The Molecular Characterization and Expression of New Human SLC26 Anion Transporters

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Helsinki University Biomedical Dissertations No. 69

The Molecular Characterization and Expression of New Human SLC26

Anion Transporters

Minna Kujala-Myllynen

Department of Medical Genetics University of Helsinki Finland

Academic dissertation

To be publicly discussed with the permission of the Faculty of Medicine, University of Helsinki, in the large lecture hall of Haartman Institute, Haartmaninkatu 3, Helsinki, on November 18^th 2005, at 12 noon.

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Supervised by:

Juha Kere, Professor

Department of Biosciences at Novum Karolinska Institute

Stockholm, Sweden and

Department of Medical Genetics University of Helsinki

Helsinki, Finland

Reviewed by:

Hannu Jalanko, MD, PhD, Adjunct Professor Hospital for Children and Adolescents University of Helsinki

Helsinki, Finland and

Anne Räisänen-Sokolowski, MD, PhD, Adjunct Professor Department of Pathology

University of Helsinki, and Helsinki University Central Hospital Helsinki, Finland

Official opponent:

Per-Henrik Groop, MD, DMSc, Adjunct Professor Folkhälsan Research Center

University of Helsinki Helsinki, Finland

ISSN 1457-8433

ISBN 952-10-2735-5 (paperback) ISBN 952-10-2736-3 (PDF)

Yliopistopaino Helsinki 2005

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To My Family

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List of Original Publications

This thesis is based on the following original publications that are referred to in the text by their Roman numerals. In addition, some unpublished data are presented.

I Lohi H., Kujala M., Kerkelä E., Saarialho-Kere U., Kestilä M. and Kere J.:

Mapping of Five New Putative Anion Transporter Genes in Human And Char- acterization of SLC26A6, a Candidate Gene for Pancreatic Anion Exchanger.

Genomics. 2000 Nov 15; 70(1):102-112.

II Lohi H., Kujala M., Mäkelä S., Lehtonen E., Kestilä M., Saarialho-Kere U., Markovich D. and Kere J.: Functional Characterization of Three Novel Tissue- speciﬁc Anion Exchangers SLC26A7, -A8 and -A9. J Biol Chem. 2002 Apr 19;

277(16):14246-54.

III Kujala M., Tienari J., Lohi H., Elomaa O., Sariola H., Lehtonen E. and Kere J.:

The SLC26A6 and SLC26A7 Anion Exchangers Have Distinct Distribution in Human Kidney. Nephron Exp Nephrol 2005;101:50-58.

IV Kujala M., Hihnala S., Tienari J., Kaunisto K., Hästbacka J., Holmberg C., Kere J.

and Höglund P.: Expression of Ion Transport Associated Proteins in Human Effer- ent and Epididymal Ducts. Submitted.

Publications I and II have also been included in the thesis of Hannes Lohi (2002).

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Abbreviations

aa amino acid

ACG1B achondrogenesis type IB

ADPKD autosomal dominant polycystic kidney disease

AE anion exchanger

AMRC apical mitochondria rich cells ATP adenosine triphosphate

AOII atelosteogenesis type II, also known as neonatal osseus dysplasia

AQP aquaporin

bp base pair

CAII carbonic anhydrase II

cAMP cyclic adenosine monophosphate CD collecting duct of the kidney

CD10 common acute lymphocytic leukemia antigen (CALLA) cDNA complementary DNA

CF cystic ﬁbrosis

CFTR cystic ﬁbrosis transmembrane conductance regulator

CK7 cytokeratin 7

CLD congenital chloride diarrhea

DCT distal convoluted tubule of the kidney

DFNB4 a form of congenital non-syndromic deafness, also known as neurosensory non-syndromic recessive deafness 4 (NSRD4) DIDS 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, an inhibitor of

several anion channels and transporters dRTA distal renal tubular acidosis

DTD diastrophic dysplasia

EMC extraglomerular mesangial cells EST expressed sequence tag

GADPH glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene IC intercalated cell of the kidney collecting duct

kb kilo base pairs

MCDK multicystic dysplastic kidney mRNA messenger RNA

NHE Na⁺/H⁺ exchanger

NHERF-1 Na⁺/H⁺ exchanger regulatory factor 1 NHE3 Na⁺/H⁺ exchanger 3

nt nucleotide

PCR polymerase chain reaction PKC protein kinase C

PNRA proximal nephrogenic renal antigen PT proximal tubule of the kidney rMED recessive multiple epiphyseal dysplasia

RT-PCR reverse transcriptase polymerase chain reaction

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SLC solute carrier

SLC26A1-A11 solute carrier family 26, members 1-11

TAL thick ascending limb of the loop of Henle in the kidney TH Tamm-Horsfall glycoprotein

UTR untranslated region

V-ATPase vacuolar proton transporting ATPase

The names refer to the human ortholog when written in all upper case (e.g. SLC26A6), and to other mammalians when in title case (e.g. Slc26a6). The gene names are written in italics (e.g. SLC26A6), the protein names in regular font (e.g. SLC26A6).

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Abstract

Appropriate control of intracellular ion concentrations is essential for every living cell. Different kinds of cells need distinct ion microenvironments, which are regulated by tissue and cell specific ion transporters. Anion transporters play key roles in several physiological processes, including control of the acid-base balance, regulation of cell growth, volume, metabolism, contractility, intracellular pH, and ion gradi- ents, among other things. When this work was initiated, only two human members of the anion transporter family SLC26 (solute carrier family 26) were known, both of which had distinct tissue specificities. Both genes were associated with rare recessively inherited diseases that belong to the Finnish disease heritage; diastrophic dysplasia and congenital chloride diarrhea. Since the simple nematode Caenorhabditis elegans was found to have seven genes encoding amino acid sequences homologous to the known human SLC26 proteins, we considered it extremely likely that several human SLC26 members were as of yet uncharacterized, and began the search for them. The ultimate goal was to find new SLC26 genes, to characterize their structure, expression and function, and to study their cell specific expression in selected physiologically relevant tissues.

New human SLC26 genes were identiﬁed utilizing a human genome-based sequence homology approach with several publicly available databases and computer programs.

The putative new gene sequences found were further analyzed by RT-PCR and sequencing, and their chromosomal location was detected by radiation hybrid mapping. Alto- gether seven novel human SLC26 genes (SLC26A1, A6-A11) were identiﬁed, their nucleotide and amino acid sequences were inspected with computer programs, and their general tissue speciﬁc expression patterns were explored by Northern blotting and PCR of multiple tissue cDNA panels (I). We then focused especially on the molecular characterization of four new genes, SLC26A6-A9.

The SLC26A6 gene, encoding a 738 aa protein, was mapped to chromosome 3p21.3.

