SLC4 Sodium-Driven Bicarbonate Transporters

The SLC4 ( s o l ute c arrier 4) family is a group of a membrane proteins that share sequence homology and in general mediate the transport of bicarbonate. It should be noted that bicarbonate transport is not unique to the SLC4 family. The structurally unrelated SLC26 family has at least three proteins that mediate Cl ⁻ –HCO ⁻ ₃ exchange. In this chapter, the biology of SLC4 Na ⁺ -dependent bicarbonate transporters will be highlighted. The outlier in the SLC4 family is the SLC4A11 gene product (NaBC1) that lacks the ability to transport bicarbonate and is currently characterized as an electrogenic sodium-borate transporter. Whether SLC4 transporters mediate bicarbonate and/or carbonate transport is unclear and therefore when the word bicarbonate or the chemical symbol HCO ⁻ ₃ is used throughout the chapter with reference to SLC4 mediated ion transport, it refers to both HCO ⁻ ₃ and/or CO ²⁻ ₃ .

In this chapter, while extra-renal studies will be highlighted, an emphasis will be placed on the biology of specific sodium-coupled SLC4 proteins in the kidney.

Key Words

SLC4, bicarbonate, kidney, renal tubular acidosis, sodium, transport, acid–base, pH

The SLC4 ( s o l ute c arrier 4) family is a group of a membrane proteins that share sequence homology and in general mediate the transport of bicarbonate. It should be noted that bicarbonate transport is not unique to the SLC4 family. The structurally unrelated SLC26 family has at least three proteins that mediate ${Cl}^{-} Unknown node type: glyph {HCO}_{3}^{-}$ ${Cl}^{-} {HCO}_{3}^{-}$ exchange. In this chapter, the biology of SLC4 Na ⁺ -dependent bicarbonate transporters will be highlighted. The outlier in the SLC4 family is the SLC4A11 gene product (NaBC1) that lacks the ability to transport bicarbonate and is currently characterized as an electrogenic sodium-borate transporter. Whether SLC4 transporters mediate bicarbonate and/or carbonate transport is unclear and therefore when the word bicarbonate or the chemical symbol ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ is used throughout the chapter with reference to SLC4 mediated ion transport, it refers to both ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ and/or ${CO}_{3}^{2 -}$ ${CO}_{3}^{2 -}$ .

In this chapter, while extra-renal studies will be highlighted, an emphasis will be placed on the biology of specific sodium-coupled SLC4 proteins in the kidney.

Functional Categorization of the SLC4 Family

A dendrogram of the SLC4 family of proteins is shown in Fig. 53.1 . The 10 different SLC4 gene products can be logically subdivided functionally and structurally into four groups that differ primarily in their Na ⁺ - and Cl ⁻ -dependence and electrogenic properties. Although various categorizations have been proposed, the following is a convenient scheme:

Figure 53.1, Dendrogram of SLC4 transporters.

Na ⁺ -independent $C l^{-} Unknown node type: glyph HC O_{3}^{-}$ $C l^{-} HC O_{3}^{-}$ exchangers: 3 homologous transporters, AE1, AE2, and AE3 ( SLC4A1, -2,-3 respectively) that mediate ${Cl}^{-} - {HCO}_{3}^{-}$ ${Cl}^{-} - {HCO}_{3}^{-}$ exchange have been well characterized. A fourth member, AE4, has also been reported to function as a ${Cl}^{-} - {HCO}_{3}^{-}$ ${Cl}^{-} - {HCO}_{3}^{-}$ exchanger. However, as will be discussed, the amino acid sequence of AE4 is more homologous to sodium-coupled SLC4 transporters.

$N a^{+} Unknown node type: glyph HC O_{3}^{-}$ $N a^{+} HC O_{3}^{-}$ cotransporters: There are 3 well-characterized members of the SLC4 family that transport Na ⁺ and ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ without concomitant net Cl ⁻ transport. NBCe1 ( SLC4A4 gene) and NBCe2 ( SLC4A5 gene) are sodium bicarbonate cotransporters that are chloride-independent and electrogenic in that their ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ to Na ⁺ transport coupling is >1 (2:1, or 3:1). NBCn1 ( SLC4A7 gene) is an electroneutral ${Na}^{+} Unknown node type: glyph {HCO}_{3}^{-}$ ${Na}^{+} {HCO}_{3}^{-}$ cotransporter with a coupling ratio of 1:1.

Na ⁺ -driven $C l^{-} Unknown node type: glyph HC O_{3}^{-}$ $C l^{-} HC O_{3}^{-}$ exchangers: Two SLC4 transporters have been reported to mediate ${Na}^{+} Unknown node type: glyph {HCO}_{3}^{-}$ ${Na}^{+} {HCO}_{3}^{-}$ cotransport in exchange for chloride. The literature regarding the functional properties of the first reported mammalian transporter, NCBE (SLC4A10), is somewhat confusing. Although this transporter is reported by several groups to mediate Na ⁺ -dependent ${Cl}^{-} Unknown node type: glyph {HCO}_{3}^{-}$ ${Cl}^{-} {HCO}_{3}^{-}$ exchange, a separate study in oocytes has shown that chloride is not transported under physiologic conditions, and accordingly the protein was renamed NBCn2 ( ${Na}^{+} - {HCO}_{3}^{-}$ ${Na}^{+} - {HCO}_{3}^{-}$ cotransporter electroneutral 2). The details of the discrepant findings in this area are discussed below. The second reported transporter in this group, NDCBE ( SLC4A8 gene), is a well-characterized Na ⁺ -driven ${Cl}^{-} - {HCO}_{3}^{-}$ ${Cl}^{-} - {HCO}_{3}^{-}$ exchanger.

