Cation-coupled bicarbonate transporters

Abstract

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.

Figure 1

Na⁺-coupled bicarbonate transporters NCBTs. Electrogenic Na,HCO₃ cotransporters mediate HCO₃⁻ efflux or HCO₃⁻ influx in a tissue-specific manner. Electroneutral Na,HCO₃ cotransporters mediate net HCO₃⁻ influx. Na⁺-driven Cl,HCO₃ exchangers electroneutrally mediate Cl⁻ influx in exchange for Cl⁻ efflux.

Figure 2

Five different modes for effective HCO₃⁻ extrusion over the basolateral membrane of the proximal tubule [adapted, with permission, from (39)]. The authors ruled out the first four modes because the transport was known to be Na⁺ dependent and electrogenic. Although this leaves option 5 (e) as the correct transport mode, it is still not decided which of the four different transport modes of (e), that is, which ion species, are actually transported by the transporter. This is not only the case for the Na⁺-dependent transport of HCO₃⁻ or a related species over the basolateral membrane of the proximal tubule, but indeed for all Na⁺-dependent transporters discussed in this article.

Figure 3

Schematic diagram showing the rationale for determination of whether HCO₃⁻ or CO₃ ²⁻ is the transported ion. It has been suggested (38, 292) that it is possible to distinguish between HCO₃⁻ and CO₃ ²⁻ transport by measuring pH in the extracellular space near the membrane following a sudden change in transport activity, before and after inhibition of the carbonic anhydrase. If CO₃⁻ is the transported species an exaggerated decrease in pH would develop when the carbonic anhydrase is inhibited, while if HCO₃⁻ is the transported species a blunted decrease in pH would be the result. From (38).

Figure 4

Model of reabsorption of HCO₃⁻ in the proximal tubules. The figure underlines the importance of basolateral HCO₃⁻ and CO₂ for the regulation of HCO₃⁻ reabsorption suggested by Zhou et al. (363) and recently documented by Fukuda et al. (102) and others. From (363).

Figure 5

Structure of NCBT proteins. (A) A generic structure of NCBTs is predicted to have an extended N-terminus, a transmembrane domain containing 14 transmembrane segments, and a relatively short C-terminal domain. The extracellular loop between segments 5 and 6 contains two N-glycosylation sites. (B) Alignment of protein sequence comprising human NCBTs. The alignment was performed using the UniProt (www.uniprot.org) with each canonical protein sequence for NBCe1 (Uniprot ID: Q9Y6R1), NBCe2 (Q9BY07), NBCn1 (Q9Y6M7), NBCn2 (Q2Y0W8), and NDCBE (Q6U841). Sequences highly conserved among NCBTs are shown in brown bars, while sequences moderately conserved are in open bars. Sequences with negligible homology are shown as a horizontal line. Internal splice cassettes are in different colors.

Figure 6

Functional characterization of NBCs expressed in Xenopus oocytes demonstrating Na⁺-dependent pH_i recovery from a CO₂/HCO3⁻-induced acidification. The electrogenic transporters NBCe1 (A) [adapted, with permission, from 70] and NBCe2 (B) [adapted, with permission, from 326] produce a large hyperpolarization due to net negative charge movement into oocytes. Activation of the electroneutral transporters NBCn1 (C) [adapted, with permission, from 75], NBCn2/NCBE (D) [adapted, with permission, from 236], and NDCBE (E) [adapted, with permission, from 117] is not associated with hyperpolarization.

Figure 7

NBCe1 (A) NBCe1 is highly expressed in the basolateral membranes of the cortical collecting ducts (arrows) but not in glomeruli (G) (209). [(B)–(D)] Functional knockout of NBCe1 in mice (199). (B) Rates of HCO₃⁻ absorption from isolated renal proximal tubules. Mice with functional knockout of NBCe1 (W516/W516×) had severely reduced reabsorption of HCO₃⁻, while heterozygous mice had a mildly reduced reabsorption. (C) Mice with functional knockout of NBCe1 were growth retarded compared to wild-type mice (+/+). NaCl in the drinking water had no effect on the growth retardation while NaHCO₃-treated mice had attenuated growth retardation. (D) Mice with functional knockout of NBCe1 had severely reduced survival rates with a sharp increase in mortality starting around 17 days, but NaHCO₃ treatment of these mice prolonged the survival time up to 81 days of age.