It was highly homologous in structure with the previously known SLC26 members transporting anions, suggesting that SLC26A6 may function as an anion exchanger as well. Its highest expression levels were in the human kidney and pancreas, but lower levels were found in several other tissues. Utilizing immunohistochemistry, the SLC26A6 protein was localized in human kidney to the distal parts of the proximal tubules, some of the thin and thick ascending segments of the Henle’s loops, macula densa cells, distal convoluted tubules and a subpopulation of intercalated cells in collecting ducts, suggesting important roles in e.g. Cl^- reabsorption and tubuloglomerular feedback. In addition, SLC26A6 was detected in a subgroup of cysts in ADPKD and multicystic dysplastic kidneys. In human epididymis, SLC26A6 was located on the luminal edge of the non-ciliated cells of the efferent ducts, together with Cl^- channel

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CFTR, Na⁺/H⁺ exchanger NHE3, and their common regulator NHERF-1, suggesting that they form an essential cooperative unit which reabsorbs Na⁺ and Cl^- and controls osmotic water absorption in these structures. Moreover, SLC26A6 was found in apical forms of AMRC in human epididymal ducts, possibly ﬁne-tuning the intralu- minal pH and osmolality (I,III,IV).

The SLC26A7 gene was located on chromosome 8q23 and encoded a 656 aa transmembrane protein. Its mRNA was found in the kidneys, testes, and placenta. When expressed in Xenopus laevis oocytes, SLC26A7 demonstrated Cl^-, SO₄^2-, and oxalate transport activity. RT-PCR and immunoblotting showed a stronger signal from medullary areas of the human kidney rather than cortical. SLC26A7 was localized to the basolateral side of type A intercalated cells of collecting ducts and extraglomerular mesangial cells in the human kidney using immunohistochemistry, imply- ing important roles in HCO₃^- reabsorption and tubuloglomerular feedback, respectively. In human epididymis, SLC26A7 was expressed in a subgroup of basal cells, possibly taking part in the regulation of the principal cells (I-IV).

SLC26A8 and A9, located on chromosomes 6p21 and 1q31-q32 respectively, both showed high tissue specific expression. Both of them were demonstrated to transport at least Cl^-, SO₄^2-, and oxalate when expressed in Xenopus laevis oocytes. SLC26A8 was expressed in testes only, more specifically in the spermatocytes and spermatids, as confirmed by in situ hybridization and immunohistochemistry. Therefore, it might have an important role in the meiotic phase of sperm development, and possibly cause impaired male fertility if mutated. Using RT-PCR, Northern blotting, and immunohistochemistry, SLC26A9 was located to the alveolar and bronchial epithelium of human lungs, suggesting a part in maintaining the airway surface liquid needed for defense against bacterial infections (I,II,IV).

The new SLC26 genes are presumed to participate in several fundamental physiologi- cal processes involving anion transport in different human organs. Furthermore, they serve as good candidates for as of yet unidentiﬁed hereditary diseases.

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Introduction

The ability to maintain the right concentration of different ions is vital for every living cell in all tissues. Strict regulation of ions is required e.g. for controlling the normal electrical potential across the cell membrane. The rapid control of the membrane potential is especially important for achieving contraction of muscle cells and signal transmission of neural cells. In addition, all polarized cells need several ion transporting systems in order to maintain their intracellular polarity and normal functions (Guyton 1991c).

In animal cells, the quantity of different ions in intracellular and extracellular spaces affects the cell volume through osmotic forces. If the concentration of ions inside a cell diminishes considerably compared to extracellular space, the osmolality of the cell diminishes. To balance the osmotic pressure across the cell membrane, the osmotic forces then drive water outside the cell. Consequently, the cell shrinks. If the concentration of intracellular ions compared to extracellular increases strongly, the osmotic pressure causes water to collect inside the cell. Thereupon, the cell swells (Guyton 1991a).

Even slight changes in pH can markedly alter the rates of chemical reactions in a cell.

Therefore, the accurate control of pH is one of the most important aspects in maintaining cellular homeostasis. The most important mechanism for maintaining the acid-base homeostasis in the human body is the bicarbonate buffer system. While HCO₃^- concentration alters when balancing the acid-base homeostasis, another anion, Cl^-, is needed to adjust the shifted anion load in body fluids. Cl^-is one of the main electrolytes in the human body, together with cations Na⁺ and K⁺. As the most abun- dant anion in extracellular fluids, Cl^- plays an important role in regulating body fluids’ osmolarity as well (Guyton 1991a, Mutanen and Voutilainen 1993).

Besides HCO₃^-, Cl^-, and PO₄^3- other inorganic anions have speciﬁc important roles in the human body as well. For example, SO₄^2- is needed for proper cell growth and development of an organism, as it is required for cell matrix synthesis and the maintenance of cell membranes. SO₄^2- is involved in many detoxiﬁcation processes of endo- and exogenous compounds as well. I^-, for its part, is an essential constituent of the thyroidal hormones thyroxine (T₄) and triiodothyronine (T₃) (Mutanen and Vouti- lainen 1993, Morris and Sagawa 2000).

The lipid bilayer of the cell membrane acts as a barrier, blocking the movement of most water molecules and water-soluble substances such as inorganic ions. The balance of intracellular ions is regulated through numerous different transporter proteins.

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The transport of ions can occur passively through ion channels by diffusion (Figure 1a) or through active transport. Active transport can be further divided to primary and secondary forms. Primary active transport happens through diverse ion pumps, requiring energy from the breakdown of high energy phosphate compounds such as adenosine triphosphate (ATP) (Figure 1b). Secondary active transport uses the concentration gradient of a certain ion to transport another ion together with it against the gradient of the latter (Figure 1c). Secondary active transport can occur as co-transport, where both ions move in the same direction through the cell membrane, or as counter-transport where the ions move in opposite directions. Usually, each transporter protein is very speciﬁc for the ions that it transports (Guyton 1991c).

This study focused on the anion transporter family SLC26 (solute carrier family 26), whose members transport negatively charged ions across the cell membrane through secondary active transport. The fact that the four ﬁrst characterized human SLC26 genes all cause severe recessively inherited diseases when mutated, conﬁrms the impor- tance of these transporters. A failure in anion transport can manifest in very different ways, depending on the SLC26 transporter affected, as mutations of this gene family can cause at least skeletal dysplasias, congenital chloride diarrhea, Pendred syndrome with deafness and goiter, and non-syndromic deafness (Hastbacka et al. 1994, Hastbacka et al. 1996, Hoglund et al. 1996b, Superti-Furga et al. 1996, Everett et al.

1997, Superti-Furga et al. 1999, Liu et al. 2003).

ATP ADP+Pi

a b

Figure 1.

c

Diagrams of

a) the function of an ion channel,

b) primary active transport through an ion pump, and

c) secondary active transport through a counter-transport type ion transporter.