Electrogenic Na-borate cotransporter: NaBC1 has been reported to mediate the electrogenic transport of ${Na}^{+} - {BO}_{4}^{-}$ ${Na}^{+} - {BO}_{4}^{-}$ in the presence of borate, or Na ⁺ OH ⁻ cotransport with coupling ratio of Na ⁺ :anion of at least 2 (electrogenic) in the absence of borate. The lack of a clear role for borate transport in various tissues where this transporter is expressed raises questions regarding the actual function of NaBC1 physiologically.

Several SLC4 transporters expressed in the kidney have been localized at the transcript/or protein level in various nephron segments and cell types ( Tables 53.1–53.3 ; Fig. 53.2 ). Studies involving the expression of SLC4 transcripts in the kidney have been done in several mammalian species including mouse, rat, rabbit and human. In addition to documented known species differences, SLC4 proteins typically have a number of specific variants (due to splicing and/or alternate promoter usage) whose localization in the kidney and functional properties have not been thus far thoroughly investigated. This fact increases significantly the complexity of addressing the actual protein expression pattern and functional characteristics of each SLC4 transporter in various renal cell types.

Table 53.1

Immunolocalization of Na ⁺ -driven SLC4 Transporters in Kidney

Transporter	Location	Apical	Basolateral	Human	Rat	Mouse
NBCe1	PT		+	+	+	+
NBCe2	OMCD	N/A	N/A	+
	P(U)	+		+
NBCn1	mTAL		+	+	+
	OMCD–αIC		+
	IMCD 2 & 3		+
	P(U) *				+
NaBC1 **	Podocytes			+	+
	PT	+	+	+	+
	tDL		+		+	+ ****
	CCD – IC ***	+		+
	OMCD – IC ***	+		+
	IMCD		+	+

PT – proximal tubule.

OMCD – outer medullary collecting duct.

P(U) – pelvis (uroepithelium).

tDL – thin descending limb.

mTAL – medullary thin descending limb.

IMCD – inner medullary collecting duct.

* LacZ staining.

** NaBC1 does not transport Na bicarbonate.

*** IC type not specified.

**** immunolocalization and LacZ staining only in the tDL in mouse.

Table 53.2

SLC4 Na ⁺ -driven Transporters that Have Not Been Immunolocalized in Kidney

Transporters	Technique	Location	Human	Rat	Mouse
NCBE/NBCn2	Northern: weak		+
NDCBE	Northern: weak		+
	RT-PCR	cortex (weak), medulla	+
	RT-PCR	inner medulla		+
	Western	cortex/CCD			+

Table 53.3

AE4 Immunolocalization in the Kidney

Location	Apical	Basolateral	Rat	Mouse	Rabbit
CCD: βIC	+				+
CCD: αIC	+	+			+
CCD: αIC, βIC		+	+
CCD: βIC		+		+

Figure 53.2, Immunlocolization of Na + -driven SLC4 transporters in the kidney. The data is derived from studies in human, rat, mouse and rabbit. Species differences exist as shown in Tables 53.1 and 53.3 .

The kidney and brain share the property of having various cell types that express different bicarbonate transporter proteins. Although most SLC4 transporters have in common the property of transporting bicarbonate, cells in the kidney and brain are likely taking advantage of the unique and specific properties of each transporter. The uniqueness of SLC4 proteins stems from the fact that these transporters differ significantly in their ion (Na ⁺ , Cl ⁻ ) dependence, coupling ratios, membrane targeting, substrate affinities, developmental expression, regulation by factors pH/phosphorylation, and protein-protein interaction. These differences also account for the fact that functionally, SLC4 transporters have various direct and indirect physiological roles in the kidney including transepithelial bicarbonate transport, intracellular pH regulation, and transport of Cl ⁻ , Na ⁺ and possibly NH ₄ ⁺ .

Because SLC4 transporters are expressed not only in the kidney but also in various extrarenal organs and cell types, diseases associated with mutations in these transporters or targeted disruption in mice result in both a renal and extrarenal phenotype ( Table 53.4 ).

Table 53.4

Phenotype with Altered SLC4 Function

Transporter	Human	Mouse
NBCe1	pRTA	pRTA
	band keratopathy	intestine obstruction
	glaucoma, cataracts	spleen abnormalities
	intracerebral calcification	enamel defect
	enamel defect
	↑ amylase, ↑ lipase
NBCe2	hypertension	hyperchloremic metabolic acidosis
		hyporeninemic hypoaldosteronism
		hypertension
NBCn1		Usher 2B- like syndrome
NDCBE		↓ CCD electroneutral NaCl transport
NCBE/NBCn2	↑ seizure propensity	↓ cerebral ventricle size
NCBE/NBCn2		↓seizure threshold
NaBC1	CHED2; Harboyan Syndrome; FECD	CHED2 – like
	CHED2; Harboyan Syndrome; FECD	↓ urine osmolarity
		↑urine volume
		↑excretion of Na ⁺ , K ⁺ , Cl ⁻ , Mg ²⁺
		↓ urinary [Ca ²⁺ ]
AE4		no phenotype

Sodium Bicarbonate Cotransporters (Electrogenic)

NBCe1 (SLC4A4 Gene)

Structural Variants

Our knowledge regarding the structure of sodium-dependant SLC4 transporters has until recently been extrapolated from the large body of data obtained from studies on the AE1 anion exchanger. More recent studies of NBCe1 reveal very interesting structural and functional differences from AE1. Three variants of the SLC4A5 gene have been well characterized. All three NBCe1 variants mediate electrogenic ${Na}^{+} - {HCO}_{3}^{-}$ ${Na}^{+} - {HCO}_{3}^{-}$ cotransport, but differ in their N- and C-terminal extremities, regulation, and intrinsic activity. In kidney, NBCe1-A (or kNBC1) is predominantly expressed in S1 and S2 proximal tubule cells where it mediates the basolateral efflux of ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ , thereby contributing to the reabsorption of ~80% of the filtered bicarbonate load. In addition to the proximal tubule, NBCe1-A is also expressed in the eye and salivary gland. NBCe1-A transcripts have also been detected in nasal submucosal glands in turbinate mucosa and nasal polyps.

