Characteristics of Mammalian Rh Glycoproteins (SLC42 transporters) and Their Role in Acid-Base Transport

Abstract

The mammalian Rh glycoproteins belong to the solute transporter family SLC42 and include RhAG, present in red blood cells, and two non-erythroid members RhBG and RhCG that are expressed in various tissues, including kidney, liver, skin and the GI tract. The Rh proteins in the red blood cell form an “Rh complex” made up of one D-subunit, one CE-subunit and two RhAG subunits. The Rh complex has a well-known antigenic effect but also contributes to the stability of the red cell membrane. RhBG and RhCG are related to the NH₄⁺ transporters of the yeast and bacteria but their exact function is yet to be determined. This review describes the expression and molecular properties of these membrane proteins and their potential role as NH₃/NH₄⁺ and CO₂ transporters. The likelihood that these proteins transport gases such as CO₂ or NH₃ is novel and significant. The review also describes the physiological importance of these proteins and their relevance to human disease.

1. Overview

Rh glycoproteins

Rh glycoproteins, belonging to solute transporter family SLC42, have long been identified and studied in human blood cells for their immunogenic characteristics and importance in pregnancy (Avent and Reid, 2000). Table 1 summarizes some of the known properties of these membrane proteins. In red blood cells, the Rh antigens exist as a hetero oligomeric “Rh complex” of membrane polypeptides that include one D subunit, one CE subunit and two glycosylated RhAG (SLC42A1) subunits each with 12 transmembrane domains TM (Avent et al., 1996; Conroy et al., 2005; Ridgwell et al., 1992). RhAG has been expressed in heterologous systems (e.g. oocytes) independently of RhCE or D subunits. In addition to its antigenic property, the Rh complex is thought to contribute to the membrane stability and structure of red blood cell. Its exact function has not been determined.

Table 1.

SLC42 - The Human Rh Ammonium Transporter Family

Human Gene Symbol	SLC Symbol	Protein Name	ORF (aa)	Predominent Substrates	Transport type Coupling ions	Tissue distribution & cellular/subcellular expression	Link to disease	Human gene locus	Sequence Accession ID	Splice Variants and their features
RHAG (Rh50A)	SLC42A1	RhAG	409	NH4+, NH3+	H+	Red blood cells, (cell membrane)	Rhnull-regulator Rhmod, OHSt*	6p12.3	AF031548
RHBG	SLC42A2	RhBG	458	NH4+, NH3, Methyl amine Methyl ammonium	Electrogenic No coupled ions	Kidney, Liver, skin, GI tract sweat glands, ovaries (basolateral membrane)	Unknown	1q21.3	AF193807	5 isoforms: Q9H310-1 Q9H310-2 Q9H310-3 Q9H310-4 Q9H310-5 (No experimental confirmation)
RHCG (RhGK)	SLC42A3	RhCG	479	NH4+, NH3	Electroneutral possibly H+	Kidney (apical membrane) Brain, Testis Placents, Pancreas, Prostate	Renal distal tubular acidosis (?)	15q25	AF193809	Unknown

^*OHSt: Dominant over-hydrated hereditary stomatocytosis

RhCG

Marini and co-workers made the original observation that human Rh antigens share sequence homology to the MEP NH₄⁺ transporters of the yeast (Marini et al., 1997). A subsequent study, (Marini et al., 2000) identified a kidney homologue originally named RhGK (for Rh glycoprotein kidney) and later RhCG (SLC42A3). The RhCG protein shows close identity to RhAG (50%) and sequence homology (24%) with the MEP/Amt ammonium transporters. RHCG cDNA was amplified from human kidney RNA and successfully expressed in yeast cells (Marini et al., 2000). Liu et al., (Liu et al., 2000) subsequently cloned human (RHCG) and mouse (Rhcg) homologues with cDNAs sequences of 1952 and 2097 bp respectively and overall identity of 68.8%. At the protein level, RHCG encodes a 53 KDa, 479 amino acid polypeptide, whereas Rhcg encodes a 55 KDa, 498 amino acid polypeptide and they are highly conserved (77.2% identity and 90.4% similarity). Hydropathy analysis indicated that they share identical topology of 12 transmembrane domains with intracellular N and C termini (Fig 1). Their 12 TM structure has conserved regions shared with other Rh homologues, especially RhAG, but differ from RhAG with a much-elongated C – terminal and a unique N – terminal. RHCG was mapped to the 15q25 chromosome whereas Rhcg was mapped by linkage analysis to a locus (D7Xrf229) on the long arm of chromosome 7.

