Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Na+/HCO3 Cotransporter NBCn1

  • Ying Liu
  • Xiao-Yu Wang
  • Zhang-Dong Xie
  • Li-Ming Chen
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101854

Synonyms

 NBC2;  NBC3;  NBCn1;  SLC4A7

Historical Background

NBCn1 is an electroneutral Na+/HCO3 cotransporter encoded by SLC4A7 gene, a member of the bicarbonate transporter family of solute carrier 4 (SLC4). In total, this family contains ten members: (1) three electroneutral anion exchangers AE1 (SLC4A1), AE2 (SLC4A2), and AE3 (SLC4A3); (2) five Na+-coupled HCO3 transporters (NCBTs), including two electrogenic Na+/HCO3 cotransporters NBCe1 (SLC4A4), NBCe2 (SLC4A5), two electroneutral Na+/HCO3 cotransporters NBCn1 and NBCn2 (SLC4A10), and an electroneutral Na+-driven Cl/HCO3 exchanger NDCBE (SLC4A8); (3) two less well-characterized members SLC4A9 and SLC4A11.

The association between the transport of Na+ and HCO3 was reported in 1970s in epithelial cells from different systems, such as proximal tubules (Burg and Green 1977) and jejunum (Turnberg et al. 1970; Podesta and Mettrick 1977). In 1983, Boron and Boulpaep clearly conceptualized for the first time Na+/HCO3 cotransporter based on their study with renal proximal tubules from Salamander (Boron and Boulpaep 1983). Proximal tubule is the major site for HCO3 reabsorption in the kidney. By microperfusion, Boron and Boulpaep demonstrated that the transport of HCO3 across the basolateral membrane of the proximal tubule is coupled to the transport of Na+. Moreover, this Na+-coupled transport of HCO3 is electrogenic and sensitive to inhibition by the stilbene disulfonate reagents, such as 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS).

Following the discovery by Boron and Boulpaep, Na+-coupled HCO3 transport was functionally characterized in many cell types from diverse systems, such as rat hepatocytes (Gleeson et al. 1989; Renner et al. 1989), retinal pigment epithelium of frog (la Cour 1989), bovine corneal endothelium (Bonanno and Giasson 1992), cardiac Purkinje fibers (Dart and Vaughan-Jones 1992), smooth muscle cells from rat arteries (Aalkjaer and Cragoe 1988; Aalkjaer and Hughes 1991) and guinea-pig ureter (Aickin 1994), rat hippocampal neurons (Schwiening and Boron 1994), and fibroblast cells (L’Allemain et al. 1985). Except for the Na+-dependence, some of these HCO3 transport activities are also dependent on Cl (L’Allemain et al. 1985; Schwiening and Boron 1994).

In 1997, Romero et al. (1997) cloned, from the kidney of Salamander, the cDNA encoding the first Na+-coupled HCO3 transporter, designated as NBCe1, literally meaning the first electrogenic Na+-bicarbonate cotransporter. The sequence of NBCe1 is homologous to the then well-established Na+-independent Cl/HCO3 exchangers AE1−3 of the SLC4 family. Based upon the successful cloning of NBCe1 by Romero et al., many more variants of NBCe1 and other NCBTs have been identified from a broad spectrum of tissues of different species.

NBCn1 is the first established electroneutral Na+/HCO3 cotransporter. Ishibashi et al. (1998) identified from human retina a cDNA encoding NBCn1 (designated as “NBC2”). However, this clone was incomplete, missing sequences for the initial amino-terminus (Nt) of NBCn1, and contained some cloning artifact. In 1999, the Kurtz group identified, from human skeletal muscle and heart, the complete cDNA encoding NBCn1, designated as “NBC3” (Pushkin et al. 1999). In 2000, the Boron group identified the cDNAs of three different NBCn1 variants from rat artery (Choi et al. 2000). This last study functionally demonstrated that the protein is an electroneutral Na+/HCO3 cotransporter and therefore designated it as NBCn1 (“n” meaning electroneutral) following the convention for the nomenclature of NBCe1 (“e” meaning electrogenic).