The second NBCe1 variant, NBCe1-B (or pNBC1), which is identical to NBCe1-A except for its unique N-terminus (85 aa replacing the 41 aa in NBCe1-A) was originally cloned from pancreas and is expressed in pancreatic duct epithelial cells where it contributes to basolateral bicarbonate flux from blood to cell, during the process of secretin-evoked pancreatic fluid secretion. NBCe1-B is also widely expressed in various other tissues including, colon, skeletal muscle, eye, airway submucosal glands, heart, gall bladder, and nasal mucosa. The third variant, NBCe1-C, has a unique C terminus (61 aa replaces 46 in NBCe1-A or B) that ends in a type I PDZ-binding motif. The N-termini of NBCe1-B and NBCe1-C are identical and unlike the N-terminus of NBCe1-A, interact with the IP3 receptor binding protein IRBIT resulting in a stimulation of transport activity. More recently additional transcript have been reported.

Topological and Structural Properties

Of the NBCe1 variants, the structural topology of NBCe1-A has been most thoroughly studied and provides a topologic framework for other Na ⁺ -driven SLC4 bicarbonate transporters ( Fig. 53.3 ). NBCe1-A is an oligomer wherein the predominant oligomeric state of the cotransporter is dimeric. The NBCe1-A monomer is a ~140-kDa glycoprotein containing 1035 amino acids and is composed of 14 transmembrane regions (TMs). The N-terminal transmembrane region has 8 TMs homologous to the SLC4 transporter AE1. Both the extreme N- and C-termini of NBCe1-A are located in cytoplasm, with a large extracellular loop between transmembrane segment (TM) 5 and 6 containing two glycosylation sites. Importantly, although NBCe1-A forms a structural oligomer, each monomeric subunit maintains its own independent transport activity.

Figure 53.3, Topology of NBCe1-A. The transporter has 14 TMs with cystoplasmic N- and C-termini. The location of the known pRTA mutations is depicted. A large extracellular loop between TM5 and 6 typifies the Na + -driven SLC4 bicarbonate transporters. Note that NBCe1-A lacks the two AE1 (SLC4A1) re-entrant loops.

Previous topological models of NBCe1 were based on the assumption that the cotransporter resembles AE1 which has been extensively studied structurally. The C-terminal transmembrane region of the two transporters share 40% sequence homology but differ structurally. NBCe1-A lacks the two AE1 reentrant loops and extensive substituted cysteine scanning mutagenesis analysis showed that the C-terminal transmembrane region of NBCe1-A is tightly folded unlike AE1 resembling in various aspects the bacterial Na ⁺ -leucine cotransporter, LeuT.

An extensive mutatgenesis study of NBCe1-A has demonstrated several key residues that are critical for its function. Domain swapping studies of the cotransporter have also shed light on its functional properties and have suggested the electrogenicity of NBCe1-A is determined by the interaction of TMs in the lipid bilayer. Arg ²⁹⁸ in the N-terminus may be involved in constructing a “ ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ tunnel”. Residue Thr ⁴⁴² in TM 1 is thought to form an external gate for the transported ions. Proposed TM8 has several residues found to be involved in forming the ion translocation pore. Interestingly, substitution of Asp ⁵⁵⁵ to glutamic acid in the proposed TM 5 of NBCe1-A induced an outward rectifying Cl ⁻ current and altered the transport substrate selectivity, indicating it plays an important role in ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ selectivity.

Inhibitors

Asp ⁵⁵⁵ is in close proximity of the proposed 4,4’-diisothiocyanatostilbene-2,2’-disulfonate DIDS (a functional inhibitor) binding site of NBCe1-A (Lys ⁵⁵⁹ ). From the extracellular side, DIDS blocks NBCe1 reversibly by binding to a KKMIK motif at the putative extracellular end of TM5. The apparent affinity of the interaction decreases at more negative voltages possibly due to alterations in the conformation of the cotransporter as the membrane voltage changes. DIDS is also capable of blocking the cotransporter from the intracellular side at an unknown site. In addition to DIDS, NBCe1-A is also blocked by the nonsteroidal inflammatory agent Tenidap from the extracellular or intracellular side. NBCe1-B is sensitive to the anion channel inhibitors phloretin, niflumic acid, NPPB, and glybenclamide. NBCe1 is inhibited by the N-cyanosulphonamide compound S0859 however the inhibition is not specific. Benzamil has also been reported to inhibit NBCe1-A.

Familial Proximal Renal Tubular Acidosis: Molecular Mechanisms Involving NBCe1

Patients with autosomal recessive mutations in NBCe1 have an unique phenotype that can be diagnosed clinically that includes severe hyperchloremic acidosis, with extra-renal manifestations including growth and mental retardation, basal ganglia calcification, cataracts, corneal opacities (band keratopathy), glaucoma, elevated serum amylase and lipase, and defects in the enamel consistent with amelogenesis imperfecta. Regarding the renal phenotype, this “experiment of nature” demonstrates the importance of NBCe1-A in mediating proximal tubule bicarbonate reclamation; the extrarenal manifestations demonstrate the importance of NBCe1- variants in eye (NBCe1-A, NBCe1-B; ), brain (NBCe1-B, NBCe1-C; ), and tooth ameloblasts (NBCe1-B; ). Thus far, the following mutants have been described: eight missense mutations (R298S, S427L, T485S, G486R, R510H, L522P, A799V, and R881C), 2 nonsense mutations (Q29X, W516X), a frameshift deletion at nucleotide 2311A, and a C-terminal 65 base-pair deletion from exon 23 to intron 23 predicted to truncate the intracytoplasmic C-terminus ( Fig. 53.3 , Table 53.5 ). Other than the NBCe1-A-Q29X mutation which only affects NBCe1-A, the NBCe1-B and -C variants are also mutated resulting in the extrarenal manifestations in this disorder.