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When this work was started, only two SLC26 gene family members in man were known (SLC26A2 and SLC26A3) (Schweinfest et al. 1993, Hastbacka et al. 1994), and soon the third human member was cloned (SLC26A4) (Everett et al. 1997). How- ever, the simple nematode Caenorhabditis elegans was noted to have seven SLC26 genes (The C. Elegans Sequencing Consortium 1998, Everett and Green 1999, Kere et al.

1999), and thus it was likely that several human SLC26 anion transporters were still uncharacterized.

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Review of the Literature

1. Anion Exchanger Genes and Proteins

1.1 The Classic Family of Anion Exchangers Belongs to Solute Carrier Family 4 (SLC4) Bicarbonate Transporters

The classic family of anion exchangers (AEs) consists of at least three distinct proteins that have important roles in the transport of CO₂ by erythrocytes, the absorption or secretion of H⁺ or HCO₃^- by various epithelia, and the regulation of intracellular volume and pH. The group of AEs form a part of the superfamily of bicarbonate transporters named solute carrier family 4 (SLC4). So far 10 mammalian SLC4 genes have been identiﬁed (SLC4A1-A5, SLC4A7-A11), and the family has been subdivided into three groups according to their functions. The ﬁrst three SLC4 members (SLC4A1- A3, also known as AE1-AE3, respectively, see Table 1) transport purely anions, predominantly the electroneutral Na⁺ independent Cl^-/HCO₃^- exchange. The remaining known members are subdivided into electrogenic and electroneutral Na⁺/HCO₃^- cotransporters. All SLC4s are structurally membrane proteins with 10-14 transmembrane segments (Romero et al. 2004, McKusick OMIM).

Table 1.

The classic family of anion exchangers.

Symbol Also

Known as Chromosomal

Location Anions

Transported Major Expression Sites

Associated Diseases SLC4A1 AE1, Band 3

protein

17q21-q22 Cl^-, HCO₃^- erythrocytes, kidney

elliptocytosis, ovalocytosis, distal renal tubular acidosis

SLC4A2 AE2 7q35-q36 Cl^-, HCO₃^- ubiquitous not known

SLC4A3 AE3 2q36 Cl^-, HCO₃^- brain, retina,

heart, smooth muscle

not known

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1.1.1 SLC4A1

Anion exchanger SLC4A1 (AE1) is located on chromosome 17q21-q22. It is also known as the band 3 protein for its position in SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins. It is the most common membrane glycoprotein in red blood cells. In addition to erythrocytes, it is also expressed in the kidney and at lower levels in the heart and colon. There are two distinct tissue speciﬁc splice variants of SLC4A1, one expressed mainly in erythrocytes and the other in the kidney (Kopito and Lodish 1985, Alper et al. 2002, Romero et al. 2004, McKusick OMIM).

SLC4A1 is one of the first transporters of any type that was physiologically characterized. It has several important roles in the human body. In erythrocytes, SLC4A1 mediated Cl^-/HCO₃^- exchange increases the CO₂ carrying capacity of the blood about five-fold. In addition, it maintains biconcave disk shape of red blood cells. In the kidney, SLC4A1 affects the acidification of the urine in the distal nephron (Romero et al. 2004, Shayakul and Alper 2004).

Distinct mutations in SLC4A1 cause two main types of diseases: morphological changes of erythrocytes (elliptocytosis or ovalocytosis) leading to hemolytic anemia, and distal renal tubular acidosis (dRTA). Interestingly, in some populations originating from malaria areas, SLC4A1 mutations leading to elliptocytosis occur at high frequencies.

These changes in SLC4A1 seem to protect individuals from malaria when inherited in heterozygous form. In the kidney, defective SLC4A1 presents either in a milder dominant or a more severe recessive form of inherited dRTA. The recessive form presents typically either with acute illness or withgrowth failure in the early years of life. dRTA is characterized by hyperchloremic metabolic acidosis of renal origin with high urinary pH (>5.5), low plasma HCO₃^-, raised plasma Cl^-, and normal anion gap (Shayakul and Alper 2004, Wagner et al. 2004, McKusick OMIM).

1.1.2 SLC4A2

SLC4A2 (AE2), with gene position 7q35-q36, is the most widely distributed of the SLC4 anion exchangers. It has been found virtually in all tissues studied, but its expression level is especially high in gastric parietal cells, choroid-plexus epithelial cells, apical enterocytes of the colon, and the renal collecting duct. In most epithelial cells, SLC4A2 is located on the basolateral membrane. SLC4A2 is believed to con- tribute to the regulation of intracellular pH by responding to alkali loads by exporting HCO₃^-. It may also have a role in the regulation of cell volume by the uptake of Cl^-. So far no genetic diseases have been associated with SLC4A2 (Demuth et al. 1986, Palumbo et al. 1986, Romero et al. 2004, McKusick OMIM).

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1.1.3 SLC4A3 Anion exchanger SLC4A3 (AE3) has been mapped to 2q36. It is mainly expressed in excitable tissues such as brain, retina, heart and smooth muscle, although it can be found, e.g., in some epithelial cells of the kidney and gastrointestinal tract as well.

SLC4A3 has not been linked to any diseases yet (Kopito et al. 1989, Su et al. 1994, Romero et al. 2004, McKusick OMIM).

1.2 The Solute Carrier Family 26 (SLC26) Anion Transporter Family

The SLC26 anion transporters were first called sulfate transporters since the first two mammalian members Slc26a1 and SLC26A2 were originally found to transport SO₄^2- (Bissig et al. 1994, Hastbacka et al. 1994, Satoh et al. 1998). As new members of this gene family were characterized, it became evident that it was not only SO₄^2- but rather several different anions that they transport with different specificities, thus the name of the protein family was changed to solute carrier family 26 (SLC26). Members of the SLC26 family form a structurally homologous group that is clearly distinct from the SLC4 family (Everett and Green 1999, Kere et al. 1999). Therefore, the SLC26 family is also known as the second anion exchanger family.

The ﬁrst three human SLC26 anion transporters SLC26A2-A4 (Table 2) were mostly found by positional cloning of rare recessive diseases: diastrophic dysplasia, congenital chloride diarrhea and Pendred syndrome (Hastbacka et al. 1994, Hoglund et al.

1996b, Everett et al. 1997), although SLC26A3 had been previously cloned as a putative tumor suppressor gene (Schweinfest et al. 1993). While these three inherited disorders are clinically very dissimilar, the associated genes turned out to belong to the same gene family encoding structurally conserved anion transporters. An intriguing fact was that two of these diseases, diastrophic dysplasia and congenital chloride diarrhea, happened to both belong to the Finnish disease heritage, a group of rare inherited diseases that are clustered in the Finnish population (Norio 2003).