Molecular Function of NBCn1

NBCn1 is a secondary active transporter that mediates coupled movement of Na+ and HCO3 across the plasma membrane. As shown in Fig. 1, the energy required to drive the operation of NBCn1 is derived from the electrochemical potential difference of Na+ across the plasma membrane. Under physiological condition, the cell maintains an inwardly directed electrochemical gradient of Na+ by the activity of Na+-K+-ATPase, a primary active transporter. This inwardly directed electrochemical gradient of sodium is used as the driving force for the transmembrane movement of many different solutes, e.g., proton, HCO3, and glucose, by different secondary active transporters.
Na+/HCO3− Cotransporter NBCn1, Fig. 1

Molecular function of NBCn1 in cells. NBCn1 mediates electroneutral Na+/HCO3 influx at an apparent stoichiometry of 1 Na+:1 HCO3 driven by the inwardly directed electrochemical gradient of Na+ established by the primary active transporter Na+-K+-ATPase. On the intracellular side, HCO3 consumes one proton to generate CO2 and H2O, both of which can leave the cell via diffusion. On the extracellular side, CO2 recreates H+ and HCO3. The latter can be recycled by NBCn1. The net effect of the whole process is equivalent to the extrusion of one proton from the cell. CA carbonic anhydrase, AQP aquaporin, Δμ Na electrochemical driving force of Na+ across plasma membrane

The cotransport of Na+ and HCO3 carried by NBCn1 is electroneutral with an apparent stoichiometry of 1 Na+ to 1 HCO3. Therefore, the ion transport by NBCn1 carries no movement of net charge across the plasma membrane and has no effect on the membrane potential of the cells. Unlike the other Na+-coupled HCO3 transporters of SLC4 family, NBCn1 is relatively insensitive to DIDS (Choi et al. 2000).

The HCO3 transport mediated by NBCn1 plays an important role in intracellular pH (pHi) regulation. Under physiological conditions, NBCn1 functions as an acid-extruder by catalyzing the inward movement of HCO3 in the cell. As shown in Fig. 1, in the cytosol, HCO3 titrates proton to form H2CO3, causing a rise in pHi. H2CO3 is then dissociated into H2O and CO2 under the influence of carbonic anhydrase (CA). H2O and CO2 flux out of the cell either through channels or by simple diffusion. In the extracellular space, the opposite process takes place to generate HCO3 and H+. The net outcome of the whole process is the extrusion of a proton from the cell.

Structure of NBCn1

NBCn1 is an N-glycosylated integral membrane protein with an apparent molecular weight of ∼180 kDa. As shown in Fig. 2a, NBCn1 contains a large amino-terminal (Nt) domain and small carboxyl terminal (Ct) domain that both localizes on the intracellular side. The transmembrane domain (TMD) contains 14 transmembrane segments (TMs). The Nt domain and the TMD each account for ∼45% of the whole polypeptide, whereas the Ct domain accounts for the remaining 10% of the polypeptide.
Na+/HCO3− Cotransporter NBCn1, Fig. 2

Topology model of NBCn1 (a) and three-dimensional structure model of Nt domain of NBCn1 (b). The overall topology of NBCn1 is predicted based upon the crystal structure of the TMD of human AE1 (Arakawa et al. 2015). The three-dimensional structure of the Nt domain of NBCn1 is generated by molecular modeling based upon the crystal structure of AE1 Nt (PDB#: 1HYN). The molecular modeling was performed with the sequence of the Nt of human NBCn1-G (NCBI accession# NP_001245308.1, see Fig. 5) using the online tool SWISS MODEL from Protein Structure Bioinformatics Group at Swiss Institute of Bioinformatics (http://swissmodel.expasy.org/). The pink trees indicate the potential N-glycosylation sites on the third extracellular loop (EL3) of NBCn1

The fine structure of NBCn1 remains to be resolved, although considerable amount of efforts have been made to explore the structures of the SLC4 family transporters during the past decades. Most of the studies were focused on AE1 (the first identified member of SLC4 family) and NBCe1 (the first identified Na+-coupled member of SLC4 family) (see reviews Liu et al. 2015; Reithmeier et al. 2016). The crystal structures of both the Nt and TMD domains of human AE1 have been resolved (Zhang et al. 2000; Arakawa et al. 2015). Both the Nt and TMD of AE1 are dimers in the crystal structure, consistent with the idea long-held in the field that the SLC4 transporters are dimers in the plasma membrane.