Figure 1. A model of the membrane topology of Mus musculus RhCG.

Solid green circles denote the hydrophobic amino acids Phe, Ile, Leu, Met, Val and Trp; orange circles, Gly, Ala and Pro; light yellow circles, polar residues Ser, Cys, Thr, Asn, Gln and Tyr; cicrles marked with + denote the positively charged Lys, Arg and His; and −, negatively charged Asp and Glu. The probability of the location of the transmembrane sequences was predicted by the N-best algorithm (Krogh et al., 2001). The N-glycosylation site (⁴⁸NIS⁵⁰), present in the first e4xoloop, is illustrated.

RhBG

Cloning and biochemical characteristics of Rh type B glycoprotein (SLC42A2) were initially accomplished by Liu et al., (Liu et al., 2001). RHBG and Rhbg have open reading frames of 1377 and 1368 bp respectively, which encode polypeptides of 458 and 455 amino acids respectively. Human and mouse RhBG are 85% identical and 94% similar at the protein level, less similar to RhCG (58% - 53%) and notably different from RhAG (43%). Both proteins have a molecular mass of 49.3 KDa, are negatively charged at physiological pH and have a single N-glycosylation motif. Like RhCG and RhAG, RhBG is a polytopic protein with 12 predicted trans-membrane domains. RHBG resides at 1q21.3 of human chromosome 1 and Rhbg is on mouse chromosome 3 on a site where many markers are similar to those of human 1q21 containing RHBG. The Rh proteins are closely related to the ammonia transporters family (Amt) and seem to have branched off from Amt ancestors in prokaryotes (Huang and Ye, 2010). The two families show different patterns of distribution but they also coexist in a variety of organisms indicating a divergent and independent evolution. Even in organisms that have Amt and Rh proteins their genes cluster independently suggesting a functional distinction between the two families.

Transport of NH₃/NH₄⁺

The original link between Rh proteins and NH₃/NH₄⁺ transport was provided by studies (Marini et al., 2000) on yeast mutants, termed (Triple-MEPΔ), rendered incapable of NH₄⁺ transport by deletions of 3 endogenous NH₄⁺-transporter genes. Yeast, like many other fungal species as well as bacteria, efficiently scavenge NH₄⁺ which is required as a nitrogen source, without which growth is greatly hampered. Expressing RhAG enabled the Triple-MEPΔ cells to grow in low NH₄⁺ medium and resulted in resistance to toxic concentrations of methyl ammonium suggesting a role in NH₄⁺ transport. Although the evidence for NH₄⁺ transport by Rh proteins is still actively debated, it is intriguing that this class of membrane proteins may be the elusive NH₄⁺ transporter. This is very important in tissues, such as the kidney where transport of NH₃/NH₄⁺ is critical for regulation of acid-base balance (Heitman and Agre, 2000).

In mammals, normal acid-base homeostasis is critically dependent on renal excretion of NH₄⁺ in the urine (Knepper et al., 1989). Renal ammoniagenesis and NH₄⁺ transport are highly regulated and NH₄⁺ excretion increases several fold during chronic acidosis (Knepper et al., 1991). In physiological fluids (pH 7.4 – 7.5), NH₃/NH₄⁺ exists predominantly as NH₄⁺ (~99%) because the pKa of the equilibrium reaction NH₃+H⁺ ↔ NH₄⁺ is high (~9.2). Whereas NH₃, a small neutral molecule, is assumed to mainly diffuse through the cell membrane, NH₄⁺ has to be transported by membrane transporters or channels (Attmane-Elakeb et al., 2001; Good et al., 1984; Knepper et al., 1989). Some K⁺ transport pathways may serve as NH₄⁺ carriers because of the similarity of the two ions in size and charge.

