Na+/HCO3− Cotransporter NBCn1
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
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
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.
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.
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.
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.
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.
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.
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−?
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?
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?
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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