Expression of the SLC26 family members differs dramatically. A good example of this are SLC26A2 and SLC26A3: while the ﬁrst is found ubiquitously in nearly all human tissues studied, the second seems to have extremely strict tissue speciﬁc expression pattern (Hastbacka et al. 1994, Haila et al. 2000, Haila et al. 2001).

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Table 2.

The characteristics of the four human SLC26s not cloned in this work.

Symbol Also Known

as Chromosomal

Location Anions

Transported Major Expression Sites

Associated Diseases SLC26A2 DTDST 5q32-q33.1 SO₄^2-, Cl^- ubiquitous achondrogene-

sis type IB, atelosteogenesis type II, diastrophic dysplasia, recessive multiple epiphyseal dysplasia SLC26A3 CLD, DRA 7q22-q31.1 Cl^-, SO₄^2-,

HCO₃^-, OH^-, oxalate

colon congenital chloride diarrhea SLC26A4 PDS, Pendrin 7q31 I^-, Cl^-, HCO₃^-,

OH^-, formate

thyroid, kidney, cochlea

Pendred syndrome, congenital non-syndromic deafness, enlarged vestibular aqueduct syndrome SLC26A5 Prestin 7q22.1 (Cl^-, HCO₃^-) cochlea recessive non-syn-

dromic neurosensory deafness

1.2.1 SLC26A1 Was Originally Found in Rats

The first mammalian member of SLC26 anion transporters, Slc26a1, was originally cloned from rat liver mRNA in 1994 (Bissig et al. 1994). Since it was found to mediate Na⁺ independent SO₄^2- transport, it was originally named sulfate anion transporter-1 (sat-1) (Bissig et al. 1994, Markovich et al. 1994). Northern blot analysis indicated a strong signal for rat Slc26a1 mRNA in the liver and kidney, and a weaker one in muscle and brain (Bissig et al. 1994). Slc26a1 protein was localized specifically to the basolateral membrane of the proximal tubules in rat kidney, and it was shown to at least mediate SO₄^2- and oxalate transport (Karniski et al. 1998). The human ortholog SLC26A1 was not characterized until our group published it in 2000 (I). Our results were confirmed and extended upon in 2003 by Regeer et al., who described the structure of human SLC26A1 in more detail (Regeer et al. 2003).

1.2.2 Mutations of SLC26A2 Cause Several Bone and Cartilage Diseases of Varying Severity

The SLC26A2 gene was found by Hastbacka et al., when they were seeking the gene responsible for diastrophic dysplasia by positional cloning using ﬁne-structure linkage

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disequilibrium mapping. Therefore the gene was ﬁrst named diastrophic dysplasia sulfate transporter (DTDST) (Hastbacka et al. 1994). Diastrophic dysplasia (DTD, DD; OMIM #222600) is a globally rare autosomal recessive osteochondrodysplasia.

However, it is prevalent in the Finnish population due to a founder effect; less than 500 patients have been described worldwide, but almost 200 of them are from Fin- land (Hastbacka et al. 1994, Norio 2003). DTD is a congenital disease with clinical features of short limbs, short stature, stiffness of the big joints, ﬂexion limitation of the ﬁnger joints, scoliosis, kyphosis, clubfoot and hitchhiker’s thumb malforma- tions, deformation of ear lobes, and often cleft palate. Generalized dysplasia of the joints frequently leads to motility restrictions and early arthrosis. The severity of the disease can even vary intrafamilially (Norio 2003, McKusick).

The SLC26A2 gene is located on chromosome 5q32-q33.1 (McKusick OMIM). It consists of 2833 nucleotides encoding a 739 aa transmembrane protein with a predicted molecular mass of 82 kDa and 12 transmembrane domains (Hastbacka et al.

1994). It is expressed widely in the human body, and the protein has been immunolocalized to cartilage, colon, eccrine sweat glands, bronchial glands, tracheal epithelium, placenta, and exocrine pancreas. In these tissues, many distinct cell types pro- duce SLC26A2, but the protein is mostly located in secretory structures (Haila et al. 2001). In addition, by PCR, SLC26A2 mRNA has been detected in several tissues that did not stain with anti-SLC26A2 antibodies in immunohistochemistry, including e.g. kidney, testis, and prostate. That probably indicates that in these tissues SLC26A2 expression level is very low or it is restricted to small areas or structures (Hastbacka et al. 1994, Haila et al. 2001).

Because of the structural similarity to the previously characterized Slc26a1, SLC26A2 was suggested to be a SO₄^2- transporter, and a defect in SO₄^2- transport could indeed be demonstrated in ﬁbroblasts of a DTD patient (Hastbacka et al. 1994). Further ver- iﬁcation was brought by Xenopus laevis oocyte experiments showing that SLC26A2 mediates DIDS-sensitive Na⁺ independent SO₄^2- transport that could be inhibited by thiosulfate, oxalate and Cl^-. The results indicated that SLC26A2 would function as a SO₄^2-/Cl^- exchanger (Satoh et al. 1998). Even though SLC26A2 is likely to transport other anions as well, direct transport studies of ions other than SO₄^2- are lacking. Thus the full combination of its substrate anions is still unknown (Mount and Romero 2004).

Soon after the cloning of SLC26A2, mutations in this gene were associated with other bone and cartilage diseases as well. Achondrogenesis type IB (ACG1B, OMIM # 600972) is a perinatally lethal autosomal recessive disease characterized by extremely short limbs caused by poor skeletal development. Typical radiological findings are defi- cient ossification in the lumbar vertebrae and absent ossification in the sacral, pubic and ischial bones (Borochowitz et al. 1988, McKusick OMIM). The finding that one ACG1B patient had impaired SO₄^2- metabolism, leading to a reduction in sulfated proteoglycans (Superti-Furga 1994), motivated Superti-Furga et al. to test the newly

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found SLC26A2 for possible mutations in ACG1B patients. Indeed, mutations were found, conﬁrming DTD and ACG1B to be allelic disorders with very different clinical severity (Superti-Furga et al. 1996).

Atelosteogenesis type II (AOII, also known as neonatal osseus dysplasia) is a neona- tally lethal chondrodysplasia that clinically and histologically resembles the less severe disease DTD. It is presumed to be an autosomal recessively inherited disease. AOII patients have strikingly short limbs, small chest, clubfeet, spinal abnormalities, cleft palate, and abducted thumbs and toes. In radiography, prevalent findings include bifid distal humerus, rounded distal femur, cervical kyphosis, scoliosis, lumbar hyperlor- dosis, and disproportion of certain metatarsals and metacarpals. Collapse of the air- ways and pulmonary hypoplasia caused by too small rib cage inflict respiratory insuf- ficiency, and AOII patients die soon after birth (Hastbacka et al. 1996, McKusick OMIM). Interestingly, it was shown that AOII is also caused by specific mutations in the SLC26A2 gene, leading to faulty uptake of inorganic SO₄^2- and inadequate sul- fation of macromolecules (Hastbacka et al. 1996).

