₃⁻ reabsorption in the proximal tubules, the mechanism of electrogenic Na/HCO₃ transport via the transporter is not well understood. Ion movement depends on loading ions at their translocation or binding sites that likely reside within the membrane field at some distance from the bath solution (11). This implies that the transmembrane domains (TMs)2 of NBCe1 and amino acid residues within TMs play critical roles in ion transport.Sequence analysis of different SLC4A proteins shows similar hydropathy plots, predicting that these proteins share structural elements of transport function (12). Such similarities have facilitated structure/function studies to define molecular domains or motifs responsible for conferring Na/HCO₃ transport of NBCe1. Abuladze et al. (13) performed a large scale mutagenesis on acidic and basic amino acids in non-TMs and found many residues affecting Na⁺-dependent base flux. McAlear et al. (14) identified amino acids in TM8 involving ion translocation. By a systematic approach of chimeric transporters between NBCe1 and the electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) (15), we and our colleagues (16) demonstrated that electrogenic Na/HCO₃ transport of NBCe1 requires interactions between the regions TM1–5 and TM6–13 of the protein. Zhu et al. (17) recently proposed TM1 as a domain lining the ion translocation pathway. On the other hand, Chang et al. (18) reported that the cytoplasmic N-terminal domain might contribute to HCO₃⁻ permeation.In the present study, we searched amino acid residues that are highly conserved among electrogenic Na/HCO₃ transporters but not among electroneutral Na/HCO₃ transporters and examined their role in electrogenic Na/HCO₃ transport. Nine candidate residues in human renal NBCe1-A (5, 19) were selected and mutated by replacement with the amino acids at the corresponding sites of NBCn1. Mutant transporters were expressed in Xenopus oocytes and assessed via two-electrode voltage clamp. Our data show that Asp⁵⁵⁵ of NBCe1 plays an important role in HCO₃⁻ selectivity.