Table 53.5

SLC4A4 Mutations: Molecular Mechanisms

Mutation	Location	Effect	Mechanism
Q29X *	N-terminus	truncation	—
R298S	N-terminus	↓ function	ion permeation pathway
S427L	TM1	↓ function	voltage sensing
T485S	TM3	↓ function	protein conformation
G486R	TM3	↓ function	protein conformation
R510H	TM4	misfolding	ER retention
W516X	TM4	truncation	—
L522P	TM4	misfolding	ER retention
2311delA	IL4	truncation	—
A799V	TM10	↓ function	protein conformation
R881C	TM12	misfolding	ER retention
65bp-del	C-terminus	truncation	—

TM Transmembrane

IL Intracellular Loop

* NBCe1-A only

The disease causing mechanism of the nonsense/frameshift mutations is due to absence of the full-length NBCe1 protein, whereas the missense mutations result from either reduced transport function and/or impaired plasma membrane processing (R510H, L522P, and R881C; (53, 114, 122)). The functional and targeting defects caused by NBCe1-A pRTA missense mutations have been analyzed in Xenopus oocyte, ECV304 cells, Madin-Darby canine kidney cells, and HEK293 cells with sometimes differing results. In general, mutant membrane transporter proteins are thought to have reduced function as a result of three possible mechanisms that are not mutually exclusive: (1) misfolding of the transporter protein resulting in ER retention; (2) alteration of the ion translocation (permeation) pathway; and (3) impairment of the transporter conformation (static/dynamic) required for substrate translocation.

Missense Mutations

Recent studies have focused on the topologic/structural/functional implications of the residues in NBCe1 that are mutated in pRTA ; ( Table 53.5 ). Arg ²⁹⁸ is located in the cytoplasmic N-terminus of NBCe1-A and resides in a tightly folded aqueous inaccessible conformation based on: (1) homology modeling to the crystallized cytoplasmic domain structure of AE1 and (2) biotin maleimide labeling assays. It has been suggested that Arg ²⁹⁸ may be involved in constructing a “ ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ tunnel” in NBCe1-A and that mutation R298S disrupts the local structure of the ion permeation pathway thereby impairing the ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ entry. The remaining missence pRTA residues have recently been localized in the transporter ( Fig. 53.3 ): Ser ⁴²⁷ to TM 1, Thr ⁴⁸⁵ , Gly ⁴⁸⁶ to TM 3, Arg ⁵¹⁰ and Leu ⁵²² to TM 4, Ala ⁷⁹⁹ to TM 10, and Arg ⁸⁸¹ to TM 12.

All pRTA residues do not appear to line an ion translocation pore and are likely located in the protein/lipid bilayer complex. Considering the structural change or charge alteration for most of the missense mutations involved in pRTA, it is conceivable that these TM residing mutations would affect protein folding/helix packing in the lipid bilayer, which could lead to significant loss of mutant protein transport function. However, other than R510H, L522P, and R881C, which cause protein intracellular retention, the remaining pRTA causing mutations (S427L, T485S, G486R, and A799V) process to the plasma membrane and retain 10–50% transport function. These findings essentially rule out the possibility that the reduced transport function of the membrane-processed pRTA causing mutations is caused by dramatic protein misfolding.

Mutation of Ser ⁴²⁷ to leucine induces a failure of the transporter to reverse its direction even at very negative membrane potentials. In addition, the transport is 10% of wild-type NBCe1-A. Ser ⁴²⁷ has been proposed to be involved in helix interaction and leucine mutation may disrupt NBCe1-A “voltage sensing,” affect a Na ⁺ coordination site, or alter the local conformation required for normal function. Ser ⁴²⁷ is located in TM 1 adjacent to Ala ⁴²⁸ , a residue that lines the substrate translocation pore. The bulky side chain of leucine may alter the geometry of the ion translocation pore and impair transport.

The T485S mutation is of interest because of the following considerations: (1) Serine and threonine both belong to the same amino acid category (nucleophilic) and have the same pK _a ; (2) structurally, serine closely resembles threonine, but lacks a CH ₃ group; (3) mutation of Thr ⁴⁸⁵ to serine/cysteine impairs NBCe1-A function by 50%. These observations suggest that the CH ₃ group of threonine at position 485 may have a unique role in maintaining the structure of NBCe1-A for normal ion translocation. Valine substitution restored NBCe1-A transport activity to 75% that of the wild-type, despite the fact that it is a hydrophobic amino acid. Moreover isoleucine substitution impaired transport function by 50% also highlighting the potential importance of the OH group at the 485 position. Based on these findings, it has been proposed that Thr ⁴⁸⁵ may reside in a space confined position involving both CH ₃ and OH chemistry that is critical for maintaining NBCe1-A in a conformation required for normal transport function. Indeed, when both CH ₃ and OH groups are removed at this particular amino acid position (alanine substitution), the transport function is decreased to 30% of the wild type. This mechanism may also be applicable to the G486R mutation, which resides adjacent to Thr. ⁴⁸⁵

The L522P mutation causing pRTA is not processed to the plasma membrane in Xenopus oocytes, ECV 304, and Madin-Darby canine kidney cells, whereas an L522C mutant does not impair membrane processing. This suggests that it is the proline residue rather than the loss of leucine that causes intracellular retention of NBCe1-A. Arg ⁵¹⁰ and Leu ⁵²² both reside in TM 4, a helix that carries signal anchor and stop transfer sequences, and has several residues whose mutation cause protein intracellular retention. TM 4 in NBCe1-A may act as a scaffolding helix that is important for the second stage folding of the transporter. Therefore, it is predictable that a helix disruption mutation (L522P) would significantly misfold the transporter.