As will be discussed in the next section, the Nt of NBCn1 contains two conserved regions: Nt-CR1 and Nt-CR2. Nt-CR1 and Nt-CR2 are highly homologous among all SLC4 members. In the crystal structure of the Nt domain of AE1, these two regions are intertwined to form a compact structure, representing the core of the Nt domain (Zhang et al. 2000; Arakawa et al. 2015). Figure 2b shows a model of the three-dimensional (3D) structure of the Nt domain of NBCn1 (lacking Nt-VR1 and cassette II, see next section) predicted by molecular modeling based upon the crystal structure of the Nt of human AE1.

The TMD of the transporter is the machinery responsible for the ion translocation across the plasma membrane. Each monomer in the dimer contains a functional unit for ion translocation. The TMD of NBCn1 shares ∼42% of sequence identities with the TMD of AE1. Figure 3 shows a model of the 3D structure of the TMD of NBCn1 obtained by molecular modeling based upon the crystal structure of the TMD of human AE1. The crystallography study on human AE1 (Arakawa et al. 2015) reveals that the TMD of SLC4 transporters contains 14 transmembrane helices (TMs). The 14 TMs fall into two inverted repeats (TM1−7 vs. TM8−14), a structural feature present in a number of membrane channels and transporters (see review Liu et al. 2015). The two inverted repeats can be superimposed on each other upon appropriate transformation. The TMD of SLC4 transporters comprises of two structural domains: the so-called core domain and gate domain that are separated by a cleft. The core domain consists of TMs 1–4 and TMs 8–11, whereas the gate domain consists of TMs 5–7 and TMs 12–14 (Arakawa et al. 2015). During ion translocation, the core domain and the gate domain undergo conformational changes to allow alternating access to the substrate binding site that resides in about the center of the membrane.
Na+/HCO3− Cotransporter NBCn1, Fig. 3

Model of three-dimensional structure of NBCn1 TMD. The structure model is created by molecular modeling with human NBCn1 (NCBI accession# NP_003606.3) using the crystal structure of human AE1 (PDB# 4YZF) as template (Arakawa et al. 2015). Molecular modeling was performed using the online tool SWISS MODEL from Protein Structure Bioinformatics Group at Swiss Institute of Bioinformatics. The overall fold of NBCn1 TMD is very similar to that of human AE1. The EL3 of NBCn1 is predicted to have a fold with five antiparallel beta sheets. However, the accuracy of this prediction about the fold of EL3 remains to be tested due to its low-sequence homology with the counter region of AE1. The pink trees in the side view represent the potential N-glycosylation sites on EL3 of NBCn1. EL3 the third extracellular loop, EL4 the fourth extracellular loop

One should note that there would be some fundamental differences in the molecular mechanisms underlying the ion translocation by AE1 and NCBTs including NBCn1. As Na+-independent Cl/HCO3 exchanger, AE1 needs no binding site for Na+. Moreover, it likely uses the same site for alternating binding of Cl and HCO3 during the ion translocation. However, NCBTs conduct cotransport of Na+ and HCO3. Therefore, in addition to a binding site for HCO3, NCBT would contain a binding site for Na+ distinct from that for HCO3.

The TMD of NBCn1 contains some specific structural features different from the TMD of AE1. The third extracellular loop (EL3) of NBCn1 (and other NCBTs as well) is much larger than that of AE1. This EL3 contains three potential N-glycosylation sites (Figs. 2 and 3a). AE1 also contains an N-glycosylation site which resides on the EL4 of AE1. The role of the large EL3 of NBCn1 in ion translocation remains unknown.

