Altered gene expression in slc4a11-/- mouse cornea highlights SLC4A11 roles.

SLC4A11 is a H+/NH3/water transport protein, of corneal endothelial cells. SLC4A11 mutations cause congenital hereditary endothelial dystrophy and some cases of Fuchs endothelial corneal dystrophy. To probe SLC4A11's roles, we compared gene expression in RNA from corneas of 17-week-old slc4a11-/- (n = 3) and slc4a11+/+ mice (n = 3) and subjected to RNA sequencing. mRNA levels for a subset of genes were also assessed by quantitative real-time reverse transcription PCR (qRT RT-PCR). Cornea expressed 13,173 genes, which were rank-ordered for their abundance. In slc4a11-/- corneas, 100 genes had significantly altered expression. Abundant slc14a1 expression, encoding the urea transporter UT-A, suggests a significant role in the cornea. The set of genes with altered expression was subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, revealing that alterations clustered into extracellular region, cytoskeleton, cell adhesion and plasma membrane functions. Gene expression changes further clustered into classes (with decreasing numbers of genes): cell fate and development, extracellular matrix and cell adhesion, cytoskeleton, ion homeostasis and energy metabolism. Together these gene changes confirm earlier suggestions of a role of SLC4A11 in ion homeostasis, energy metabolism, cell adhesion, and reveal an unrecognized SLC4A11 role in cytoskeletal organization.

Large conformational dynamics in Band 3 protein: Significance for erythrocyte senescence signalling.

Band 3 (Anion Exchanger 1, AE1), the predominant protein of erythrocyte membranes, facilitates Cl-/HCO3- exchange and anchors the plasma membrane to the cytoskeleton. The Band 3 crystal structure revealed the amino acid 812-830 region as intracellular, conflicting with protein chemical data that suggested extracellular disposition. Further, circulating senescent cell auto-antibody that cannot enter erythrocytes, binds two regions of Band 3: residues 538-554 and 812-830. To reconcile this discrepancy, we assessed localization of residues 812-830 with Band 3 expressed in HEK293 cells and human erythrocytes, using chemical labeling probes and an antibody against residues 812-830. Antibody and chemical probes revealed reorientation of 812-830 region between extracellular and intracellular. This dramatic conformational change is an intrinsic property of the Band 3 molecule, occurring when expressed in HEK293 cells and without the damage that occurs during erythrocyte circulation. Conditions used to crystallize Band 3 for structural determination did not alter conformational dynamics. Collectively, these data reveal large Band 3 conformational dynamics localized to a region previously identified as an erythrocyte senescence epitope. Surface exposure of the senescence epitope (812-830), limited by conformational dynamics, may act as the "molecular clock" in erythrocyte senescence.

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal Dystrophies.

The cornea, the eye's outermost layer, protects the eye from the environment. The cornea's innermost layer is an endothelium separating the stromal layer from the aqueous humor. A central role of the endothelium is to maintain stromal hydration state. Defects in maintaining this hydration can impair corneal clarity and thus visual acuity. Two endothelial corneal dystrophies, Fuchs Endothelial Corneal Dystrophy (FECD) and Congenital Hereditary Endothelial Dystrophy (CHED), are blinding corneal diseases with varied clinical presentation in patients across different age demographics. Recessive CHED with an early onset (typically age: 0-3 years) and dominantly inherited FECD with a late onset (age: 40-50 years) have similar phenotypes, although caused by defects in several different genes. A range of molecular mechanisms have been proposed to explain FECD and CHED pathology given the involvement of multiple causative genes. This critical review provides insight into the proposed molecular mechanisms underlying FECD and CHED pathology along with common pathways that may explain the link between the defective gene products and provide a new perspective to view these genetic blinding diseases.

Human red blood cell uptake and sequestration of arsenite and selenite: Evidence of seleno-bis(S-glutathionyl) arsinium ion formation in human cells.

Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]-, which undergoes biliary and fecal elimination. [(GS)2AsSe]-, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS)2AsSe]-. 75SeIV accumulation was temperature and Cl--dependent, inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (IC50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS)2AsSe]- in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS)2AsSe]- in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease.

Defective cell adhesion function of solute transporter, SLC4A11, in endothelial corneal dystrophies.

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity.

Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies.

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Membrane-anchored carbonic anhydrase IV interacts with monocarboxylate transporters via their chaperones CD147 and GP70.

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.

Role of N-glycosylation in the expression of human SLC26A2 and A3 anion transport membrane glycoproteins 1.

