Structural model of the anion exchanger 1 (SLC4A1) and identification of transmembrane segments forming the transport site.
ABSTRACT: The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na(+) and K(+) conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.
Project description:Previous results suggested that specific point mutations in human anion exchanger 1 (AE1) convert the electroneutral anion exchanger into a monovalent cation conductance. In the present study, the transport site for anion exchange and for the cation leak has been studied by cysteine scanning mutagenesis and sulfhydryl reagent chemistry. Moreover, the role of some highly conserved amino acids within members of the SLC4 family to which AE1 belongs has been assessed in AE1 transport properties. The results suggest that the same transport site within the AE1 spanning domain is involved in anion exchange or in cation transport. A functioning mechanism for this transport site is proposed according to transport properties of the different studied point mutations of AE1.
Project description:AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.
Project description:Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of a-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400-Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin-Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of a-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.
Project description:Anion exchanger 1 (AE1) is responsible for the exchange of bicarbonate and chloride across the erythrocyte plasma membrane. Human AE1 consists of a cytoplasmic and a membrane domain joined by a 33-residue flexible linker. Crystal structures of the individual domains have been determined, but the intact AE1 structure remains elusive. In this study, we use molecular dynamics simulations and modeling to build intact AE1 structures in a complex lipid bilayer that resembles the native erythrocyte plasma membrane. AE1 models were evaluated using available experimental data to provide an atomistic view of the interaction and dynamics of the cytoplasmic domain, the membrane domain, and the connecting linker in a complete model of AE1 in a lipid bilayer. Anionic lipids were found to interact strongly with AE1 at specific amino acid residues that are linked to diseases and blood group antigens. Cholesterol was found in the dimeric interface of AE1, suggesting that it may regulate subunit interactions and anion transport.
Project description:Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3- and Cl- exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl-/HCO3- exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3- and outward Cl- gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3- for stomatin-deficient patients. Similarly, kinetics of Cl- efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.
Project description:Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na(+)-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation. Introduction of single and double mutations at the equivalent arginine (Arg(283)) and at an interacting glutamate (Glu(85)) in the cytosolic domain of human AE1 (cdAE1) had no effect on the cell surface expression or the transport activity of AE1 expressed in HEK-293 cells. In addition, the membrane domain of AE1 (mdAE1) efficiently mediated anion transport. A 2.1-? resolution crystal structure of cd?54AE1 (residues 55-356 of cdAE1) lacking the amino-terminal and carboxyl-terminal disordered regions, produced at physiological pH, revealed an extensive hydrogen-bonded network involving Arg(283) and Glu(85). Mutations at these residues affected the pH-dependent conformational changes and stability of cd?54AE1. As these structural alterations did not impair functional expression of AE1, the cytosolic and membrane domains operate independently. A substrate access tunnel within the cytosolic domain is not present in AE1 and therefore is not an essential feature of the SLC4 family of bicarbonate transporters.
Project description:Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1-4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (DeltaA400-A408) in TM1. These results suggest that the TM2-3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1-2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2-3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2-3 suggest that these segments form a re-entrant loop in human AE1.
Project description:Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the ?-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.
Project description:It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in ?-intercalated cells and the basolateral anion exchanger (AE1) in ?-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and ?-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6-20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base.
Project description:We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na(+) efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na(+) efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl(-) transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl(-) transport activity, but R730 substitution with I, E, or H inactivated Cl(-) transport. AE1 R730C expression substantially increased endogenous oocyte Na(+)-K(+)-ATPase-mediated (86)Rb(+) influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH(o)) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH(o)-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH(o) insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl(-) influx by acid pH(o). Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb(+) and Li(+) influxes. Selective rescue of acid pH(o)-stimulated sulfate uptake and conferral of acid pH(o)-stimulated Cl(-) influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.