Lack of Charge Interaction in the Ion Binding Site Determines Anion Selectivity in the Sodium Bicarbonate Cotransporter NBCe1.

The Na/HCO3 cotransporter NBCe1 is a member of SLC4A transporters that move HCO3- across cell membranes and regulate intracellular pH or transepithelial HCO3 transport. NBCe1 is highly selective to HCO3- and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp555 with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl-. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO3-. In this study, we tested whether the Cl- transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl- was assessed by two-electrode voltage clamp. The results show that the Cl- transport is induced by a charge interaction between Glu555 and Lys558. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO3- selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.

The colorful mantle of the giant clam Tridacna squamosa expresses a homolog of electrogenic sodium: Bicarbonate cotransporter 2 that mediates the supply of inorganic carbon to photosynthesizing symbionts.

Giant clams live in symbiosis with phototrophic dinoflagellates, which reside extracellularly inside zooxanthellal tubules located mainly in the colourful and extensible outer mantle. As symbiotic dinoflagellates have no access to the ambient seawater, they need to obtain inorganic carbon (Ci) from the host for photosynthesis during illumination. The outer mantle has a host-mediated and light-dependent carbon-concentrating mechanism to augment the supply of Ci to the symbionts during illumination. Iridocytes can increase the secretion of H+ through vacuolar H+-ATPase to dehydrate HCO3- present in the hemolymph to CO2. CO2 can permeate the basolateral membrane of the epithelial cells of the zooxanthellal tubules, and rehydrated back to HCO3- in the cytoplasm catalysed by carbonic anhydrase 2. This study aimed to elucidate the molecular mechanism involved in the transport of HCO3- across the apical membrane of these epithelial cells into the luminal fluid surrounding the symbionts. We had obtained the complete cDNA coding sequence of a homolog of electrogenic Na+-HCO3- cotransporter 2 (NBCe2-like gene) from the outer mantle of the fluted giant clam, Tridacna squamosa. NBCe2-like gene comprised 3,399 bp, encoding a protein of 1,132 amino acids of 127.3 kDa. NBCe2-like protein had an apical localization in the epithelial cells of zooxanthellal tubules, denoting that it could transport HCO3- between the epithelial cells and the luminal fluid. Furthermore, illumination augmented the transcript level and protein abundance of NBCe2-like gene/NBCe2-like protein in the outer mantle, indicating that it could mediate the increased transport of HCO3- into the luminal fluid to support photosynthesis in the symbionts.

Heterologous expression and purification of the bicarbonate transporter BicA from Synechocystis sp. PCC 6803.

The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-ß-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.

Structural mechanism of the active bicarbonate transporter from cyanobacteria.

Bicarbonate transporters play essential roles in pH homeostasis in mammals and photosynthesis in aquatic photoautotrophs. A number of bicarbonate transporters have been characterized, among which is BicA-a low-affinity, high-flux SLC26-family bicarbonate transporter involved in cyanobacterial CO2-concentrating mechanisms (CCMs) that accumulate CO2 and improve photosynthetic carbon fixation. Here, we report the three-dimensional structure of BicA from Synechocystis sp. PCC6803. Crystal structures of the transmembrane domain (BicATM) and the cytoplasmic STAS domain (BicASTAS) of BicA were solved. BicATM was captured in an inward-facing HCO3--bound conformation and adopts a '7+7' fold monomer. HCO3- binds to a cytoplasm-facing hydrophilic pocket within the membrane. BicASTAS is assembled as a compact homodimer structure and is required for the dimerization of BicA. The dimeric structure of BicA was further analysed using cryo-electron microscopy and physiological analysis of the full-length BicA, and may represent the physiological unit of SLC26-family transporters. Comparing the BicATM structure with the outward-facing transmembrane domain structures of other bicarbonate transporters suggests an elevator transport mechanism that is applicable to the SLC26/4 family of sodium-dependent bicarbonate transporters. This study advances our knowledge of the structures and functions of cyanobacterial bicarbonate transporters, and will inform strategies for bioengineering functional BicA in heterologous organisms to increase assimilation of CO2.

Trafficking, localization and degradation of the Na+,HCO3- co-transporter NBCn1 in kidney and breast epithelial cells.

The Na+;HCO3- co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1. Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal NBCn1 C-tail. Proximity Ligation Assay and co-immunoprecipitation confirmed that native NBCn1 interacts with RACK1 in a cellular context. Consistent with a functional role of this complex, RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression. Notably, only non-confluent cells exhibited detectable NBCn1-RACK1 plasma membrane co-localization, suggesting that RACK1 regulates the trafficking of NBCn1 to the membrane. Whereas total NBCn1 degradation was slow, with a half-life of more than 24 h, one-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min. This suggests that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). Our findings have important implications for understanding NBCn1 regulation as well as its dysregulation in disease.