The R510H and R881C mutants are also misfolded causing ER retention. Although R881C was partially expressed on the plasma membrane in the Xenopus oocytes and had significant transport activity it was fully retained intracellularly in Madin-Darby canine kidney cells and HEK cells. Arg ⁵¹⁰ and Arg ⁸⁸¹ are localized to TM 4 and 12, respectively, and do not reside in surface or re-entrant loops as was previously thought. Misfolding of NBCe1-A caused by these two mutations suggests that the arginine at positions 510 and 881 is involved in forming ionic interactions within the TMs to maintain the overall folding of the protein.

Potential Mutant–Specific Therapy in Proximal Renal Tubular Acidosis

In one of the nonsense mutations of NBCe1-A causing proximal RTA, a wt-CAG sequence encoding glutamine has been replaced by a UAG stop codon sequence resulting in premature truncation. Deciphering the rules dictating read-through efficiency is of primary importance for pharmacological treatment of renal tubular acidosis resulting from mutations causing premature stop codons (PSC). Aminoglycosides offer a potential therapeutic approach to treat PSC mutations by inducing ribosomal read-through. Aminoglycosides bind to the internal loop of helix 44 of the 16S ribosomal RNA, a region termed the decoding site. In both prokaryotes and eukaryotes, aminoglycosides induce miscoding by mimicking the conformation change in 16S rRNA that would be induced by a correct codon–anticodon pair, thereby compromising the integrity of codon–anticodon proofreading during translation. As a general rule, glutamine is inserted at nonsense UAG or UAA read-through sites, whereas UGA sites miscode to tryptophan.

Recent studies have shown that the NBCe1-A-Q29X mutation can be rescued in vitro by treatment with aminoglycoside antibiotics. These findings represent the first evidence that in the presence of the NBCe1-A-Q29X mutation that causes proximal renal tubular acidosis, full-length functional NBCe1-A protein can be produced. In particular these studies offer the opportunity to treat the eye phenotype locally without systemic toxicity. Recently the aminoglycoside derivative NB54 was designed which has significantly less toxicity, with stop-codon read-through potency that is significantly greater than gentamicin likely due to the presence of the flexible N-1-AHB group ( S) -2-hydroxy-4-aminobutyl group at the N-1 position). In addition non-aminoglycoside compounds such PTC124 may prove efficacious.

Additional Lessons Learned from Mice with Targeted Disruption of SLC4A4

Mice with disruption of the Slc4a4 gene (NBCe1 ^−/− mice) have a more severe (and fatal) phenotype than patients. In addition to severe hyperchloremic metabolic acidosis, they have severe volume depletion, hyponatremia, splenomegaly, intestinal obstructions, and die before weaning. In addition, NBCe1 ^−/− mice also have abnormal dentition as do patients. Paine et al. showed that enamel producing ameloblast cells express AE2a apically and NBCe1-B basolaterally. Based on this data, they hypothesized that ameloblasts mediate regulated transcellular bicarbonate secretion during the various phases of enamel formation. Results in NBCe1 ^−/− mice demonstrated that normal enamel development in mammals requires the NBCe1-B variant.

The physiologic importance of NBCe1 in mediating intestinal bicarbonate secretion is exemplified by the intestinal obstruction phenotype in NBCe1 ^−/− mice. NBCe1-B is widely expressed throughout the gastrointestinal tract. In the possum ileum, the transporter is predominantly expressed in the mid region of the villi, with lower levels of expression in the crypts, whereas in the murine colon its expression is higher in crypts than in surface cells. Although NBCn1 (SLC4A7) is also thought to function as a basolateral bicarbonate uptake mechanism in the intestine, NBCe1 is expressed at significantly higher levels than NBCn1, and in addition mice with targeted disruption of SLC4A7 don’t have an obvious intestinal phenotype.

Gain of Function

No gain of function mutations have been thus far described.

Systemic Factors Affecting NBCe-1 Function in the Kidney

Mechanism of enhanced bicarbonate excretion in Na ⁺ loading . In rats NaCl and NaHCO ₃ loading reduces the expression of NHE3 and NBCe1 in the proximal tubule potentially contributing to prevention of volume overload and metabolic alkalosis (during bicarbonate loads).

Metabolic alkalosis-potassium depletion: enhanced bicarbonate reclamation : Potassium depletion results in an increase in renal bicarbonate reabsorption proximally and distally. Amlal et al. suggested that the upregulation of NBCe1 in the proximal tubule could contribute to the maintenance of metabolic alkalosis. Of note although NBCe1 is normally not expressed in the mTAL and IMCD, the authors reported an increase in the expression of NBCe1 in these nephron segments.

Sympathetic nervous system: norepinephrine : Sonalker reported that long-term (15 days) infusions of noradrenaline (600 ng/min) in rats led to an ~2.5 fold increase in the expression of NBCe1 on Western blotting of rat cortex. The underlying mechanism was not characterized but may have relevance to proximal tubule handling of sodium during alterations in sympathetic outflow to the kidney.

Dopamine : Dopamine decreased the activity of NBCe1 in rabbit and rat proximal tubules but not in spontaneously hypertensive SHR rats. Interestingly, dopamine inhibited the activity of NBCe1 when the cotransporter operated with the 3 ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ :1 Na ⁺ stoichiometry. In SHR a defect of DA1 receptor signaling could contribute to the development of hypertension.

PTH : Functional activity of NBCe1 is decreased by PTH in rat tubules in vivo . In rabbit tubules perfused in vivo under normal conditions, PTH has no effect. In rat tubules pre-incubated in DMEM (Delbecco’s modified Eagles medium)+norepinephrine, PTH inhibits the cotransporter possibly via a cAMP dependent mechanism.