The renal proximal tubule generates NH₃/NH₄⁺ which is then secreted predominantly into the luminal fluid (Hamm and Simon, 1990). This occurs by both NH₃ diffusion into the more acid luminal fluid and by NH₄⁺ transport in exchange for sodium on the Na⁺- H⁺ exchanger (Knepper et al., 1989). In the loop of Henle, total ammonia may be secreted into the descending limb of Henle but is then reabsorbed in the thick ascending limb (Good and Knepper, 1985; Knepper et al., 1989). NH₄⁺ is reabsorbed from the lumen into the cell via substitution for K⁺ on both the Na-K-2Cl co-transporter and the apical membrane potassium channel (see (Attmane-Elakeb et al., 2001; Good et al., 1984). In the collecting duct, total ammonia is secreted along the length of the tubule. Most, if not all, of this secretion has been thought to occur by non-ionic diffusion of NH₃ driven by the progressively increasing concentrations of ammonia in the medullary interstitium (Hamm, 1986). Concurrent acid secretion along the length of the collecting duct (by H-ATPase for example) keeps luminal concentrations of NH₃ low, maintaining a favorable NH₃ gradient from interstitium to lumen. The apical membrane of collecting duct cells is one site where rate-limiting rapid NH₃ diffusion has to occur. The basolateral membrane, on the other hand, is the site where NH₄⁺ transport occurs presumably by one or more membrane transporters (Hamm et al., 1985; Knepper et al., 1989).

2. Expression of Rh Glycoproteins

In red blood cells, RhAG is expressed in the membrane as a component of the “Rh complex”, as mentioned above. The “Rh Complex” consists of RhAG in association with the nonglycosylated Rh proteins RhD and RhCE in humans or with Rh30 in non-human mammals. RhAG is an erythrocyte-specific protein not found in other tissues (Cartron, 1999; Liu and Huang, 1999). Expression in heterologous systems showed that RhAG was fully glycosylated and properly trafficked to the plasma membranes of oocytes (Westhoff et al., 2002), yeast cells (Marini et al., 2000) and HeLa cells (Benjelloun et al., 2005). Cell surface expression of the “Rh complex” was shown to be linked to RhAG interaction with spectrin-based ankyrin (Nicolas et al., 2006). It was also implied that in red blood cells (RBC) Rh protein, Band-3 and ankyrin form an integral membrane complex that may modulate transport of HCO₃⁻ and possibly NH₄⁺ (Nicolas et al., 2006).

Expression of the RHCG and Rhcg genes has been assessed by Northern blot analysis, in situ hybridization and immunohistochemistry (Eladari et al., 2002; Liu et al., 2000; Verlander et al., 2003). These studies indicated that in human adult tissues RhCG was abundantly expressed in the kidneys, brain, testis, placenta, skeletal muscle, liver, GI tract, pancreas, and prostate (Handlogten et al., 2005; Liu et al., 2000; Marini et al., 2000; Weiner et al., 2003). In human fetal tissues, RhCG was expressed only in the kidney. In mouse adult tissues, RhCG was expressed in kidney, testis, liver and other tissues. Using RT – PCR on rat micro dissected tubules, RhCG was found in distal convoluted tubules, the connecting segment (CNT), cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) but not the proximal tubule and the thick ascending limb of Henle’s loop. Immuno-localization studies showed labeling of the apical membrane of cells within the cortex, outer medulla and upper portion of inner medulla. RhCG was present in all CCD cells and the intercalated cells of OMCD and IMCD (inner medullary collecting duct). Another study, however, indicated that RhCG is restricted to the intercalated cells of the CCD cells (Eladari et al., 2002). RhCG was also present in the principal cells of the outer stripe of OMCD. The subcellular distribution of RhCG seemed to differ depending on tissue and species. In the mouse kidney, RhCG was reported to be at the apical membrane (Eladari et al., 2002; Verlander et al., 2003) whereas in the rat RhCG was reported to be either exclusively apical (Eladari et al., 2002) or at both apical and basolateral membranes (Seshadri et al., 2006). In the human kidney, RhCG semed to be restricted to the apical membrane (Eladari et al., 2002; Verlander et al., 2003) but another study reported basolateral expression as well (Han et al., 2006).