The EL4 between TM7 and TM8 is of particular interest for the ion translocation machinery of NCBTs. As shown in Fig. 3b, the EL4 of NBCn1 exhibits a flexible structure connecting the core domain and the gate domain. This EL4 plays an important role in determining the electroneutrality vs. electrogenicity of NCBTs. Replacing EL4 of NBCe1 with that of NBCn1 abolishes the electrogenicity of NBCe1, whereas the opposite substitution converts an electroneutral transporter into electrogenic (Chen et al. 2011).

Structural Variations of NBCn1

Most mammalian genes often contain more than one promoter controlling the initiation of transcription. Moreover, most mammalian genes contain optional sequences that can be alternatively included in or excluded from the final messenger RNA during pre-RNA splicing. The optional sequences are termed as cassette exons herein. The presence of alternative promoters and cassette exons enables mammals to give rise to multiple expression variants from one single gene.

Shown in Fig. 4a and b are the structures of human SLC4A7 and mouse Slc4a7 genes, respectively. Both human SLC4A7 and mouse Slc4a7 contain two promoters, the distal promoter P1 and the proximal promoter P2. Human SLC4A7 contains five known cassette exons (shown in purple). In mouse, the Slc4a7 gene contains an additional cassette exon that appears to be species specific, i.e., not present in human genome. As shown in Fig. 4c, the mechanism of alternative splicing of the cassette exons in SLC4A7 fall into three different categories: (1) exon skipping, such as exons 8, 10, 15, and 27 (in human SLC4A7), (2) alternative 5′-donor sites, such as exon 7 in human SLC4A7 (exon 8 in mouse Slc4a7), (3) intron retention, such as exon 3 in mouse Slc4a7 which contains a cryptic intron. The entire exon 3 can be skipped in some transcripts of Slc4a7. Note that the transcription of SLC4A7/Slc4a7 from alternative promoters P1 or P2 and the expression of their cassette exons are tissue specific.
Na+/HCO3− Cotransporter NBCn1, Fig. 4

Structures of human SLC4A7 (a) and mouse Slc4a7 genes (b). The human SLC4A7 spans for ~110 kb at 3p22 in the genome, and the mouse Slc4a7 spans for ~98 kb. P1 and P2 indicate the two promoters of SLC4A7/Slc4a7. Exon 3 in mouse Slc4a7 appears species specific. Sequence homologous to mouse exon 3 is not identified in human SLC4A7. The exons in grayed box indicate the region encoding the TMD of NBCn1. Purple indicate the exons that can be alternatively spliced. Splicing-out exon 15 (exon 16 in mouse Slc4a7) causes the production of the isolated Nt domain of NBCn1 (see Fig. 5)

As summarized in Fig. 5, the mammalian SLC4A7 gene is able to produce at least 18 full-length NBCn1 variants plus two specialized products with the isolated Nt domain only. These NBCn1 variants contain four different extreme Nts. The variants with an extreme Nt starting with “MERF” or “MIPL” or “MDEL” are derived from promoter P2, whereas those starting with “MEAD” are derived from promoter P1. The Nt domain of NBCn1 contains three splicing cassettes: cassettes I, II, and IV, whereas the small Ct domain of NBCn1 contains one splicing cassette: cassette III. No structural variation is present in the TMD of NBCn1. The alternative Nts and splicing cassettes are collectively referred to as optional structural elements (OSEs) hereafter in the following discussion.
Na+/HCO3− Cotransporter NBCn1, Fig. 5

Expression variants of NBCn1. NBCn1 variants are alphabetically designated according to the order of discovery (for NCBI accession numbers, see review Liu et al. 2015). The variants with the same initial Nt sequence, e.g., those starting with “MEAD,” are grouped together. Nt domain contains two variable regions Nt-VR1 and Nt-VR2 and two conserved regions Nt-CR1 and Nt-CR2. The Nt domain is connected to the TMD via a linker peptide “Nt-linker.” Two AE1 variants (the erythrocytic variant eAE1 and the kidney variant kAE1) are included in the diagram for reference

According to the localization of the OSEs, the Nt domain of NBCn1 can be divided into two variable regions (Nt-VR1 and Nt-VR2) and two conserved regions (Nt-CR1 and Nt-CR2; Fig. 5). The whole Nt domain is connected to the TMD by a linking peptide “Nt-linker.” As discussed in the above section, the conserved regions Nt-CR1 and Nt-CR2 are highly homologous to the counterparts of other SLC4 members and form a structure representing the core domain of the Nt domain (Fig. 2b). The variable regions (Nt-VR1 and Nt-VR2) are appendages to this core domain. The structure of Nt-VR1 and Nt-VR2 and their relationships with the core domain of NBCn1 Nt remains unknown.