Figure 8

(A) Double-labeling immunofluorescence microscopic analysis of NBCe2 (red) and NCBE/NBCn2 (green) localization in rat choroid plexus (46). The fluorescence image was overlaid a differential interference contrast image and shows apical localization of NBCe2 (arrows) and basolateral localization of NCBE/NBCn2. Panels B and C show ventricular volume and (D) intracranial pressure in wild-type (WT) and NBCe2 knockout (Slc4a5^−/−) mice (152). Panel B shows MRI imaging (horizontal plane) of the lateral ventricles in WT and Slc4a5^−/− mice. The ventricular volume and intracranial pressure are significantly reduced in Slc4a5^−/− mice.

Figure 9

Effect of intracellular pH on vascular tone (35). In the NBCn1 (Slc4a7) knockout mouse (red) vascular endothelial cell (A) and smooth muscle cell (C) pH is reduced (vertical bars show SEM, n = 5 and 6, respectively). This is associated with a reduced endothelial cell mediated smooth muscle cell relaxation to acetylcholine (ACh) of norepinephrine (NE) activated mesenteric small arteries (B) and reduced tension development to NE in the presence of 100 μmol/L nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) E), (vertical bars show SEM, n = 10). In the presence of 10 μmol/L Rho-kinase inhibitor fasudil, the tension development to NE is similar in arteries from wild type and knockout mice (D) (n = 5), consistent with the Rho-kinase being pH sensitive allowing for an effect of intracellular pH on smooth muscle cell tone.

Figure 10

NBCn1 expression in cancer cells. Micrographs of normal human breast (A) and breast cancer (B) immunostained (brown) for NBCn1. (C) Average (with SEM, n = 5) membrane density of NBCn1 immunostaining in normal breast, primary breast carcinomas, and metastases as indicated. (D) Na⁺-dependent ethylispropylamilroide (EIPA) insensitive recovery of intracellular pH in a biopsy of human breast cancer following washout of NH₄Cl (vertical bars show SEM, n = 5). The recovery of intracellular pH was Na⁺ and HCO₃⁻ dependent and thus demonstrates a NCBT transport activity in breast cancer. The authors also showed that the NCBT activity was modestly DIDS-sensitive (34±9%), consistent with NBCn1 being responsible. Scale bars = 20 μm. Adapted, with permission, from (32).

Figure 11

Localization of NDCBE to the hippocampus of the human brain (adapted, with permission, from 81). (B) Effects of knockout of NDCBE (Slc4a8) on Na⁺-dependent pH_i changes in intercalated cells in mice cortical collecting ducts (adapted, with permission, from 187). Traces are the average of pH_i changes recorded when luminal Na⁺ was removed and readded, in the presence of extracellular HCO₃⁻. The data provides functional evidence for the presence of NDCBE in the apical membrane, which is important for reabsorption of Na⁺ by these cells.

Figure 12

NBCn2 versus NCBE. In Xenopus oocytes and HEK 293 cells, SLC4A10 encoding NBCn2/NCBE produces Cl⁻ flux. This flux is either due to Cl⁻ self-exchange uncoupled to Na/HCO₃ transport (236) or Na,HCO₃-dependent Cl⁻ transport (79).

Figure 13

NBCn2/NCBE (Slc4a10) is present in the basolateral membrane of the choroid plexus epithelial cells and Slc4a10 knockout mice have reduced decreased volume of the brain ventricles (adapted, with permission, from 143). (A) Slc4a10 (green) localizes to the basolateral, but not the apical, membrane of the choroid plexus epithelium. The apical membrane is stained for the Na⁺/K⁺-ATPase (red) and the blue stain is nuclei (scale bar 30 μm). (B) In Slc4a10 knockout mice no NBCn2/NCBE staining was seen. [(C) and (D)] MRI scans of mice brain show the brain ventricles. (E) The volume of the brain ventricles from knockout mice is reduced to ≈25% of the volume in wild-type mice.