RhBG expression was confirmed in non-erythroid tissues only. RhBG was expressed in kidney, skin, sweat glands, GI tract (from duodenum to the colon), liver and, to a lesser extent, ovaries. Using immunohistochemistry in mouse kidney, Verlander et al., (Verlander et al., 2003), localized RhBG to the majority of cells of the connecting segment (CNT) and the cortical collecting duct (CCD). In the OMCD and IMCD, only a subpopulation of cells was labeled. Co-localization studies with carbonic anhydrase II, Na-Cl co-transporter and the anion-exchanger (AE1) demonstrated that RhBG was expressed in all CNT cells and in principal cells of CCD. In CCD, RhBG was also labeled in A-type intercalated cells but not B-type intercalated cells. In OMCD and IMCD only intercalated cells exhibited immunoreactivity to RhBG (Quentin et al., 2003; Verlander et al., 2003). It was demonstrated by double immunostaining that RhCG and RhBG were expressed in the same cell but with distinct apical and basolateral localizations, suggesting different transport properties of the two homologous membrane proteins. In a study on mouse liver, RhBG was localized to the basolateral membrane of hepatocytes surrounding central veins (perivenous) but not in periportal or midzonal hepatocytes (Weiner et al., 2003). RhBG expression has been found to be exclusively restricted to the basolateral membrane of CCD cells (Weiner et al., 2003).

3. Molecular Structure of Rh glycoproteins

An important advancement in the field has been the crystallographic structure of AmtB, the bacterial homologue of Rh (Khademi et al., 2004; Li et al., 2006). The high resolution crystals indicate a trimeric complex of subunits made up of 11 TM helices, each forming a central channel where substrate transport presumably occurs. The crystals showed no conformational changes in the presence or absence of NH₄Cl. The proposed tertiary structure indicates that the individual pore of each monomer is composed of an extracellular vestibule followed by a narrow (1.2 A°) and long pore (20 A°) which is terminated at the cytoplasmic side by a wider vestibule. The upper vestibule is lined by aromatic residues that recruit NH₄⁺. The central hydrophobic pore has 2 conserved His residues that promote hydrophobic conduction of substrate. In the absence of resolved structure of RhBG or RhCG the AmtB crystal structure serves as the closest and best model for the Rh protein.

Based on analysis of the 3-dimensional structure of AmtB, a novel transport model was proposed (Fig 2). In AmtB, the upper vestibule recruits NH₄⁺ by a cation - π interactions provided by the residues Trp 148 and Ser 219. As NH₄⁺ moves toward the hydrophobic pore, it gives up its proton predominately on the side of entry (extracellularly) and NH₃ is transported through the pore. NH₃ in the pore is stabilized by two highly conserved histidines (H168 and H318), which interact with NH₃ at 3 specific locations in the pore. On the cytoplasmic side, NH₃ acquires an intracellular H⁺ to re-equilibrate with NH₄⁺.

Figure 2. Proposed architecture of the ammonium pore of RhBG based on the crystal structure of AmtB.

The model shows the equivalent amino acid residues important for transport of NH₃/NH₄⁺ based on alignment of RhBG and AmtB sequences.

The degress to which the tertiary structure of Amt B resembles that of Rh glycoproteins remains an open question. Constructing homology models for Rh proteins based on AmtB structure must take into account critical differences in sequences that render proper alignment tricky. Among these differences are putative 12 TM domains in Rh proteins compared to 11 TM domains in Amt B; a long intracellulara C-Terminal α-helix in Rh proteins; and altered critical amino acid residues at sites that that are thought to be key for transport function (Huang and Ye; Lupo et al., 2007). Nevertheless, in the absence of resolved crystal structures or RhCG or RhBG, the structure of AmtB is one of the best models to determine structural determinants of function. Initial studies based on this approach point to important characteristics of function including: a role of C-terminal in gating function and potential binding partners to Rh proteins among cellular proteins (e.g. carbonic anhydrase) and other membrane proteins.