A fourth disease has been associated with SLC26A2. An autosomal recessive form of multiple epiphyseal dysplasia (rMED, MED4; OMIM # 226900) can clinically manifest as clubfoot only, with normal body height. With X-rays epiphyseal dysplasia can be detected. However, rMED patients may have short limbs, short stature, movement limitations or stiffness of joints, double-layered patella, and hip dysplasia as well (Superti-Furga et al. 1999, Huber et al. 2001, Makitie et al. 2003, McK- usick OMIM).

Over 30 mutations of SLC26A2 have been detected so far. Interestingly, genotype – phenotype correlations are observable, since patients with the most severe disease ACG1B tend to be homozygous, and AOII patients heterozygous for loss-of-function mutations, which cause a truncated protein or a non-conservative amino acid substitu- tion in a transmembrane domain. Respectively, individuals with milder diseases DTD or rMED are typically homozygous for alleles encoding a protein with some residual activity, having non-transmembrane amino acid substitutions and splice site mutations (Hastbacka et al. 1996, Karniski et al. 1998, Rossi and Superti-Furga 2001).

1.2.3 SLC26A3 is Defective in Congenital Chloride Diarrhea SLC26A3 was ﬁrst identiﬁed by Schweinfest et al. as a gene that was down-regulated in colon adenomas and adenocarcinomas, hence it received the name DRA (down- regulated in adenoma) (Schweinfest et al. 1993). It is located on chromosome 7q22- q31.1 spanning over 39 kb (Taguchi et al. 1994, McKusick OMIM). The SLC26A6 mRNA includes 2881 bp corresponding to a 764 aa open reading frame of a 84,5 kDa transmembrane protein with predicted 10, 12 or 14 transmembrane domains (Schweinfest et al. 1993, Byeon et al. 1996, Haila et al. 1998).

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The SLC26A3 protein has a strict tissue speciﬁc expression pattern, and so far it has been immunolocalized only in epithelium of colon, eccrine sweat glands, and seminal vesicles in humans (Haila et al. 2000). It has been shown to function as a Na⁺ independent anion exchanger transporting at least SO₄^2-, Cl^-, HCO₃^-, OH^-, and oxalate. Its function can be inhibited by the anion transporter inhibitor DIDS (Silberg et al. 1995, Byeon et al. 1998, Melvin et al. 1999, Moseley et al. 1999, Jacob et al. 2002).

Congenital chloride diarrhea (CLD, CCD; OMIM #214700) is a rare autosomal recessive disease with voluminous watery diarrhea that already begins in utero. Clin- ical ﬁndings include polyhydramnios, premature birth, excessive weight loss, byper- bilirubinemia and dehydration of the affected newborn. These are caused by exten- sive loss of Cl^- through the stool caused by defective Cl^-/HCO₃^- exchange in the colon.

The treatment of CLD by compensating for the lost Cl^- is lifelong. However, if the right diagnosis is not made and treatment not started, the affected individuals die of severe electrolyte perturbation within the ﬁrst weeks of life (Gamble et al. 1945, Hol- mberg et al. 1975, Norio 2003). Worldwide, CLD is an extremely rare disease. How- ever, it is part of the Finnish disease heritage, and the incidence is about 1:20,000 in Finland. It is also common in some Arabic countries, e.g. Kuwait, where as many as 1:3,200 newborns may have CLD (Badawi et al. 1998, Makela et al. 2002). Interest- ingly, it was noted recently, as the ﬁrst properly treated CLD patients have reached reproductive age, that the male patients appear to be subfertile. Despite normal sper- matogenesis, CLD patients were found to have oligoasthenoteratozoospermia, and high Cl^- but low pH in seminal plasma as well as spermatoceles. This suggests that the gene responsible for CLD might have a role in the proximal male reproductive tract in addition to the seminal vesicles (Hoglund et al. 2005).

Using a candidate gene approach, Kere et al. found the gene responsible for CLD to locate near, but to be different from, the cystic fibrosis transmembrane regulator (CFTR) gene on chromosome 7 (Kere et al. 1993). The localization of the CLD gene was further refined by a linkage disequilibrium study in the Finnish founder population and on a physical map (Hoglund et al. 1995, Hoglund et al. 1996a), and finally mutations in SLC26A3 were shown to cause CLD (Hoglund et al. 1996b). Hence the gene is also known as CLD. The SLC26A3 mutations identified in CLD patients have been shown to cause loss-of-function in vitro assays with Xenopus laevis oocytes (Moseley et al. 1999). No diseases other than CLD have been associated with the SLC26A3 gene (Makela et al. 2002).

1.2.4 Certain Forms of Deafness Result from Flawed SLC26A4 SLC26A4 was found by positional cloning of the gene mutated in Pendred syndrome (OMIM # 274600). Thus the gene, and the protein it encodes, ﬁrst received the names PDS and Pendrin, respectively (Everett et al. 1997). Pendred syndrome is the most common form of inherited syndromic hearing loss, accounting for up to 10%

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of hereditary deafness. It is an autosomal recessive disease, characterized by sensorineural hearing loss and euthyroid goiter. Deafness may exist at birth or develop during the early years of childhood. Some Pendred syndrome patients have vestibular dysfunction or mental retardation. Development of thyroid carcinoma has also been reported (Everett et al. 1997, McKusick OMIM).

In addition to Pendred syndrome, mutations in SLC26A4 have been associated with other forms of recessive hearing impairment. Both a form of congenital non-syndromic deafness (DFNB4, also known as neurosensory nonsyndromic recessive deafness 4 NSRD4; OMIM #600791), and enlarged vestibular aqueduct syndrome (EVA;

OMIM #603545) characterized by ﬂuctuating and sometimes progressive sensorineural hearing loss and disequilibrium symptoms, are in some cases caused by mutations in this gene (Li et al. 1998, Pryor et al. 2005, McKusick OMIM).

SLC26A4 maps to chromosome 7q31, less than 50 kb from SLC26A3. These genes are also structurally very similar, even within the SLC26 family, suggesting evolutionary relationship (Everett et al. 1997, Kere et al. 1999). SLC26A4 is a 780 aa transmembrane protein with highly tissue speciﬁc expression. It has been located to the thyroid gland and the inner ear, as expected from the Pendred syndrome phenotype, but also to Sertoli cells, fetal brain and both fetal and adult kidneys (Everett et al. 1997, Lac- roix et al. 2001). Interestingly, the exact expression of SLC26A4 in distinct speciﬁc kidney tubules is debated, and differences between species possibly exist in cell types producing this anion transporter (Lacroix et al. 2001, Royaux et al. 2001, Soleimani et al. 2001, Kim et al. 2002, Wall et al. 2003). Since none of the Pendred syndrome patients have been reported to have any kidney symptoms, it is possible that some other anion transporters may replace its function in the kidney.