The human solute carrier 26 (SLC26) gene family of anion transporters consists of 10 members (SLC26A1-A11, A10 being a pseudogene) that encode membrane glycoproteins with 14 transmembrane segments and a C-terminal cytoplasmic sulfate transporter anti-sigma antagonist domain. Thus far, mutations in eight members of the SLC26 family (A1-A6, A8, and A9) have been linked to diseases in humans. Our goal is to characterize the role of N-glycosylation and the effect of mutations in SLC26A2 and A3 proteins on their functional expression in transfected HEK-293 cells. We found that certain mutants were retained in the endoplamic reticulum via an interaction with the lectin chaperone calnexin. Some could escape protein quality control and traffic to the cell surface upon removal of the N-glycosylation sites. Furthermore, we found that loss of N-glycosylation reduced expression of SLC26A2 at the cell surface. Loss of N-glycosylation had no effect on the stability of SLC26A3, yet resulted in a profound decrease in transport activity. Thus, N-glycosylation plays three roles in the functional expression of SLC26 proteins: (1) to retain misfolded proteins in the endoplamic reticulum, (2) to stabilize the protein at the cell surface, and (3) to maintain the transport protein in a functional state.

Ophthalmic Nonsteroidal Anti-Inflammatory Drugs as a Therapy for Corneal Dystrophies Caused by SLC4A11 Mutation.

Purpose: SLC4A11 is a plasma membrane protein of corneal endothelial cells. Some mutations of the SLC4A11 gene result in SLC4A11 protein misfolding and failure to mature to the plasma membrane. This gives rise to some cases of Fuchs' endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). We screened ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) for their ability to correct SLC4A11 folding defects. Methods: Five ophthalmic NSAIDs were tested for their therapeutic potential in some genetic corneal dystrophy patients. HEK293 cells expressing CHED and FECD-causing SLC4A11 mutants were grown on 96-well dishes in the absence or presence of NSAIDs. Ability of NSAIDs to correct mutant SLC4A11 cell-surface trafficking was assessed with a bioluminescence resonance energy transfer (BRET) assay and by confocal microscopy. The ability of mutant SLC4A11-expressing cells to mediate water flux (SLC4A11 mediates water flux across the corneal endothelial cell basolateral membrane as part of the endothelial water pump) was measured upon treatment with ophthalmic NSAIDs. Results: BRET-assays revealed significant rescue of SLC4A11 mutants to the cell surface by 4 of 5 NSAIDs tested. The NSAIDs, diclofenac and nepafenac, were effective in moving endoplasmic reticulum-retained missense mutant SLC4A11 to the cell surface, as measured by confocal immunofluorescence. Among intracellular-retained SLC4A11 mutants, 20 of 30 had significant restoration of cell surface abundance upon treatment with diclofenac. Diclofenac restored mutant SLC4A11 water flux activity to the level of wild-type SLC4A11 in some cases. Conclusions: These results encourage testing diclofenac eye drops as a treatment for corneal dystrophy in patients whose disease is caused by some SLC4A11 missense mutations.

Molecular phenotype of SLC4A11 missense mutants: Setting the stage for personalized medicine in corneal dystrophies.

SLC4A11 mutations cause cases of congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). Defective water reabsorption from corneal stroma by corneal endothelial cells (CECs) leads to these corneal dystrophies. SLC4A11, in the CEC basolateral membrane, facilitates transmembrane movement of H2 O, NH3 , and H+ -equivalents. Some SLC4A11 disease mutants have impaired folding, leading to a failure to move to the cell surface, which in some cases can be corrected by the drug, glafenine. To identify SLC4A11 mutants that are targets for folding-correction therapy, we examined 54 SLC4A11 missense mutants. Cell-surface trafficking was assessed on immunoblots, by the level of mature, high molecular weight, cell surface-associated form, and using a bioluminescence resonance energy transfer assay. Low level of cell surface trafficking was found in four out of 18 (20%) of FECD mutants, 19/ out of 31 (61%) of CHED mutants, and three out of five (60%) of HS mutants. Amongst ER-retained mutants, 16 showed increased plasma membrane trafficking when grown at 30°C, suggesting that their defect has potential for rescue. CHED-causing point mutations mostly resulted in folding defects, whereas the majority of FECD missense mutations did not affect trafficking, implying functional impairment. We identified mutations that make patients candidates for folding correction of their corneal dystrophy.

Molecular mechanism for the red blood cell senescence clock.

Lacking protein synthesis machinery and organelles necessary for autophagy or apoptosis, aged red blood cells (RBCs) are marked by circulating auto-antibodies for macrophage-mediated clearance. The antigen recognized by these auto-antibodies is the major protein of the RBC membrane, Band 3. To ensure regulation and specificity in clearance, the molecular "clock" must mark senescent cells in a way that differentiates them from younger cells, to prevent premature clearance. Predominant models of Band 3 senescence signaling are reviewed, and merits are discussed in light of the recently published crystal structure of the Band 3 membrane domain. © 2017 IUBMB Life, 70(1):32-40, 2018.