4. Function of Rh Glycoproteins

The role of Rh proteins as NH₄⁺ transporters

Most studies provide strong evidence for a role of the Rh proteins in NH₃/NH₄⁺ transport. The initial studies to characterize the functions of RhAG were done in yeast mutant cells deficient in NH₄⁺ transporter genes (Triple mepΔ). Triple mepΔ cells expressing RhAG were able to grow in media containing very low concentrations of NH₄⁺ (Marini et al., 2000). Another study reported that RhAG expression in yeast cells resulted in resistance to toxic concentrations of methyl ammonium (200mM). Also the rate of extracellular NH₄⁺ accumulation was higher in cells expressing RhAG (or RhGK) suggesting that RhAG promotes efflux of NH₄⁺. Measurement of methyl-amine uptake in oocytes expressing RhAG suggested that RhAG serves as an electro neutral counter-transporter of methyl amine (and NH₄⁺) coupled to H⁺ exit from the cell (Westhoff et al., 2002). As such RhAG would result in transport of net NH₃ equivalents. At least one study on HeLa cells suggested that RhAG transports NH₃ and NH₄⁺ (Benjelloun et al., 2005). However, RhAG is an erythrocyte membrane protein and its contribution to NH₄⁺ transport in other tissues is unlikely.

Several studies on RhBG expressed in oocytes or mammalian cells report that it can transport NH₃/NH₄⁺. However different results are reported by different labs. Some studies report that NH₄⁺ transport is electroneutral and coupled to the H⁺ gradient and behaving as a NH₄⁺- H⁺ exchanger (Ludewig, 2004; Mak et al., 2006; Zidi-Yahiaoui et al., 2005). Recent studies indicate that RhBG mediates electrogenic NH₄⁺ transport and that MA/MA⁺ is transported differently than NH₃/NH₄⁺ (Nakhoul et al.; Nakhoul et al., 2005; Nakhoul et al., 2006). The reported affinity of RhBG to NH₄⁺ is 2–4 mM. This concentration is within reported values of interstitial NH₄⁺ concentrations in the renal medulla which vary between 2.5 and 9 mM and is considerably increased in acidosis. In the kidney, the expression of RhBG at the basolateral membrane and the electrochemical gradient for NH₄⁺, are consistent with electrogenic influx of NH₄⁺ although electroneutral NH₃ transport may still occur.

Similar uncertainties about RhCG also persist. Some of the earlier studies did confirm a role in NH₃/NH₄⁺ transport. However the questions of electrogenic NH₄⁺ transport (Nakhoul et al., 2005), or electroneutral NH₄⁺- H⁺ exchange (Ludewig, 2004) or simultaneous NH₃ and NH₄⁺ transport (Bakouh et al., 2004) are not yet resolved. Studies on reconstituted RhCG in liposomes demonstrated increased NH₃ permeability but had no effect on NH₄⁺ permeability (Mouro-Chanteloup et al.). Surface pH measurements in oocytes expressing RhCG indicate transport of NH₃ (Musa-Aziz et al., 2009). The presence of RhCG at the apical membrane of the collecting duct, where electroneutral NH₃/NH₄⁺ transport is likely, seems to indicate that the mode of transport by RhCG may be different from that of basolateral RhBG.

The role of Rh glycoproteins as gas channels

Historically it was assumed that gases such as CO₂ and NH₃ cross cell membranes by diffusing through the lipid bilayer. However the discovery that some membranes have low permeability to NH₃ or CO₂ challenged this view (Kikeri et al., 1989; Singh et al., 1995; Waisbren et al., 1994) and suggested that specific membrane proteins may facilitate transport of these gases. Rh and related proteins were investigated as potential transporters of CO₂ or NH₃.