The OSEs play important roles in modulating the function of NBCn1. Firstly, some OSEs have profound effects on the intrinsic activity (the transport activity per molecule) of NBCn1. Although the differences in the alternative Nts (MEAD vs. MERF, and likely MIPL) has little effect on the intrinsic transport activities of NBCn1, splicing cassettes II, III, and IV elicit strong stimulatory effect on the intrinsic transport activity of NBCn1 (Liu et al. 2013). Depending on the structural context, the intrinsic transport activity of one NBCn1 variant, e.g., NBCn1-N containing cassettes III and IV, can be five times higher than that of another, e.g., NBCn1-E lacking cassettes III and IV (Liu et al. 2013).

Secondly, some OSEs of NBCn1 contain binding sites for regulatory partners. One example is Nt-VR1 which contains structural determinants for the binding of IRBIT (IP3R binding protein released with IP3), also known as S-adenosyl homocysteine hydrolase-like 1 (AHCYL1). The interaction of IRBIT can stimulate the activity of NBCn1 (Parker and Boron 2013). IRBIT can also interact with and inhibit the activity of inositol 1,4,5 trisphosphate (IP3) receptor (IP3R), a Ca2+ channel on endoplasmic reticulum that plays a critical role in cellular Ca2+ signaling (Yang et al. 2011). The interaction between IRBIT and IP3R is abolished upon binding of IP3 to IP3R, a process that would cause a rise in intracellular Ca2+ concentration. Another well-studied example is cassette II which contains binding determinants for calcineurin A (CnA) (see review Parker and Boron 2013). Unique to NBCn1, this cassette contains 124 residues in human NBCn1 (123 aa in rodent NBCn1). Interaction of CnA with cassette II stimulates the transport activity of NBCn1 (Danielsen et al. 2013). CnA is a Ca2+/calmodulin-activated serine/threonine-specific phosphatase that plays an important role in the regulation of a series of channels and transporters in the cardiovascular system (Wang et al. 2014).

Note that, both signaling pathways involving IRBIT or CnA are related to Ca2+. The relationship between NBCn1 and Ca2+ signaling in the cell is of interest. For more details, see discussion in section “NBCn1 in the Cardiovascular System.”

NBCn1 in the Central Nervous System

NBCn1 plays important physiological and pathological roles in diverse systems. Discussed in this section is NBCn1 in the central nervous system (CNS). The role of NBCn1 in the kidney, cardiovascular system, and breast cancer will be discussed in the following sections.

CNS is likely the system where NBCn1 is most abundantly expressed in the body. Here, NBCn1 is predominantly expressed in neurons and the epithelial cells in choroid plexus. Together with other acid-base transporters, NBCn1 is involved in the maintenance of pH homeostasis in the brain.

pHi is a fundamental regulator of the excitability of CNS. It is generally appreciated that neurons in the CNS is stimulated by intracellular alkalosis and is inhibited by intracellular acidosis. On the other hand, neuronal activities, such as presynaptic transmitter release, GABAA receptor activities, action potential firing, can cause transients in both pHi and extracellular pH (pHo) in CNS. Thus, it is critically important to maintain the pH homeostasis in the nervous system.

Dysfunction of acid-base transporters is associated with multiple neural diseases, such as epilepsy, migraine, autism, and mental retardation. Specifically, SLC4A7 is shown to be associated with drug addiction in human. In addition, genetic disruption of Slc4a7 affects the development of retina and inner ears, resulting in blindness and auditory impairment in mouse, suggesting that NBCn1 plays an important role in sensory systems (Bok et al. 2003; Lopez et al. 2005).