Hypertension : In the SHR rat with spontaneous hypertension, Sonalker reported that NBCe1 protein expression was increased ~2 fold in comparison to control WKY rats. However in a separate study, in immortalized proximal tubule SHR cells, NBCe1 activity was reported to be less than normal.

Renal transplant rejection : In a rat model of acute renal transplant rejection, Velic reported down-regulation of NHE-3 but up-regulation of NBCe1-A. These findings may account for changes in proximal tubule bicarbonate transport following renal transplantation.

FK506 : The calcineurin inhibitor FK506 has been reported to cause pRTA. Chronic metabolic acidosis per se has no effect on NBCe1-A expression in the kidney. In rats FK506 administration was found to decrease the protein abundance of NBCe1-A in the cortex without a change in acid–base status.

Lithium Toxicity : Chronic lithium toxicity is associated with histologic changes in the kidney and distal renal tubular acidosis. In rats, lithium administration leads to an increase in NBCe1 expression perhaps as a compensatory mechanism.

Ureteral Obstruction : Chronic ureteral obstruction is associated with chronic renal failure and a hyperkalemic distal renal tubular acidosis. In neonatal rats chronic ureteral obstruction resulted in a significant decrease in NBCe1-A expression in the proximal tubule.

Extrarenal Tissues: Role of NBCe-1-B/C in Various Organs and Selective Disease States

Cardiac physiology, ischemia, and reperfusion injury : Several studies have discussed the importance of electrogenic NBC transport in ventricular electrophysiology and function. The data in the literature is sometimes difficult to interpret because NBCe1, NBCe2, and NBCn1 are expressed in the heart and the molecular basis for a given functional effect is not often clearly established. Villa-Abrille showed electrogenic ${Na}^{+} - {HCO}_{3}^{-}$ ${Na}^{+} - {HCO}_{3}^{-}$ cotransport modulates resting membrane potential and action potential duration in cat ventricular myocytes. Yamamoto et al. emphasized the functional diversity of electrogenic NBC activity in various species. Schafer et al. demonstrated that following reoxygenation of rat myocytes initially exposed to anoxia, sodium bicarbonate cotransport is responsible for ~50% of the pH _i recovery. DIDS (an NBCe1 and NBCe2 inhibitor) blocked calcium oscillations that cause hypercontracture of the cells. Khandouri et al. have reported an increase in NBCe1-B in myocardium from humans with heart failure. In addition, NBCe1 mRNA and protein was reported to be increased in the rat heart following myocardial infarction. An antibody against NBCe1 significantly improved the post-ischaemic cardiac function. Verdonck hypothesized that the NBCe1-B transporter might contribute to the incidence of arrhythmias in heart failure.

Cystic Fibrosis : NBCe1-B is expressed on the basolateral membrane of pancreatic duct cells in humans. In cystic fibrosis pancreatic bicarbonate secretion is decreased and it has been hypothesized that an alteration in basolateral membrane voltage (resulting from defective CFTR transport), impairs NBCe1-B basolateral bicarbonate uptake. IRBIT has been recently shown to be a key coordinator of epithelial fluid and ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ secretion in murine intralobular pancreatic ducts via independent activation of NBCe1-B and CFTR. Interestingly, NBCe1 knockout mice and patients with NBCe1 loss of function mutations do not have a cystic fibrosis phenotype although evidence for pancreatic involvement (elevated amylase or lipase) has been reported in patients.

Duodenal ulcers : NBCe1-B is expressed in the duodenum, and contributes to transepithelial HCO ₃ secretion and potential duodenal cell pHi regulation. Duodenal bicarbonate secretion is thought to protect the duodenal mucosa from acid-related injury. However patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, paradoxically don’t develop duodenal ulcers. Studies in rats support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from ulcer formation.

CNS . Upregulation of NBCe1 (NBCe1-B/C) mediated HCO ₃ transport in specific neurons of the brain may prevent excessive extracellular acidosis and inhibit neuronal activity as part of a negative feedback mechanism. In seizure-prone gerbils, at 30 minutes postictally, NBCe1 immunoreactivity is substantially elevated in the hippocampus (CA1-, -2, and -3 regions). By three hours, elevated NBCe1 staining is also detectable in the dentate gyrus (and the granule layer).

Molecular Regulation of NBCe1 Function and Membrane Expression

Cytosolic N- and C-terminus : Truncation mutant studies of heterologously expressed NBCe1 variants in oocytes have shown that unique amino terminus of NBCe1-A stimulates transporter activity (autostimulatory domain (ASD)), whereas the unique amino terminus of the B and C variants inhibits activity. The N-terminus in NBCe1-B and NBCe1-C has accordingly been termed an autoinhibitory domain (AID). The mechanism involved is currently unknown but likely involves interaction(s) between the specific amino terminus and the transport pathway and/or binding to cytosolic factors. In contrast, the carboxy termini affect the plasma membrane expression without affecting cotransporter function.

In mammalian cells however, Espiru et al. reported that truncation of the N- or C- termini failed to prevent targeting to the plasma membrane (HEK293 cells) or to the basolateral membrane of OK cells. These results differ from Li et al. who showed mistargeting of the C-terminal mutant to the apical membrane with residual basolateral expression in MDCK cells and identified a QQPFLS carboxyl-terminal motif as a basolateral targeting sequence. Horita et al. reported that C-terminal truncation resulted in intracellular NBCe1 localization. Length of truncation, duration of heterologous expression, and cell type may account for these differences. Moreover single point mutation analysis may provide a more informative approach to deciphering the residues that are required for proper folding of the transporter and escape from the ER.