Studies on yeast demonstrated dependence of yeast growth in NH₃/NH₄⁺ deficient media on pH and concluded that MEP proteins facilitate diffusion of NH₃ and not NH₄⁺(Soupene et al., 2001). Other studies of E. coli also indicated facilitated NH₃ diffusion by AmtB (Soupene et al., 2002). It was also suggested that RhAG mediates facilitated transport of NH₃ (Ripoche et al., 2004) leading to trapping of NH₄⁺ at higher concentrations in RBC than in plasma.

Other evidence indicates that Rh proteins transport CO₂ (Kaplan et al., 2004; Soupene et al., 2004). Direct studies of CO₂/HCO₃⁻ transport in RBC indicated that DIDS markedly decreased transport of CO₂ and suggested that Rh and Band-3 may be coupled functionally. Another study on CO₂ permeability in normal and Rh_null RBC concluded that CO₂ permeation is mediated in part by Rh/RhAG and by AQP1 (Endeward et al., 2006). Another study demonstrated that expressing RhCG in oocytes increased CO₂ permeability compared to H₂O-injected oocytes (Bakouh et al., 2006). A recent study proposed that NH₃ moves through the monomeric pores of AmtB and RhAG whereas CO₂ could move through the central pores of the trimeric structure (Musa-Aziz et al., 2009). Expressing RhCG in oocytes seemed to enhance CO₂ transport (Bakouh et al., 2006). A possible role of Rh glycoproteins in transport of gases such as CO₂ and NH₃ is physiologically critical and studies to resolve the identity of the transported substrate (CO₂, NH₃ or NH₄⁺) are needed.

Substrate specificity and pH dependence

Candidate substrates that can be transported by Rh proteins include NH₄⁺ (Liu et al., 2000; Ludewig, 2004; Marini et al., 2000); NH₃ (Palkova et al., 1997), CO₂ (Kustu and Inwood, 2006) or methyl ammonium (Ludewig, 2004; Mak et al., 2006). Methyl amine hydrochloride is often used instead of ammonium chloride because of the similarity of the molecules and because it can be easily radiolabeled. The pH sensitivity of murine RhCG and other Rh glycoproteins is important for several reasons. First, as putative NH₄⁺ transporters, a change in pH will shift the equilibrium of NH₃ to NH₄⁺ and thus may affect transport of NH₄⁺. Second, it has been proposed that murine RhCG may function as an NH₄⁺-H⁺ exchanger that is driven by the H⁺ gradient (Ludewig, 2004; Mak et al., 2006). (Note that functionally, NH₄⁺-H⁺ exchange is equivalent to NH₃ transport, a notable feature complicating many studies of total ammonia transport. However, NH₄⁺-H⁺ exchange and NH₃ transport are not the same and will respond differently to physiologic stimuli.) Therefore, a pH change will also affect transport of NH₄⁺ by RhCG. Third, pH sensitivity of these transporters is probably highly significant in acid-base disturbances where renal RhBG and RhCG, for example, may play an important role in the adaptive response to acidosis. Finally, based on the crystal structure of AmtB, a model of NH₃/NH₄⁺ transport is proposed where deprotonation of NH₄⁺ is critical for NH₃ conduction through the pore. This step is also pH sensitive. A recent study showed that NH₄⁺ transport by RhBG in mice was activated by raising extracellular pH but was completely inhibited by decreasing extracellular or intracellular pH (Nakhoul et al.).

5. Physiological Significance and Relevance to Human Diseases

Rh and related proteins are highly expressed in mammalian tissues and primitive species. This suggests that Rh proteins are evolutionarily preserved under high selective pressure, yet their biological function remains poorly defined. Of significance, yet not fully understood, are the following observations: The erythroid RhAG is needed for cell surface expression of the “Rh complex” but it may also be involved in NH₃ or CO₂ transport. The non-erythroid RhBG and RhCG are expressed in tissues that transport or metabolize NH₃/NH₄⁺ and their renal distribution correlates well with NH₄⁺ excretion. However, it was proposed (Li et al., 2007) that they could be linked to other membrane proteins to form a “transport complex” and may transport CO₂. It was also suggested that RhBG and RhCG may represent a new class of NH₄⁺ sensing receptors or “ammonium sensors” acting as mediators to regulate different cellular processes (Van Kim et al., 2006).

