The crystal structure of the dimeric membrane domain of human Band 3(1), the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.

Background To date, only monoclonal antibodies have been shown to be effective for outpatients with COVID-19. Interferon lambda-1 is a type III interferon involved in innate antiviral responses with activity against respiratory pathogens. We aimed to investigate the safety and efficacy of peginterferon lambda in the treatment of outpatients with mild-to-moderate COVID-19. MethodsIn this double-blind, placebo-controlled trial, outpatients with laboratory-confirmed COVID-19 were randomly assigned to a single subcutaneous injection of peginterferon lambda 180 µg or placebo within 7 days of symptom onset or first positive swab if asymptomatic. Participants were randomly assigned (1:1) using a computergenerated randomisation list created with a randomisation schedule in blocks of four. At the time of administration, study nurses received a sealed opaque envelope with the treatment allocation number. The primary endpoint was the proportion of patients who were negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on day 7 after the injection, analysed by a χ² test following an intention-to-treat principle. Prespecified analysis of the primary endpoint, adjusted for baseline viral load, using bivariate logistic regression was done. The trial is now complete. This trial is registered with ClinicalTrials.gov, NCT04354259.Findings Between May 18, and Sept 4, 2020, we recruited 30 patients per group. The decline in SARS-CoV-2 RNA was greater in those treated with peginterferon lambda than placebo from day 3 onwards, with a difference of 2•42 log copies per mL at day 7 (p=0•0041). By day 7, 24 (80%) participants in the peginterferon lambda group had an undetectable viral load, compared with 19 (63%) in the placebo group (p=0•15). After controlling for baseline viral load, patients in the peginterferon lambda group were more likely to have undetectable virus by day 7 than were those in the placebo group (odds ratio [OR] 4•12 [95% CI 1•15-16•73; p=0•029). Of those with baseline viral load above 10⁶ copies per mL, 15 (79%) of 19 patients in the peginterferon lambda group had undetectable virus on day 7, compared with six (38%) of 16 in the placebo group (OR 6•25 [95% CI 1•49-31•06]; p=0•012). Peginterferon lambda was well tolerated, and adverse events were similar between groups with mild and transient aminotransferase, concentration increases more frequently observed in the peginterferon lambda group. Two individuals met the threshold of grade 3 increase, one in each group, and no other grade 3 or 4 laboratory adverse events were reported.Interpretation Peginterferon lambda accelerated viral decline in outpatients with COVID-19, increasing the proportion of patients with viral clearance by day 7, particularly in those with high baseline viral load. Peginterferon lambda has potential to prevent clinical deterioration and shorten duration of viral shedding.

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50 -60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 ؎ 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.Carbon dioxide, the metabolic end product of oxidative respiration, must be effectively cleared from the human body. CO 2 diffuses out of cells into the blood stream and into erythrocytes, where it is hydrated by cytosolic carbonic anhydrase (CA). 1 The resulting membrane-impermeant HCO 3 Ϫ is exported into the plasma by the plasma membrane Cl Ϫ /HCO 3 Ϫ anion exchanger (AE1), thus increasing the blood capacity for carrying CO 2 . Upon returning to the lungs the process is reversed; HCO 3 Ϫ is transported into the erythrocyte in exchange for Cl Ϫ by AE1 and dehydrated by CA, and the resulting CO 2 diffuses across the erythrocyte and alveolar membranes to be expired from the body. The 5 ϫ 10 4 s Ϫ1 turnover rate of AE1 (1) and the high content of AE1 in the membrane (2) facilitate completion of bicarbonate transport within 50 ms during passage of an erythrocyte through a capillary (3).AE1 is a 911-amino acid polytopic glycoprotein that facilitates the one for one electroneutral exchange of Cl Ϫ for HCO 3

Plasma membrane anion exchangers constitute a multigene family that contributes to the regulation of intracellular pH and chloride concentration in many cell types. We have characterized two polypeptide isoforms of the AE3 gene that are expressed in the rat retina. Using antipeptide antibodies specific for defined NH2-terminal and COOH- terminal epitopes, we have identified a 165 kDa polypeptide whose expression is restricted to the primary glial cell type of the retina, the Muller cell, and a 125 kDa polypeptide that is expressed in horizontal neurons. Expression of the Muller cell isoform exhibits a polarized distribution and is highest in basal endfoot processes. These AE3 isoforms exhibit a distinct developmental expression pattern in postnatal rat retina. The neuronal isoform is undetectable in neonatal retina until postnatal day 10–15, correlating strongly with the onset of retinal function.

terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 Ϯ 1 mM H ϩ equivalents/min was inhibited 53 Ϯ 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 Ϯ 4% without effect on DRA transport activity (105 Ϯ 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII. metabolon; chloride/bicarbonate exchanger; downregulated in adenoma BICARBONATE METABOLISM is essential in humans, because carbon dioxide is the metabolic end product of respiratory oxidation and CO 2 /HCO 3 Ϫ is the body's primary pH buffer system. The bicarbonate transport superfamily of genes (SLC4 and SLC26 gene families), responsible for transmembrane movement of membrane-impermeant HCO 3 Ϫ , comprises the Cl Ϫ /HCO 3 Ϫ anion exchanger (AE) family (1,19,20), the Na ϩ /HCO 3 Ϫ cotransporter proteins (NBC) (3, 4), and the recently identified proteins pendrin (9, 32, 37) and downregulated in adenoma (DRA) (23, 27, 48).Several lines of evidence have demonstrated an interaction between cytosolic carbonic anhydrase II (CAII) and the AE1, AE2, and AE3 anion exchanger isoforms. Binding of erythrocyte membranes to CAII increased CAII enzymatic activity (25), which suggests an interaction between these two proteins. CAII can be coimmunoprecipitated with solubilized AE1 and incubation with an extracellular lectin-caused agglutination of AE1 and a similar redistribution of CAII on the cytosolic surface of the erythrocyte membrane (45). A sensitive microtiter binding assay, using truncation and point mutation of the AE1 COOH terminus, led to the identification of the binding site of CAII in AE1 as LDADD (amino acids 886-890) (46) and the basic amino-terminal region of CAII as the binding site for AE1 (44).The functional consequences of the AE/CAII interaction have been studied (42). Using HEK-293 cells transiently transfected with AE1 cDNA, we determined that inhibition of endogenous CAII activity with acetazolamide resulted in a decrease of AE1 transport activity. Mutation of the AE1, LDADD, and CA...

Abstract-Plasma membrane anion exchangers (AEs) regulate myocardial intracellular pH (pH i ) by Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchange. Angiotensin II (Ang II) activates protein kinase C (PKC) and increases anion exchange activity in the myocardium. Elevated anion exchange activity has been proposed to contribute to the development of cardiac hypertrophy. Our Northern blots showed that adult rat heart expresses AE1, AE2, AE3fl, and AE3c. Activity of each AE isoform was individually measured by following changes of pH i , associated with bicarbonate transport, in transfected HEK293 cells. Exposure to the PKC activator, PMA (150 nmol/L), increased the transport activity of only the AE3fl isoform by 50Ϯ11% (PϽ0.05, nϭ6), consistent with the increase observed in intact myocardium. Cotransfection of HEK293 cells with AE3fl and AT1 a -Ang II receptors conferred sensitivity of anion transport to Ang II (500 nmol/L), increasing the transport activity by 39Ϯ3% (PϽ0.05, nϭ4). PKC inhibition by chelerythrine (10 mol/L) blocked the PMA effect. To identify the PKC-responsive site, 7 consensus PKC phosphorylation sites of AE3fl were individually mutated to alanine. Mutation of serine 67 of AE3 prevented the PMA-induced increase of anion transport activity. Inhibition of MEK1/2 by PD98059 (50 mol/L) did not affect the response of AE3fl to Ang II, indicating that PKC directly phosphorylates AE3fl. We conclude that following Ang II stimulation of cells, PKC⑀ phosphorylates serine 67 of the AE3 cytoplasmic domain, inducing the Ang II-induced increase in anion transport observed in the hypertrophic myocardium. Key Words: hypertrophy Ⅲ anion exchange Ⅲ pH regulation Ⅲ angiotensin II Ⅲ protein kinase C I ntracellular pH (pH i ) regulates excitation-contraction coupling in cardiac cells through ionic conductances, 1 metabolic pathways, 2 Ca 2ϩ homeostasis, 3 contractility, 4 and electrical conduction. 5 Four well-characterized membrane ion transporters regulate cardiomyocyte pH i . Acid loads activate Na ϩ /H ϩ exchange (NHE) and Na ϩ /HCO 3 Ϫ symport (NBC), 6,7 whereas after an alkaline load, Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchangers (AEs) 8 and a second dual acid-loading mechanism 9 are activated. Anion exchange proteins, which facilitate the reversible electroneutral exchange of Cl Ϫ for HCO 3 Ϫ across the plasma membrane, regulate pH i , intracellular chloride concentration, bicarbonate metabolism and cell volume. The AE family comprises 3 members: AE1, AE2, and AE3. 10 Heart and retina coexpress 2 different isoforms of AE3, AE3 full length (AE3fl, the most abundant AE protein expressed in the brain) and AE3 cardiac (AE3c), which result from alternative promoter usage. 11,12 Rat AE3fl is 1227 amino acids long and AE3c contains 1034 amino acids. The C-terminal 957 amino acids of both polypeptides are identical, but the AE3c protein contains a unique N-terminal sequence of 73 amino acids, which replaces the unique first 270 amino acids of the AE3fl form. 11 A truncated form of AE1 was recently characterized in rat ventricular my...

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Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.