SLC4A11 Three-Dimensional Homology Model Rationalizes Corneal Dystrophy-Causing Mutations.

We studied the structural effects of point mutations of a membrane protein that cause genetic disease. SLC4A11 is a membrane transport protein (OH- /H+ /NH3 /H2 O) of basolateral corneal endothelium, whose mutations cause some cases of congenital hereditary endothelial dystrophy and Fuchs endothelial corneal dystrophy. We created a three-dimensional homology model of SLC4A11 membrane domain, using Band 3 (SLC4A1) crystal structure as template. The homology model was assessed in silico and by analysis of mutants designed on the basis of the model. Catalytic pathway mutants p.Glu675Gln, p.His724Arg, and p.His724Ala impaired SLC4A11 transport. p.Ala720Leu, in a region of extended structure of the proposed translocation pore, failed to mature to the cell surface. p.Gly509Lys, located in an open region at the core domain/gate domain interface, had wild-type level of transport function. The molecular phenotype of 37 corneal dystrophy-causing point mutants was rationalized, based on their location in the homology model. Four map to the substrate translocation pathway, 25 to regions of close transmembrane helix packing, three to the dimeric interface, and five lie in extramembraneous loops. The model provides a view of the spectrum of effects of disease mutations on membrane protein structure and provides a tool to analyze pathogenicity of additional newly discovered SLC4A11 mutants.

Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies.

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na+ independent. Studies in oocytes and HEK293 cells could not detect Na+-coupled HCO3- transport or Cl-/HCO3- exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltage-dependent OH- or H+ movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH-/H+ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H.

Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context.

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.

The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11.

Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy-Causing Mutants of SLC4A11.

PURPOSE: Protein misfolding, causing retention of nascent protein in the endoplasmic reticulum (ER), is the most common molecular phenotype for disease alleles of membrane proteins. Strategies are needed to identify therapeutics able to correct such folding/trafficking defects. Mutations of SLC4A11, a plasma membrane transport protein of the human corneal endothelial cell layer, cause cases of congenital hereditary endothelial dystrophy, Harboyan syndrome, and Fuchs' endothelial corneal dystrophy. Most SLC4A11 mutations induce SLC4A11 misfolding and retention in the ER. METHODS: An assay amenable to high-throughput screening was developed to quantify SLC4A11 at the plasma membrane, enabling a search for potential traffic-correcting small molecules. The assay was validated by comparing cell surface abundance of SLC4A11 mutants measured in the assay to observations from confocal immunofluorescence and values from cell surface biotinylation. Functionality of mutant proteins was assessed, using a confocal microscopic green fluorescent protein (GFP) water flux assay where relative rates of cell swelling are compared. RESULTS: A small-scale screen revealed that the nonsteroidal anti-inflammatory drugs (NSAIDs), glafenine, ibuprofen, and acetylsalicylic acid dissolved in 0.2% dimethyl sulfoxide (DMSO), partially rescued the trafficking defect in some SLC4A11 mutants, expressed in HEK293 cells. These SLC4A11 mutants retained functional activity when rescued to the plasma membrane by glafenine treatment. Glafenine was effective with an EC50 of 1.5 ± 0.7 µM. CONCLUSIONS: These data suggest that glafenine, and perhaps other NSAIDs, hold potential as therapeutics for misfolded membrane proteins, like SLC4A11. The high throughput approach described here can be modified to identify correctors of other misfolded plasma membrane proteins that cause eye disease.

Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification.

The bicarbonate ion (HCO3(-)) is involved in two major physiological processes in corals, biomineralization and photosynthesis, yet no molecular data on bicarbonate transporters are available. Here, we characterized plasma membrane-type HCO3(-) transporters in the scleractinian coral Stylophora pistillata. Eight solute carrier (SLC) genes were found in the genome: five homologs of mammalian-type SLC4 family members, and three of mammalian-type SLC26 family members. Using relative expression analysis and immunostaining, we analyzed the cellular distribution of these transporters and conducted phylogenetic analyses to determine the extent of conservation among cnidarian model organisms. Our data suggest that the SLC4γ isoform is specific to scleractinian corals and responsible for supplying HCO3(-) to the site of calcification. Taken together, SLC4γ appears to be one of the key genes for skeleton building in corals, which bears profound implications for our understanding of coral biomineralization and the evolution of scleractinian corals within cnidarians.

Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3.

Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.