NBCn1 in the Cardiovascular System

The physiological and pathophysiological significance of NBCn1 in the cardiovascular system is becoming increasingly recognized. In human, an SNP in SLC4A7 gene is associated with increased risk of hypertension (Ehret et al. 2011). In mouse, genetic disruption in Slc4a7 causes mild hypertension at rest, suggesting NBCn1 playing an important role in modulating the vascular tone (Boedtkjer et al. 2011). On the other hand, disruption of NBCn1 attenuates blood pressure increase induced by angiotensin II administration in mouse (Boedtkjer et al. 2011). NBCn1 appears to be the primary acid extruder expressed in vascular smooth muscle cells and heart endothelial cells. Knockout of NBCn1 causes mild intracellular acidosis in smooth muscle and endothelial cells under steady-state condition.

As shown in Fig. 6, the NBCn1-related phenotypes of the cardiovascular system might be the result of multiple different processes given the pH sensitivity of many soluble enzymes, membrane ion channels, and transporters in the blood vessel (see review Boedtkjer and Aalkjaer 2012). For example, the activities of the endothelial nitride oxide synthase (eNOS) and Rho kinase, myofilaments, and troponin complex in vascular smooth muscle cells are stimulated by elevating pHi. In endothelial cells, eNOS is responsible for the production of nitric oxide (NO) from L-arginine. NO plays a critically important role in blood vessel relaxation. Rho kinase is involved in the regulation of the Ca2+ sensitivity of vascular smooth muscle cells, probably due to the phosphorylation and deactivation of myosin light chain phosphatase, which plays an important role in the development of hypertension in response to angiotensin II.
Na+/HCO3− Cotransporter NBCn1, Fig. 6

Role of NBCn1 in vascular system. NBCn1 is expressed in both endothelial cells and vascular smooth muscle cells (VSMCs). In the VSMCs, NBCn1 is regulated by IRBIT and calcineurin (CnA). Disruption in NBCn1 causes intracellular acidification, which in turn would affect the function of a number of proteins involved in the regulation of smooth muscle contraction. The activity of eNOS is reduced in endothelial cells due to knockout of NBCn1. Green arrows indicate stimulation, blue line indicates inhibition and dashed line indicates presumed effect. BK Ca Ca2+-activated potassium channel, CaM calmodulin, IP 3 inositol 1,4,5 trisphosphate, NCX Na+/Ca2+ exchanger, IP 3 R IP3 receptor, SR sarcoplasmic reticulum, PMCA plasma membrane Ca2+ ATPase

Raising pHi stimulates the activity of the Ca2+-activated potassium (BKCa) channel, which plays a pivotal role in modulating the excitability of vascular smooth muscle cells (VSMCs). Activation of the BKCa channel would render the VSMCs hyperpolarized and therefore inhibit smooth muscle contraction.

Excitation and contraction of VSMCs are accompanied with transients in intracellular Ca2+ content (Bolton 2006). The release and clearance of cytosolic Ca2+ are mediated by a group of Ca2+ channels and transporters on the plasma and sarco-endoplasmic membranes. The activities of these channels and transporters are affected by the changes in pHi. For example, the sarcolemmal Na+/Ca2+ exchanger (NCX), sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), and sarcolemmal L-type Ca2+ channels are stimulated by raising pHi.

Consistent with the hypertension induced by disruption of Slc4a7 in mouse, Boedtkjer et al. have shown that the activities of eNOS, Rho kinase, Ca2+-activated potassium (BKCa) channel in mouse arteries are inhibited by intracellular acidosis derived from disruption of Slc4a7 (Boedtkjer et al. 2011).

The regulation of pHi and Ca2+ signaling is obviously reciprocally related. On one hand, as discussed above, the activities of machineries involved in intracellular Ca2+ homeostasis is affected by changes in intracellular pH. On the other hand, the activity of acid-base transporters like NBCn1 is regulated by pathways related to Ca2+ signaling. As discussed in section “Structural Variations of NBCn1” and shown in Fig. 6, NBCn1 is stimulated by IRBIT and CnA, both of which are related to or affected by the intracellular Ca2+ signaling. The release of IRBIT from IP3R relies on the binding of IP3 to the Ca2+ channel IP3R. In the meantime, the binding of IP3 activates IP3R, causing an intracellular Ca2+ transient due to the release of Ca2+ store in the SR lumen. The rise in the concentration of cytosol Ca2+ stimulates calmodulin (CaM), which in turn activates CnA, which in turn stimulates the HCO3 uptake by NBCn1.