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1.

Na+-coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

The role of disease-linked residue glutamine-913 in support of the structure and function of the human electrogenic sodium/bicarbonate cotransporter NBCe1-A.

Mutations in the sodium bicarbonate cotransporter NBCe1 (SLC4A4) cause proximal renal tubular acidosis (pRTA). We recently described a novel pRTA mutation p.Gln913Arg (Q913R), inherited in compound heterozygous form with p.Arg510His (R510H). Q913R causes intracellular retention of NBCe1 and a 'gain of function' Cl- leak. To learn more about the importance of glutamine at position 913, we substituted a variety of alternative amino-acid residues (Cys, Glu, Lys, Leu, Ser) at position 913. Studying cRNA-injected Xenopus oocytes by voltage clamp, we find that most de novo mutants exhibit close-to-normal NBCe1 activity; only Q913K expresses a Cl- leak. Studying transiently-transfected, polarised kidney cells by fluorescence microscopy we find that most de novo mutants (except Q913E) are intracellularly retained. A 3D homology model predicts that Gln913 is located in the gating domain of NBCe1 and neighbours the 3D space occupied by another pRTA-associated residue (Arg881), highlighting an important and conformationally-sensitive region of NBCe1. We conclude that the intracellular retention of Q913R is caused by the loss of Gln at position 913, but that the manifestation of the Cl- leak is related to the introduction of Arg at position 913. Our findings will inform future studies to elucidate the nature and the consequences of the leak.

The crystal structure of the regulatory domain of the human sodium-driven chloride/bicarbonate exchanger.

The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the SLC4 family. A novel conserved Zn2+-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.

Molecular characterization of Pacific white shrimp (Litopenaeus vannamei) sodium bicarbonate cotransporter (NBC) and its role in response to pH stress.

The sodium bicarbonate cotransporter (NBC) is an integral membrane ion transporter that can transport HCO3- (or a related species, such as CO32-) across the plasma membrane. Previous researches revealed that NBC might play an important role in the regulation of intracellular pH in vertebrates. In the present study, an NBC cDNA was identified from Pacific white shrimp (Litopenaeus vannamei) and designated as Lv-NBC. The full-length Lv-NBC cDNA is 4479 bp in size, containing a 5'-untranslated region (UTR) of 59 bp, a 3'-UTR of 835 bp and an open reading frame (ORF) of 3585 bp that encodes a protein of 1194 amino acids with a deduced molecular weight of 134.34 kDa. The Lv-NBC protein contains two functional domains (Band_3_cyto and HCO3_cotransp) and twelve transmembrane (TM) domains. Expression of the Lv-NBC mRNA was ubiquitously detected in all selected tissues, with the highest level in the gill. By in situ hybridization (ISH) with Digoxigenin-labeled probe, the Lv-NBC positive cells were shown mainly located in the secondary gill filaments. After low or high pH challenge, the transcript levels of Lv-NBC in the gill were found to be up-regulated. After knockdown of the Lv-NBC level by siRNA, the mortality of shrimp significantly increased under pH stress. Our study, as a whole, may provide evidences for the role of NBC in shrimp responding to pH stress, and give a new insight of the acid/base homeostasis mechanism in crustaceans.

Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers.

Boron is essential for plant growth because of its incorporation into plant cell walls; however, in excess it is toxic to plants. Boron transport and homeostasis in plants is regulated in part by the borate efflux transporter Bor1, a member of the solute carrier (SLC) 4 transporter family with homology to the human bicarbonate transporter Band 3. Here, we present the 4.1-Å resolution crystal structure of Arabidopsis thaliana Bor1. The structure displays a dimeric architecture in which dimerization is mediated by centralized Gate domains. Comparisons with a structure of Band 3 in an outward-open state reveal that the Core domains of Bor1 have rotated inwards to achieve an occluded state. Further structural comparisons with UapA, a xanthine transporter from the nucleobase-ascorbate transporter family, show that the downward pivoting of the Core domains relative to the Gate domains may access an inward-open state. These results suggest that the SLC4, SLC26, and nucleobase-ascorbate transporter families all share an elevator transport mechanism in which alternating access is provided by Core domains that carry substrates across a membrane.

Effects of Nt-truncation and coexpression of isolated Nt domains on the membrane trafficking of electroneutral Na+/HCO3- cotransporters.