The physiological roles of RhBG and RhCG in renal NH₃/NH₄⁺ handling are becoming more evident. Studies on RhCG in mice indicated that chronic metabolic acidosis increased RhCG protein expression in the medullary CD (Seshadri et al., 2006) and that the increase was in both intercalated cells and the principal cells (Seshadri et al., 2006). The same study indicated an increase in apical and basolateral RhCG expression and a decrease in cytoplasmic RhCG expression. Metabolic studies demonstrated that mice lacking RhCG had impaired urinary NH₄⁺ excretion in response to acid loads (Biver et al., 2008). The same study concluded that Rhcg knockout mice had reduced apical NH₃ permeability and transepithelial NH₃/NH₄⁺ transport. A recent study on mice with renal collecting duct-specific Rhcg deletion (CD-KO) (Lee et al., 2009) showed that under basal conditions urinary NH₄⁺ excretion was less in KO mice than in control mice and that after acid loading CD-KO mice developed more severe metabolic acidosis than controls.

Studies on RhBG showed that genetic deletion of pendrin, an apical Cl⁻-HCO₃⁻, decreased RhBG expression. Although earlier studies on Rhbg knockout mice (Chambrey et al., 2005) did not elicit abnormal acid-base balance or ammonium handling, later studies proved differently. Recently, Bishop et al., (Bishop, 2009) generated intercalated cell-specific Rhbg KO mice and demonstrated that urinary ammonium excretion was significantly less in KO mice vs controls. The same study showed that in control mice, HCl-induced acidosis increased RhBG protein expression significantly in three days.

Most studies provide strong evidence for a role of the Rh proteins in NH₃/NH₄⁺ transport. Renal NH₄⁺ excretion is critical for acid-base homeostasis and the mechanism includes NH₃ and NH₄⁺ transport components. Yet simple diffusion of NH₃ is limited and NH₄⁺-specific transporters had not been known to fully account for the significant renal NH₃/NH₄⁺ transport. As shown in this figure (Fig 3), it is likely that RhBG and RhCG, working in tandem, may actually function as the elusive NH₄⁺ -specific transporters and/or gas channels for NH₃ to explain how the distal nephron achieves transepithelial NH₃/NH₄⁺ transport. Characterizing the functions of these proteins is essential to explain these novel mechanisms and will shed light on understanding acid-base homeostasis and its regulation by the mammalian kidney.

Figure 3. Schematic diagram of a medullary collecting duct cell indicating pathways of NH₃ and NH₄⁺ transport.

Transporters labeled RhBG and RhCG show the membrane location and putative role of these proteins in transport of NH₃ or NH₄⁺.

In relevance to human diseases, the association of Rh proteins with red blood cell (RBC) disorders is well established (Huang et al., 2000; Van Kim et al., 2006). Mutations in the RH locus leading to complete absence of RBC RH antigens cause Rh null syndrome (Cherif-Zahar et al., 1998; Huang et al., 1998). Dominant over-hydrated hereditary stomatocytosis (OHSt) or Rh deficiency syndrome is caused by mutations of RHAG (Cherif-Zahar et al., 1996; Huang and Ye). OHSt is reported to cause increased permeability of RBC to monovalent cations. A recent study (Stewart et al.) reported a heterozygous RhAG missense mutation (F65S) in patients with OHSt that caused loss of function of RhAG for amine transport (NH₃/NH₄⁺ and MA/MA⁺). The physiological importance of RhBG and RhCG are mostly evident in KO mice studies as described above. These studies suggest that loss of function mutation, as shown for Rhcg, may cause distal renal acidosis and male infertility (Biver et al., 2008). Moreover, RhBG and RhCG have been suggested to act as tumor suppressor factors given their down-regulation in human esophageal cancers (Chen et al., 2002) and mouse brain tumors (Johansson et al., 2004).