Functional experiments have revealed that SLC26A4 does not transport SO₄^2-, as the three first mammalian SLC26 members do. Instead, it is capable of mediating I^-, Cl^-, HCO₃^-, OH^-, and formate transport (Kraiem et al. 1999, Scott et al. 1999, Bogazzi et al. 2000, Scott and Karniski 2000, Soleimani et al. 2001). The mutations associated with Pendred syndrome cause complete loss of Cl^- and I^- transport in the Xenopus lae- vis oocyte experimental model, while the alleles leading to DFNB4 only reduce the transport efficiency of these anions. It is probable that the residual transport capability is sufficient to avert the onset of goiter in DFNB4 (Scott et al. 2000).

1.2.5 The Cochlear Motor Protein SLC26A5 is Associated with Non-syndromic Deafness

Slc26a5 was ﬁrst cloned from a gerbil by Zheng et al., when seeking the motor protein of the outer hair cells of the cochlea. It was ﬁrst designated Prestin after the musical notation ‘presto’ (Zheng et al. 2000). The outer hair cells are non-neuronal epithelial cells that mechanically amplify the acoustic signals entering the inner ear. When

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their membrane potential changes, the outer hair cell stiffness and length is altered rapidly, causing variation in the cell shape of up to 5% (Kachar et al. 1986, Ashmore 1987, Zheng et al. 2000). Slc26a5 was located speciﬁcally to the outer hair cells only.

Most importantly, it was able to cause cell shape changes when expressed in cultured human kidney cells (Zheng et al. 2000). Intracellular Cl^- and HCO₃^- anions were shown to act as extrinsic voltage sensors, binding to Slc26a5, thereby triggering con- formational changes in outer hair cells (Oliver et al. 2001). Thus, Slc26a5 was con- cluded to act as the motor protein of the outer hair cells (Zheng et al. 2000, Oliver et al. 2001). This suggestion was supported by the ﬁnding that the targeted deletion of Slc26a5 caused the loss of outer hair cell electromotility and hearing impairment in mice (Liberman et al. 2002).

Human SLC26A5 is located on chromosome 7q22.1 and contains 21 exons. It has at least four different splicing variants, encoding proteins having 7-12 predicted transmembrane domains, and varying between 335 and 746 aa in size. SLC26A5 mRNA expression was found only in the cochlea of the 13 human tissues tested. Interest- ingly, mutations in SLC26A5 have been found in some Caucasian probands with recessive non-syndromic neurosensory deafness (DFNB61; OMIM +604943) (Liu et al. 2003, NCBI Entrez).

1.2.6 The New Members SLC26A6-A11 In this thesis, the identification and molecular characterization of six new human SLC26 anion transporters is described (I-II) and the specific expression of selected proteins in human kidney and epididymis is studied in detail (III-IV). Simultaneously with our work, other groups have cloned some of these genes as well. A few months after our article tentatively describing and mapping these six genes for the first time came out in Genomics (I), Waldegger et al. published identification of human SLC26A6 in the same journal (Waldegger et al. 2001). While we were preparing our second man- uscript presenting SLC26A7-A9 in more detail (II), Touré et al published the human SLC26A8 and named it Tat1 for Testis Anion Transporter 1 (Toure et al. 2001). An article about molecular cloning of human SLC26A7 by Vincourt et al. was printed in February 2002 (Vincourt et al. 2002), the same month when our SLC26A7-A9 article was published on the Web (II). A year later Vincourt et al. reported the further molecular and functional characterization of human SLC26A11 (Vincourt et al.

2003). So far, no totally new mammalian members of the SLC26 family have been delineated since our ﬁrst article (I).

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2. Interactions of Several Proteins Define Overall Ion Transport

Net ion transport to and from a cell is a sum of the actions of several distinct transporters, channels, and their regulators. Several proteins have been shown either to interact directly with SLC26 members or to share the same interaction partners, suggesting that they form functional cooperation units. The interaction partners identi- ﬁed so far, and one functionally reasonable potential interaction partner of the SLC26 anion transporters are introduced below (Table 3).

Table 3.

The presumable interaction partners of the SLC26 proteins.

Symbol Also

Known as Chromosomal

Location Protein Type Major Expression Sites

Associated Diseases

CFTR 7q31.2 Cl^- channel ubiquitous cystic ﬁbrosis

NHE3 SLC9A3 5p15.3 Na⁺/H⁺

exchanger

kidney, intestine not known V-ATPases ATP6 multiple subunits

located in different chromosomes

H⁺ pump ubiquitous recessive distal renal tubular acidosis, recessive infantile malignant osteopetrosis

CAII 8q22 zinc metallo-

enzyme

ubiquitous osteopetrosis with renal tubular acidosis and cerebral calciﬁcation NHERF-1 SLC9A3R1,

EBP50

17q25.1 apical PDZ protein

ubiquitous not known

2.1 Cystic Fibrosis Transmembrane Conductance Regulator

Cystic ﬁbrosis (CF; OMIM # 219700) is an autosomal recessive condition and the most common lethal genetic disease in Caucasian populations, with an incidence of 1:2,000 live births. However, it is much rarer in Finland, with only 1-2 new cases per year. CF is associated with a widespread dysfunction in secretory processes of several exocrine glands all over the human body (Savilahti 1997, Kinnunen et al. 2005, McKusick OMIM). It is caused by mutations in the cystic ﬁbrosis transmembrane conductance regulator (CFTR) gene encoding a Cl^- channel (Riordan et al. 1989).

Clinical manifestation of CF varies enormously, from a life threatening lung and pancreas insufﬁciency at early age to mild forms not detected before middle age. Possible

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symptoms are, inter alia, meconium ileus, liver disease with biliary duct obstruction, pancreatic insufﬁciency, recurrent pulmonary infections, congenital absence of vas deferens, and infertility. There is also evidence that CFTR mutations can cause poor sperm quality without any other clinical symptoms. CFTR genotype determines the biochemical abnormality, which further deﬁnes the clinical phenotype of CF (Bien- venu et al. 1993, Sheppard et al. 1993, van der Ven et al. 1996, Savilahti 1997, Wong et al. 2002, McKusick OMIM).