The SLC4 genes are all capable of producing multiple variants by alternative splicing or using alternative promoters. The physiological consequences of such diversity are of great interest to investigators. Here, we identified two novel variants of the electroneutral Na(+)/HCO3- cotransporter NBCn1, one full-length starting with "MIPL" and the other Nt-truncated starting with "MDEL". Moreover, we identified a new promoter of Slc4a10 encoding NBCn2 and a novel type of Nt-truncated NBCn2 starting with "MHAN". When heterologously expressed, the new NBCn1 variants were well localized to the plasma membrane and exhibited characteristic NBCn1 activity. However, MHAN-NBCn2 was poorly localized on the plasma membrane. By deletion mutations, we identified the Nt regions important for the surface localization of NBCn2. Interestingly, coexpressing the full-length NBCn2 greatly enhances the surface abundance of the Nt-truncated NBCn2. Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells. Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2. The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

Rescue of the temperature-sensitive, autosomal-recessive mutation R298S in the sodium-bicarbonate cotransporter NBCe1-A characterized by a weakened dimer and abnormal aggregation.

BACKGROUND: Band keratopathy, an ocular disease that is characterized by hypercalcemia and opaque bands across the cornea, has been associated with kidney disease. Type-II renal tubular acidosis (RTA), a condition in which the kidneys fail to recover bicarbonate (HCO3-) in the proximal tubule of the nephron, results in HCO3- wastage in the urine and low blood pH. The development of these diseases is associated with autosomal-recessive mutations in the Na+-coupled HCO3- cotransporter NBCe1-A located at the basolateral membranes of either cell type. METHODS: We provide insight into the devastating R298S mutation found in type-II RTA-afflicted individuals using confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in human corneal endothelial and proximal tubule cells and from in-depth biophysical studies of their cytoplasmic N-terminal domains (Nt and Nt-R298S), including Nt crystal structure, melting-temperature, and homodimer dissociation constant (KD) analyses. RESULTS: We illuminate and rescue trafficking defects of the R298S mutation of NBCe1-A. The KD for Nt monomer-dimer equilibrium is established. The KD for Nt-R298S is significantly higher, but immeasurable due to environmental factors (pH, temperature, concentration) that result in dimer instability leading to precipitation. The crystal structure of Nt-dimer shows that R298 is part of a putative substrate conduit and resides near the dimer interface held together by hydrogen-bond networks. CONCLUSIONS: The R298S is a temperature-sensitive mutation in Nt that results in instability of the colloidal system leading to abnormal aggregation. GENERAL SIGNIFICANCE: Our findings provide new perspectives to the aberrant mechanism of certain ocular pathologies and type-II RTA associated with the R298S mutation.

Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.

Cl(-) is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl(-) (Cl(-) in) as a signaling ion has not been previously evaluated. Here we report that Cl(-) in functions as a regulator of cellular Na(+) and HCO3 (-) concentrations and transepithelial transport through modulating the activity of several electrogenic Na(+)-HCO3 (-) transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl(-) in concentrations highlighting the role of GXXXP motifs in Cl(-) sensing. Regulation of the ubiquitous Na(+)-HCO3(-) co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl(-) in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl(-) in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl(-) in interacting site. NBCe1-A is unaffected by Cl(-) in between 5 and 140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl(-) in. These findings reveal a cellular Cl(-) in sensing mechanism that plays an important role in the regulation of Na(+) and HCO3 (-) transport, with critical implications for the role of Cl(-) in cellular ion homeostasis and epithelial fluid and electrolyte secretion.

Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

Cation-coupled bicarbonate transporters.

Cation-coupled HCO3(-) transport was initially identified in the mid-1970s when pioneering studies showed that acid extrusion from cells is stimulated by CO2/HCO3(-) and associated with Na(+) and Cl(-) movement. The first Na(+)-coupled bicarbonate transporter (NCBT) was expression-cloned in the late 1990s. There are currently five mammalian NCBTs in the SLC4-family: the electrogenic Na,HCO3-cotransporters NBCe1 and NBCe2 (SLC4A4 and SLC4A5 gene products); the electroneutral Na,HCO3-cotransporter NBCn1 (SLC4A7 gene product); the Na(+)-driven Cl,HCO3-exchanger NDCBE (SLC4A8 gene product); and NBCn2/NCBE (SLC4A10 gene product), which has been characterized as an electroneutral Na,HCO3-cotransporter or a Na(+)-driven Cl,HCO3-exchanger. Despite the similarity in amino acid sequence and predicted structure among the NCBTs of the SLC4-family, they exhibit distinct differences in ion dependency, transport function, pharmacological properties, and interactions with other proteins. In epithelia, NCBTs are involved in transcellular movement of acid-base equivalents and intracellular pH control. In nonepithelial tissues, NCBTs contribute to intracellular pH regulation; and hence, they are crucial for diverse tissue functions including neuronal discharge, sensory neuron development, performance of the heart, and vascular tone regulation. The function and expression levels of the NCBTs are generally sensitive to intracellular and systemic pH. Animal models have revealed pathophysiological roles of the transporters in disease states including metabolic acidosis, hypertension, visual defects, and epileptic seizures. Studies are being conducted to understand the physiological consequences of genetic polymorphisms in the SLC4-members, which are associated with cancer, hypertension, and drug addiction. Here, we describe the current knowledge regarding the function, structure, and regulation of the mammalian cation-coupled HCO3(-) transporters of the SLC4-family.