NBCn1 in Renal Acid-Base Transport

The kidney plays a central role in maintaining the systemic acid-base balance in the body by reabsorbing bicarbonate in the renal filtrate and excreting net acid into the urine. The major site for bicarbonate reabsorption is the proximal tubule, where the electrogenic Na+/HCO3 cotransporter NBCe1 plays an important role in HCO3 reabsorption. The role of NBCe1 in HCO3 reabsorption is not discussed here.

A large amount of the acid is excreted as ammonium in the urine (Weiner and Hamm 2007). As shown in Fig. 7a, the ammonium is primarily produced by glutamine metabolism in the proximal tubule epithelial cells. The ammonium is then secreted into the tubule lumen. A large fraction of the ammonium is reabsorbed by the medullary thick ascending limb (mTAL) of the Henle’s loop and secreted into the interstitial space; a small fraction of the ammonium is delivered to the distal convoluted tubule. While some of the ammonium secreted into the interstitial space might flux back into the descending limb of the Henle’s loop, a large part eventually enters into the collecting duct and finally appears in the urine.
Na+/HCO3− Cotransporter NBCn1, Fig. 7

Role of NBCn1 in ammonium transport in the kidney. (a) Overview of ammonium transport in the kidney. Ammonium is primarily derived from glutamine metabolism in the proximal tubule epithelial cells. In the Henle’s loop, the ammonium undergoes a recycling process, resulting in medullary accumulation of ammonium. Numbers in red indicate the ammonium delivered to specific sites as percentage of the final ammonium excreted in the urine (see review Weiner and Hamm 2007). (b) Molecular mechanism for ammonium reabsorption by the epithelia in the mTAL. The luminal ammonium enters into the epithelial cells via the apical Na+-K+-Cl cotransporter NKCC or potassium channel ROMK. In the cell, NH4+ is dissociated to form NH3 and H+, resulting intracellular acidification. The basolateral NBCn1 mediates HCO3 uptake to attenuate the acidification. The NH3 derived from NH4+ and the CO2 derived from titration of HCO3 diffuse into the interstitial space

NBCn1 at the basolateral membrane of mTAL plays an important role in the ammonium reabsorption by the mTAL epithelial cells (Fig. 7b). Here, the uptake of ammonium from the tubular lumen could produce a profound acidification in the pHi of the mTAL epithelial cells. NBCn1 could attenuate the intracellular acidification by mediating HCO3 influx to titrate the intracellular acid load due to the apical NH4+ influx. Moreover, the titration of NH4+ increases the inward driving force for NH4+ across the apical membrane and therefore promotes the NH4+ reabsorption by the mTAL.

NBCn1 and Breast Cancer

In solid tumors, the cancer cells undergo profound changes in cellular metabolism. The tumor tissues are usually poorly vascularized, resulting in inadequate blood supply and therefore hypoxic condition in the microenvironment of the tumors. On the other hand, the tumor tissues are metabolically highly active due to the high demand in energy. Compared to the normal tissues, tumor tissues have a much higher rate of aerobic glycolysis. The high rate of metabolism and increased aerobic glycolysis result in increased production of metabolic acids, e.g., lactate, from nonoxidative breakdown of glucose in the tumor tissues. This is the so-called Warburg effect (Webb et al. 2011).

As an adaptive response to the increased acid production, the acid-extruders, such as proton pumps, Na+-H+ exchangers, and monocarboxylate trasnporters, are often upregulated to promote acid clearance in the cancer cells. However, the poor vascularization in the tumor tissues impairs the disposal of the metabolic acids, resulting in an acidic microenvironment in the extracellular space in tumor tissues. The upshot is an outwardly-directed pH gradient in tumor tissues (pHi > 7.2 vs. pHo = ∼6.7–7.1), which is in striking contrast to the inwardly directed pH gradient in normal tissues (pHi ≈ 7.2 vs. pHo ≈ 7.4) (Webb et al. 2011). The inverted pH gradient can promote tumor growth in several perspectives. The enhanced pHi is stimulatory to the proliferation of tumor cells and is inhibitory to cell apoptosis. Moreover, the acidic pHo promotes cell-matrix remodeling and therefore increases metastasis and evasion of tumor cells.