CFTR is located on chromosome 7q31.2 and is expressed in numerous different epi- thelia. It encodes a cAMP-regulated Cl^- channel that controls the regulation of other ion transport processes as well (Riordan et al. 1989, Anderson et al. 1991a, Ander- son et al. 1991b, Lee et al. 1999, Mendes et al. 2004, McKusick OMIM). Recently, it was shown that the CFTR and SLC26 transporters have a reciprocal activation capability for each other. This interaction is mediated by the binding of the STAS (sulphate transporter and anti-sigma antagonist) domain of SLC26 family members with the regulatory (R) domain of CFTR, and is facilitated by their PDZ ligands (Ko et al. 2004).

2.2 Sodium/Hydrogen Exchanger 3

Na⁺/H⁺ Exchanger 3 (NHE3, SLC9A3) is an apical Na⁺/H⁺ exchanger involved in transepithelial electroneutral Na⁺ absorption (Tse et al. 1992, Tse et al. 1993). It belongs to the gene family of Na⁺/H⁺ exchangers (NHE, also known as SLC9) that all catalyze the exchange of one extracellular Na⁺ for one intracellular H⁺ across the plasma membrane. Today, nine human members of the NHE family have been iden- tiﬁed (Zachos et al. 2005, McKusick OMIM).

NHE3 is located on human chromosome 5p15.3 and expressed in renal and intestinal epithelia (Brant et al. 1993, McKusick OMIM). So far, no human disease has been associated with this gene (McKusick OMIM). NHE3 activity is controlled by recy- cling it between the cell membrane and subapical endosomes (Janecki et al. 1998, Zachos et al. 2005). There is an indication that NHE3 may operate together with some of the SLC26 proteins, since expression of Slc26a3 mRNA was modestly up- regulated in the colon of mice lacking NHE3, suggesting that these transporters normally act together to absorb Na⁺ and Cl^- (Melvin et al. 1999).

2.3 Vacuolar H

⁺

ATPase

The vacuolar-type proton ATPases (V-ATPases, ATP6) are ubiquitous multi-subunit proteins. They are expressed practically in every eukaryotic cell, located on the membranes of intracellular organelles, such as endosomes and lysosomes. Their main function is the pH regulation of these structures by acidiﬁcation. In addition, V-ATPases

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are also involved in hydrogen ion transport across the plasma membrane into the extracellular space in some specific cell types, e.g., in the epididymis where seminal fluid is acidified, or in the kidney where V-ATPases are involved in acid-base transport, thus contributing to overall body homeostasis (Breton et al. 1996, Sun-Wada et al. 2004, Wagner et al. 2004, McKusick OMIM).

V-ATPases consist of two main domains, a peripheral catalytic V₁ domain (640 kDa;

ATP6V1) and a membrane-bound V₀ domain (240 kDa; ATP6V0), together forming a protein complex of approximately 900 kDa. The cytosolic V1 domain is com- posed of eight subunits, named A-H (ATP6V1A-H), containing three catalytic sites for ATP hydrolysis.The V₀ sector, containing up to ﬁve subunits (a, c, cV, cU and d;

ATP6V0A-D), translocates protons across the membranes. Some of the subunits further have several distinct isoforms. The genes encoding for the different subunits and isoforms are spread all over the human genome and show different expression patterns (Sun-Wada et al. 2004, Wagner et al. 2004, McKusick OMIM).

At least three different inherited diseases are associated with distinct isoforms of V- ATPase subunits. V-ATPase subunit B1 (ATP6V1B1) is located on chromosome 2p13, and expressed in the inner ear, intercalated cells of the kidney, epididymis, and cil- iar body. When mutated, it causes a syndrome of recessive distal renal tubular acidosis with sensorineural hearing loss (dRTA with SNHL). This disorder inﬂicts severe metabolic acidosis, together with perturbations of K⁺ balance, urinary Ca²⁺ solubility, bone physiology, and growth. The hearing loss is progressive in nature, with the age at the diagnosis of hearing loss quite variable (Karet et al. 1999, Alper 2002, Wagner et al. 2004). Mutations in the V-ATPase subunit a4 encoding gene (ATP6V0A4) cause recessive distal renal tubular acidosis dRTA. This subunit is located almost solely on the apical surface of type A intercalated cells in the human kidney (Smith et al. 2000, Alper 2002, Wagner et al. 2004).

The a3 subunit of V-ATPase (ATP6V0A3), expressed chieﬂy in osteoclasts, has been shown to be essential for the maintenance of normal bone turnover. Mutations in the a3 subunit cause a subset of autosomal recessive infantile malignant osteopetrosis (OMIM # 259700). This disease is characterized by macrocephaly, progressive deafness and blindness, hepatosplenomegaly, and severe anemia beginning in early infancy or in utero (Frattini et al. 2000, McKusick OMIM).

2.4 Carbonic Anhydrase II

Carbonic anhydrases (CAs) form a large family of zinc-containing metalloenzymes that catalyze the reversible hydration of carbon dioxide and water to H⁺ and HCO₃^-, thus playing roles in acid-base regulation. The tissue distribution of the different CAs shows a high degree of species variation. CAs are encoded by members of 3 independent CA gene families; alpha-CA, beta-CA, and gamma-CA.

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Carbonic anhydrase II (CAII) is a highly active cytoplasmic member of the alpha- CA family. It is ubiquitously expressed in human tissues (Alper 2002, McKusick OMIM). CAII interacts with the carboxyl terminus of anion exchanger SLC4A1, forming a membrane protein complex involved in the regulation of HCO₃^- metabolism and transport. This interaction enhances anion transport activity and allows maximal transport (Vince et al. 2000, Sterling et al. 2001). In addition, CAII may be needed for full-efﬁcacy HCO₃^- transport by anion transporter SLC26A3 (Sterling et al. 2002). Interestingly, CAII was very recently shown to form a HCO₃^- transport metabolon complex with SLC26A6 (Alvarez et al. 2005).

Mutations in CAII lead to the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calciﬁcation. In this syndrome, renal tubular acidosis appears in a mixed form, showing both proximal and distal components. Bone marrow stem cell transplantation can correct the osteopetrosis and halt progression of cerebral calciﬁcation, but not the renal symptoms (Sly et al. 1985, McMahon et al. 2001, Alper 2002, McKusick OMIM).

2.5 PDZ Domain Containing Proteins

For any epithelium to gain directional secretory and absorptive functions, correct localization of distinct transporters to speciﬁc membranes and their strict regulation is crucial. Recent studies have shown that PDZ (PSD-95/Disc-large/ZO-1, named after the ﬁrst three proteins found to contain PDZ domains) domain containing proteins (PDZ proteins) have important roles in both of these tasks. They retain proteins on the correct membrane and bring them close to each other, enabling their interactions and the formation of transducisomes, which are spatially restricted units of functionally connected proteins such as transporters, receptors or kinases. Several different PDZ proteins targeted either for the apical or basolateral membrane have been characterized so far. Human genome analysis suggested the presence of 540 distinct PDZ domains in 306 different proteins, making the PDZ domain one of the most abun- dant modular domains found in human proteins. Multiple PDZ domains, together with other protein-interaction domains, within a single protein further expand the possible functional variety of protein networks. PDZ ligands, for their own part, are sequences a few amino acids long usually located at the target proteins’ carboxyl terminus which are needed for interactions with PDZ domain containing proteins (She- nolikar et al. 2004, Brone and Eggermont 2005).