PKA-dependent phosphorylation : Given the prevailing Na ⁺ , ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ and basolateral membrance voltage in proximal tubule cells, the Nerst equation predicts that for NBCe1-A to mediate the flux of sodium bicarbonate from cell to blood, the ion transport stoichiometry of NBCe1-A is 1 Na ⁺ :3 ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ . Interestingly, the ratio is 1:2 in certain heterologous expression systems such as HEK293 cells and oocytes. Studies in a mouse proximal and distal tubule cells lines have reported that the stoichiometry of both NBCe1-A and NBCe1-B is cell-type dependent and variable suggesting that unknown cytosolic factors in the proximal tubule interact with NBCe1-A (and NBCe1-B) and modulate the stoichiometry. In a proximal tubule cell line, the shift in NBCe1-A stoichiometry from 1:3 to 1:2 is mediated by protein kinase A-dependent phosphorylation of Ser ⁹⁸² . Thr in the unique amino-terminus of NBCe1-B was found to play an important role in modulating the cAMP-induced increase in cotransporter current without altering its stoichiometry. Accordingly, cAMP has also been found to increase intestinal NBCe1-B mediated transport in part via a change in membrane expression.

PKC-dependent phosphorylation : ANG II has a biphasic effect on NBCe1 transport in both oocytes and renal proximal tubule cells. Studies in oocytes have reported that the ANG II-induced inhibition of NBCe1-A is mediated by the Ca ²⁺ -insensitive PKCε isoform that increases its association with NBCe1-A in the membrane, and AII-induced Ca ²⁺ mobilization may trigger decreased NBCe1-A surface expression via the AT _1B receptor. In addition to affecting cell membrane expression, intracellular calcium modulates the stoichiometry of the cotransporter. Specifically, in oocytes expressing NBCe1-A it has been reported that an increase in cytosolic Ca ²⁺ shifts the transport stoichiometry from 1:2 to 1:3, possibly via a phosphorylation (PKC) event. Data in a rat parotid cell line suggest that NBCe1-A and NBCe1-B participate in constitutive and stimulated (carbachol) endocytosis regulated by conventional PKCs (PKCαβγ) and by a novel PKCδ. This data may only have relevance in secretory epithelia involving NBCe1-B transport such as salivary duct, ileum, and colon.

Phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) : PIP ₂ is noteworthy because in addition to its role as a precursor of the Ca ²⁺ -mobilizing inositol triphosphate (IP ₃ ) and the kinase-activating diacylglycerol (DAG), PIP ₂ can regulate solute movement. NBCe1 and other members of the SLC4 family have a stretch of C-terminal lysines that may interact electrostically with PIP ₂ . In voltage-clamped oocytes heterologously expressing NBCe1 (-A, -B, and -C) variants, PIP ₂ increased both the B and C variant currents. It has been hypothesized that the NBCe1-A may not respond to exogenous PIP ₂ because it may have a higher affinity for endogenous PIP ₂ compared to the B and C variants or that a putative autoinhibitory domain in the B and C variants might be masked by PIP ₂ .

Intracellular Mg ²⁺ : When heterologously expressed in oocytes, a phenomenon of NBCe1-A-rundown occurs. The majority of NBCe1-A rundown is likely due to Mg ²⁺ -dependent phosphatase (5′-lipid phosphatase) activity because the rundown is largely inhibited by removing bath Mg ²⁺ , both in the presence or absence of vanadate and F ⁻ . It has been suggested that the 5′-lipid phosphatase dephosphorylates PIP ₂ to PIP. While removing bath Mg ²⁺ inhibits NBCe1-A rundown, raising bath Mg ²⁺ appears to inhibit NBCe1-A directly. Yamaguchi and Ishikawa reported that intracellular Mg ²⁺ inhibits NBCe1-B expressed in mammalian HEK 293 and NIH3T3 cells. An N-terminal deletion mutant was less sensitive to Mg ²⁺ compared to wild-type NBCe1-B, consistent with the view that the inhibition may be associated, directly or indirectly, with the N-terminal region. Inhibition of NBCe1-mediated transport via an increase in cytosolic Mg ²⁺ during ischemia may reduce post-ischemic dysfunction in kidney (NBCe1-A) and heart (NBCe1-B) possibly by keeping the intracellular Na ⁺ concentration lower and thereby preventing Ca ²⁺ overload.

IRBIT : The NBC1-B variant is activated by IRBIT whereas the NBCe1-A is not. IRBIT binds to the N-terminal 1–42 residues of NBCe1-B that are not present in NBCe1-A. Phosphorylation of the PEST (Pro-Glu-Ser-Thr) domain is required for IRBIT binding and activation. Regulation of NBCe1-B by IRBIT suggests that IRBIT may regulate fluid and ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ secretion in secretory tissues such as the pancreatic duct.

ATP : In oocytes expressing NBCe1-A, when ATP is applied to the intracellular surface of oocyte membrane patches with low transport activity, the cotransporter current increased. This activation is not due to active ATP-driven transport. The non-hydrolysable ATP analog AMP-PNP does not mimic the effect of ATP. It has been hypothesized that ATP may phosphorylate NBCe1-A by means of an unidentified protein kinase.

Carbonic anhydrase II (CAII) : It has been proposed the CAII interacts with an NBCe1 C-terminal a D ⁹⁸⁶ NDD ⁹⁸⁹ motif forming a transport metabolon that transfers bicarbonate intra-molecularly between the two proteins. This hypothesis is of interest given the known expression of CAII in the cytosol of proximal tubule cells. In addition, direct interaction between NBCe1 and membrane bound CAIV has been reported. The data in the literature is both in favor and against the existence of a “transport metabolon;” in part due to difference in techniques employed and the sensitivity/specificity of the measurements utilized. Recently, the question was readdressed using the substituted cysteine accessibility method which showed that the C-terminal tails of the NBCe1-A dimer are highly structured and may sterically prevent CA II binding. Against the quantitative importance of a transport metabolon with CAII is an experiment of nature wherein patients and mice with loss of proximal tubule CAII function do not have as severe proximal renal tubular acidosis as might be predicted; or in comparison to patients with NBCe1 mutations. Moreover, neither an alteration in either renal bicarbonate handling or hypobicarbonatemia has thus far been reported in patients or mice with loss of CA IV function.