NBCn1 is a major acid extruder in breast cancer cells and plays an important role in breast cancer. Disruption of NBCn1 greatly delays the development of breast cancer in mouse (Lee et al. 2016). The expression of NBCn1 is upregulated in breast carcinomas and metastases (Boedtkjer et al. 2013). In breast cancer cells, the expression of NBCn1 is regulated by the signaling pathway involving receptor tyrosine kinase ErbB2 (see review by Gorbatenko et al. 2014). A constitutively active variant of ErbB2 truncated in the Nt is associated with increased risk of breast cancer. This Nt-truncated ErbB2 can greatly stimulate the transcription of SLC4A7. Interestingly, a single-nucleotide polymorphism (SNP) in the 3′-untranslated region of transcripts of human SLC4A7 is strongly associated with increased risk of breast cancer in human. The effect of this SNP on the expression of NBCn1 in breast cancer remains unknown.

Summary

The electroneutral Na+/HCO3 cotransporter NBCn1 is widely expressed in a wide spectrum of tissues. It plays an important role in the regulation of intracellular pH and the transepithelial transport of electrolytes. The physiological and pathological significance of NBCn1 has been emerging during the past years. The SLC4A7 gene encoding NBCn1 has been associated with multiple diseases, including breast cancer, hypertension, and drug addiction.

Although great progress has been made during the past years in understanding the structure and function, functional regulation, physiological and pathological roles of NBCn1, many issues remain unclear. The following questions are of particular interest for future study.
  1. 1.

    Molecular mechanism underlying the ion transport by NBCn1. TMD is the structural unit directly carrying out the ion translocation across the plasma membrane. The resolution of the crystal structure of AE1 TMD represents a major step forward towards understanding the structure and function of the SLC4 transporters. However, there are also major differences between the AE1 which is Na+-independent and NBCn1 (other NCBTs as well) which is Na+-dependent. What is the mechanism for the energy coupling underlying the cotransport of Na+ and HCO3? What is the structural mechanism for determining the stoichiometry of Na+ and HCO3?

     
  2. 2.

    Functional relationship between the Nt domain and TMD. The Nt domain is connected to TMD by a flexible linker. It has been well recognized that the Nt domain plays an important role in the regulation of the transporter. Specific structural elements (e.g., cassettes II and IV) in the Nt domain can stimulate the intrinsic activity of NBCn1. It remains unclear how the Nt domain affect the efficiency of the ion transport by the TMD. What is the physical interaction between the Nt domain and TMD of NBCn1?

     
  3. 3.

    Signaling pathways underlying the regulation of NBCn1. The activity of NBCn1 is stimulated by IRBIT and CnA. However, the signaling pathway underlying the regulation of IRBIT and CnA remains not so clear. What is the relationship between the regulation by the IRBIT pathway and that by the CnA pathway? As IRBIT and CnA are both related to Ca2+, what is the role of Ca2+ signaling in the regulation of NBCn1?

     
  4. 4.

    Crosstalk between proton signaling and Ca2+ signaling. NBCn1 activity affects the dynamic concentration of protons in the cell. Fluctuation in cellular proton concentration affects the activities of the cellular machineries involved in the maintenance of cellular Ca2+ homeostasis. On the other hand, the Ca2+ signaling regulates the activity of NBCn1 via the CnA pathway. The crosstalk between proton signaling and Ca2+ signaling is of great interest.

     

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Ying Liu
    • 1
  • Xiao-Yu Wang
    • 1
  • Zhang-Dong Xie
    • 1
  • Li-Ming Chen
    • 1
  1. 1.Department of Biophysics and Molecular Physiology, Key Laboratory of Molecular Biophysics of Ministry of EducationHuazhong University of Science and Technology School of Life Science and TechnologyWuhanChina