Direct evidence for calcineurin binding to the exon-7 loop of the sodium-bicarbonate cotransporter NBCn1.

The NaHCO3 cotransporter NBCn1 plays a role in neutralizing intracellular acid loads at the basolateral membrane in cells of the medullary thick ascending limb (mTAL). Calcineurin inhibitors (Cn-Is) are known to both downregulate NBCn1 expression in the distal nephron and cause renal tubular acidosis (RTA), a risk factor for nephrocalcinosis and nephrolithiasis. In this report, we provide a new perspective on concurrent studies of NBCn1 in various tissues by using cell-free binding assays to investigate the interaction of NBCn1 with the calcineurin (Cn) isoform PPP3CA. Surface plasmon resonance (SPR) analyses show that the protein domain Exon 7 (translated from cassette II of NBCn1) binds Cn with an equilibrium dissociation constant (KD) of 30 +/- 15 nm. Linked-reaction tests suggest that the binding involves a conformational change. Nested PCR reactions were used to show that NBCn1-Exon 7 splice variants with alternative N-termini regions are expressed in the kidney, as well as other tissues. Additionally, we discuss NBCn1-Exon 7 implication in acid-base balance and calcium crystallization in the kidney.

NBCe1A dimer assemble visualized by bimolecular fluorescence complementation.

Mutations in the electrogenic Na(+)/HCO3(-) cotransporter (NBCe1) that cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic although their tissues should make some mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An enhanced yellow fluorescent protein (EYFP) fragment (1-158, EYFP(N)) or (159-238, EYFP(C)) was fused to the NH2 or COOH terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the NH2 terminus of NBCe1A (EYFP(N)-N-NBCe1A w/ EYFP(C)-N-NBCe1A), and the electrophysiology data demonstrated this EYFP-NBCe1A coexpressed pair have wild-type transport function. These data suggest NBCe1A forms dimers and that NH2 termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% wild-type function individually): S427L (naturally occurring) and E91R (for NH2-terminal structure studies). When we coexpressed S427L and E91R, we measured 50% wild-type function, which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.

Carbonic anhydrases and their interplay with acid/base-coupled membrane transporters.

Carbonic anhydrases (CAs) have not only been identified as ubiquitous enzymes catalyzing the fast reversible hydration of carbon dioxide to generate or consume protons and bicarbonate, but also as intra- and extracellular proteins, which facilitate transport function of many acid/base transporting membrane proteins, coined 'transport metabolon'. Functional interaction between CAs and acid/base transporters, such as chloride/bicarbonate exchanger (AE), sodium-bicarbonate cotransporter (NBC) and sodium/hydrogen exchanger (NHE) has been shown to require both catalytic CA activity as well as direct binding of the enzyme to specific sites on the transporter. In contrast, functional interaction between different CA isoforms and lactate-proton-cotransporting monocarboxylate transporters (MCT) has been found to be isoform-specific and independent of CA catalytic activity, but seems to require an intramolecular proton shuttle within the enzyme. In this chapter, we review the various types of interactions between acid/base-coupled membrane carriers and different CA isoforms, as studied in vitro, in intact Xenopus oocytes, and in various mammalian cell types. Furthermore, we discuss recent findings that indicate the significance of these 'transport metabolons' for normal cell functions.

Validation of miRNA-mRNA interactions by electrophoretic mobility shift assays.

BACKGROUND: MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3'-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3'-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA. FINDINGS: RNA molecules corresponding to miR-224 and to the 3'-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors. CONCLUSIONS: In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA.

A substrate access tunnel in the cytosolic domain is not an essential feature of the solute carrier 4 (SLC4) family of bicarbonate transporters.

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-Å resolution crystal structure of cdΔ54AE1 (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 cdΔ54AE1. 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.