NBCe2: (SLC4A5 Gene)

Structural Variants and Tissue Distribution

NBCe2 (or NBC4) is an electrogenic sodium bicarbonate cotransporter encoded by SLC4A5. Interestingly, NBCe2 and p150 ^Glued were originally reported to be encoded by the same locus, DCTN1-SLC4A5, that spans ~230 kilobases on chromosome 2p13 and contains 66 exons. p150 ^Glued is a component of the dynactin heteromultimeric complex of proteins which is required for dynein-mediated vesicle and organelle transport by microtubules. In eukaryotes, it is rare for a single gene to encode two functionally unrelated proteins. More recent studies suggest that SLC4A5 has its own promoter. Six variants have been reported; 4 that are valid splice variants from full-length transcripts (NBCe2-A, -B, -C, and -D; ) and 2 additional variants that are invalid given they are not found in the SLC4A5 genomic sequence. The most widely expressed variant is NBCe2-C (also called NBC4-C) consists of 1121 aa and is ~145 kDa.

NBCe2 transcripts are expressed in several tissues including brain (human cerebellum, cerebrum, hippocampus, and choroid plexus), heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. At the protein level, NBCe2 has been immunolocalized to the apical membrane of uroepithelial cells and unspecified collecting duct cells in rats. In humans, NBCe2 is expressed on the apical membrane of a subset of collecting duct cells in the renal outer medulla. In extra-renal tissues the transporter is expressed in the basolateral membrane of hepatocytes, the apical membranes of cholangiocytes, apical membrane of rat choroid plexus epithelial cells (not detectable in human choroid plexus ), skeletal muscle, and the basolateral membrane of polarized Calu-cells (suggesting the transporter might be expressed in airway submucosal glands).

Functional Properties

When expressed in mammalian cells NBCe2-C elicits, a Na ⁺ -driven, DIDS-sensitive- bicarbonate flux in the absence of chloride. Similar findings were subsequently reported in oocytes expressing the transporter. NBCe2-C is electrogenic and mediates a hyperpolarization when cells are exposed to bicarbonate and a depolarization when Na ⁺ is removed. These responses are analogous to those induced by NBCe1. Like NBCe1, NBCe2 appears to function with a ${Na}^{+} {: HCO}_{3}^{-}$ ${Na}^{+} {: HCO}_{3}^{-}$ stoichiometry of 1:2 or 1:3. In mouse choroid plexus epithelial cells, Millar et al. measured a small DIDS-sensitive, Na ⁺ -dependent, ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ -induced outward current. Based on the reversal potential obtained from I-V plots, and the known localization of NBCe2 to the apical membrane of choroid epithelial cells, it was concluded the measured function was mediated by NBCe2 functioning with a 1:3 ${Na}^{+} {: HCO}_{3}^{-}$ ${Na}^{+} {: HCO}_{3}^{-}$ stoichiometry. Given that the membrane potential of the choroid plexus epithelial cells is −35 mV to −60 mV, NBCe2 would mediate bicarbonate secretion. In a study addressing the potential importance in apical choroid plexus bicarbonate transport, in a mouse model where basolateral bicarbonate influx mediated by NCBE/NBCn2 (see below) was disrupted, apical NHE activity but not NBCe2 was up-regulated. Moreover, DIDS-sensitive ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ recovery (presumably assaying NBCe2) in these mice did not function in an inward transport mode. Thus far in native kidney tubules, there are no studies which have addressed the functional contribution of NBCe2 to either transcellular bicarbonate transport and/or intracellular pH regulation.

Hypertension

The Family Blood Pressure Program in 2002 found an association between the SLC4A5 gene and hypertension. In African Americans 5 SNPs in the SLC4A5 gene showed significant association with pulse pressure. In Utah pedigrees, at baseline and after a 10-year follow-up period, the association of SLC4A5 polymorphisms and hypertension was subsequently reported. A meta-analysis of three studies (HyperGEN, HERITAGE, and Family Study and Genetics of Hypertension in Blacks) was done to determine LOD scores for systolic and diastolic blood pressure in African-American and Nigerian families, and linkage evidence showed an association with 2p14–p13.1 where SLC4A5 is located. In another meta-analysis, the region flanking the SLC4A5 locus (2p12–q22) was also suggested to be important in whites for blood pressure variation. Finally, SLC4A5 has been proposed as a promising candidate gene for variations in the blood pressure response to drugs that inhibit the renin-angiotensin system. In a recent analysis, the observed association with resting and submaximal-exercise cardiovascular and metabolic traits in the HERITAGE Family Study were not found to be due variation in the promoter or coding SNPs of the SLC4A5 gene. Further studies are therefore required to clarify the role of NBCe2 in human hypertension.

Targeted Disruption in the Mouse

Targeted disruption of the Slc4a5 gene results in significant abnormalities in intracerebral ventricle volume, intracranial pressure, and CSF electrolyte levels. In the retina, there is loss of photoreceptors, ganglion cells, and retinal detachment. In a separate study, mice with loss of NBCe2 were shown to have increased blood pressure, a compensated metabolic acidosis and hyporeninemic hypoaldosteronism, elevated fluid intake and urine excretion and an increased glomerular filtration rate. It was suggested that the loss of sodium bicarbonate reabsorption by SLC4A5 initiates compensatory bicarbonate reabsorption via other sodium-bicarbonate transporters.

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