Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE.

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

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.

Basolateral NBCe1 plays a rate-limiting role in transepithelial intestinal HCO3- secretion, contributing to marine fish osmoregulation.

Although endogenous CO2 hydration and serosal HCO3- are both known to contribute to the high rates of intestinal HCO3- secretion important to marine fish osmoregulation, the basolateral step by which transepithelial HCO3- secretion is accomplished has received little attention. Isolated intestine HCO3- secretion rates, transepithelial potential (TEP) and conductance were found to be dependent on serosal HCO3- concentration and sensitive to serosal DIDS. Elevated mucosal Cl- concentration had the unexpected effect of reducing HCO3- secretion rates, but did not affect electrophysiology. These characteristics indicate basolateral limitation of intestinal HCO3- secretion in seawater gulf toadfish, Opsanus beta. The isolated intestine has a high affinity for serosal HCO3- in the physiological range (Km=10.2 mmol l(-1)), indicating a potential to efficiently fine-tune systemic acid-base balance. We have confirmed high levels of intestinal tract expression of a basolateral Na+/HCO3- cotransporter of the electrogenic NBCe1 isoform in toadfish (tfNBCe1), which shows elevated expression following salinity challenge, indicating its importance in marine fish osmoregulation. When expressed in Xenopus oocytes, isolated tfNBCe1 has transport characteristics similar to those in the isolated tissue, including a similar affinity for HCO3- (Km=8.5 mmol l(-1)). Reported affinity constants of NBC1 for Na+ are generally much lower than physiological Na+ concentrations, suggesting that cotransporter activity is more likely to be modulated by HCO3- rather than Na+ availability in vivo. These similar functional characteristics of isolated tfNBCe1 and the intact tissue suggest a role of this cotransporter in the high HCO3- secretion rates of the marine fish intestine.

Expression and localization of Na-driven Cl-HCO(3)(-) exchanger (SLC4A8) in rodent CNS.

The Na(+)-driven Cl-HCO(3) exchanger (NDCBE or SLC4A8) is a member of the solute carrier 4 (SLC4) family of HCO(3)(-) transporters, which includes products of 10 genes with similar sequences. Most SLC4 members play important roles in regulating intracellular pH (pH(i)). Physiological studies suggest that NDCBE is a major pH(i) regulator in at least hippocampal (HC) pyramidal neurons. We generated a polyclonal rabbit antibody directed against the first 18 residues of the cytoplasmic N terminus (Nt) of human NDCBE. By Western blotting, the antibody distinguishes NDCBE-as a purified Nt peptide or a full-length transporter (expressed in Xenopus oocytes)-from other Na(+)-coupled HCO(3)(-) transporters. By Western blotting, the antiserum recognizes an approximately 135-kDa band in several brain regions of adult mice: the cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and HC. In CX, PNGase F treatment reduces the molecular weight to approximately 116 kDa. By immunocytochemistry, affinity-purified (AP) NDCBE antibody stains the plasma membrane of neuron cell bodies and processes of rat HC neurons in primary culture as well as freshly dissociated mouse HC neurons. The AP antibody does not detect substantial NDCBE levels in freshly dissociated HC astrocytes, or astrocytes in HC or CB sections. By immunohistochemistry, the AP antibody recognizes high levels of NDCBE in neurons of CX, HC (including pyramidal neurons in Cornu Ammonis (CA)1-3 and dentate gyrus), substantial nigra, medulla, cerebellum (especially Purkinje and granular cells), and the basolateral membrane of fetal choroid plexus. Thus, NDCBE is in a position to contribute substantially to pH(i) regulation in multiple CNS neurons.

Expression and purification of the cytoplasmic N-terminal domain of the Na/HCO3 cotransporter NBCe1-A: structural insights from a generalized approach.

The cytoplasmic, N-terminal domain (Nt) of the electrogenic sodium/bicarbonate cotransporter--NBCe1--over-expresses in Escherichia coli and yields a large amount of soluble protein. A novel purification strategy, which involves a streptomycin precipitation, overcomes obstacles of instability and copurifying proteins, and leads to the first seen Nt-NBCe1 crystals. The purification procedure generally lends itself to the purification of Nts from other classes of the SLC4 family. Size-exclusion chromatography suggests that the Nt of NBCe1 as well as the Nt of other SLC4 members form dimers. A comparison of Nt-NBCe1 to SLC4 member Nt-AE1, based on purification properties and predicted secondary-structure sequence alignments, suggests a similar mechanism for dimer stabilization.

A sodium bicarbonate transporter from sea urchin spermatozoa.

Bicarbonate (HCO3-) transporters play crucial roles in cell-signaling pathways and are essential for cell viability. Here we describe the first cloning and localization of a HCO3- transporter from sperm of the sea urchin, Strongylocentrotus purpuratus. The deduced protein is 1214 amino acids and has a calculated molecular mass of 135 kDa. The annotated protein coding region of the transporter gene consists of 24 exons. The most similar human protein is the Na+/HCO3- cotransporter-2 (NBC2), which has 53% identity and 68% similarity to the sea urchin protein. The sea urchin protein shares the major structural features of HCO3- transporters, including 13 transmembrane segments, a DIDS (4,4-diiodothiocyanatostilbene-2, 2-disulfonic acid) binding motif and N-linked glycosylation sites. It has longer N- and C-terminal cytoplasmic domains compared to human HCO3- transporters. The sea urchin protein possesses a relatively long 3rd extracellular loop with four conserved cysteine residues. This is characteristic for Na+/HCO3- cotransporters, but not for anion exchangers, suggesting that the sea urchin protein is a Na+/HCO3- cotransporter. It is therefore designated as Sp-NBC. A neighbor-joining tree shows that Sp-NBC branches closer to the electroneutral type of HCO3- transporters. Western immunoblots and immunoflourescence show that Sp-NBC is concentrated in the flagellar plasma membrane, suggesting a role in motility regulation.