One of the most studied apical PDZ domain containing proteins is the Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1). It was originally found to be a regulator of NHE3 (SLC9A3) (Weinman et al. 1995), and thus it is also called SLC9A3 regulatory factor 1 (SLC9A3R1). A third name for NHERF-1 is 50kDa ezrin-radixin- moesin binding phosphoprotein (EBP50) (Reczek et al. 1997, McKusick OMIM).

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NHERF-1 is expressed widely in human tissues, however most abundantly in polarized epithelia tissues such as kidney, small intestine, placenta and liver. NHERF-1 is needed at least for the cAMP mediated inhibition of NHE3, but NHE3 is able to function as a transporter even without NHERF-1. In addition, NHERF-1 has been shown to interact e.g. with SLC26A6, CFTR, and H⁺V-ATPase subunit B1, that all include carboxyl terminal consensus PDZ binding sequences (Weinman et al. 1995, Yun et al. 1997, Wang et al. 1998, Breton et al. 2000, Weinman et al. 2000, Lohi et al. 2003, Shenolikar et al. 2004, Brone and Eggermont 2005).

Thus far, no human diseases have been directly associated with NHERF-1. However, psoriasis, a chronic inflammatory dermatosis of multifactorial ethiology, has been associated in or near the NHERF-1 gene in one study (Helms et al. 2003), but that asso- ciation could not be replicated by another group (Stuart et al. 2005). Interestingly, some mutations in human disease genes that encode NHERF-1 target proteins have been associated with impaired NHERF-1 binding. For example, some mutations in CFTR cause decreased affinity for NHERF-1, possibly inflicting reduced targeting of the mutant CFTR proteins to the apical surface of epithelial cells and affecting the regulation of Cl^- transport (Moyer et al. 2000, Shenolikar et al. 2004).

3. The Human Kidney

3.1 Structure of the Human Kidney

A human kidney contains over one million functional units called nephrons. A single nephron consists of a glomerulus, which ﬁltrates primary urine, and a uriniferous tubule, which modulates the primary urine into the ﬁnally excreted urine. The uriniferous tubule is further divided into distinct segments according to the tubule shape and epithelium: proximal tubule (PT) consisting of proximal convoluted tubule and proximal straight tubule, thin segment including descending and ascending thin limbs, thick ascending tubule, macula densa, distal convoluted tubule, connecting tubule, and collecting duct (Figure 2). The descending proximal straight tubule, the thin segment and the distal thick ascending tubule together form the loop of Henle.

The collecting duct can be further subdivided into cortical, outer medullary, and inner medullary segments (Ross and Romrell 1989).

While the proximal segments consist mainly of one cell type each, there are at least three distinct cell types in the connecting tubules and collecting ducts: principal cells, and at least two subtypes of intercalated cells (IC), namely types A, and B (or alpha, and beta, respectively). Intercalated cells make up approximately 40% of the overall cell population in the cortical collecting duct and in the outer medulla, while in the inner medulla they are only found in the initial segment (Wagner and Geibel 2002).

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Figure 2.

A schematic diagram of the nephron segments.

1 = afferent arteriole; 2 = efferent arteriole; 3 = glomerulus; 4 = urinary space;

5 = Bowman’s capsule; 6 = proximal convoluted tubule; 7 = thick descending limb (proximal straight tubule); 8 = thin descending limb; 9 = thin ascending limb; 10 = thick ascending limb (distal straight tubule); 11 = macula densa; 12

= extraglomerular mesangial cells; 13 = distal convoluted tubule; 14 = connect- ing tubule; 15 = collecting duct. Units consisting of several segments: 3 + 4 + 5 = renal corpuscle; 6 + 7 = proximal tubule; 7 + 8 + 9 + 10 = the loop of Henle;

10 + 11 + 13 = distal tubule.

3.2 Kidneys Have Many Functions

Kidneys have several important physiological functions, including the regulation of water and electrolyte balance, controlling of acid-base homeostasis, excretion of metab- olites and foreign chemicals, as well as participation in the regulation of the blood pressure. Different nephron segments have specific roles in these tasks. The glomeruli together filtrate about 180 liters of primary urine within 24 hours. Over 99% of the water filtered through the glomeruli is reabsorbed in the nephron tubules. Since water transport in the kidney tubules occurs by osmotic diffusion, successful reabsorption of

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osmotic particles such as ions is of great signiﬁcance. Active secretion of ions into the produced urine occurs during its ﬂow through the tubules as well (Guyton 1991a).

3.2.1 Kidneys Regulate pH of the Body

The normal pH of arterial blood is 7.4, whereas in venous blood and interstitial ﬂuids it is about 7.35. If the pH is lower, a person is considered to have acidosis, and with higher pH values alkalotic. The normal intracellular pH ranges between 6.0 and 7.4 in various cell types. The human body is normally able to maintain body liquid pH values with the help of three compensatory mechanisms. The quickest way, acting within a fraction of a second, is the acid-base buffer systems present in all body liquids, of which HCO₃^- is the most common. According to the physiochemical equilibrium, HCO₃^- is able to rapidly balance the pH by fusing with hydrogen to form water and carbon dioxide, or vice versa, in the reaction catalyzed by carbonic anhydrases:

H⁺ + HCO₃^- ⇌ H₂CO₃ ⇌ H₂O + CO₂

If the amount of hydrogen is changed substantially, it stimulates the respiratory center, leading to an adjustment in the breathing rate within minutes. As a consequence, excretion of carbon dioxide from the body is changed (Guyton 1991a).

The third, and most powerful, compensatory mechanism is the excretion of either acidic or alkaline urine by the kidneys, requiring from several hours to many days for restoring the overall body pH balance.This is achieved by three mechanisms: reabsorption of most of the ﬁltered HCO₃^-, titration of non-bicarbonate buffers, mainly phosphate, and generation and excretion of ammonium cations (Guyton 1991a, Capasso et al. 2002).

Distinct metabolic processes of the body yield acidic end products, in other words, surplus H⁺. Therefore under normal conditions kidneys actively excrete H⁺. The ﬁnal urine pH is a consequence of the original pH in the glomerular ﬁltrate and a series of several complex transport processes occurring along the diverse nephron segments.

Approximately 70-90% of HCO₃^-is reabsorbed in the proximal tubule, predominantly in the initial parts of them, coupled to H⁺ secretion. Large amounts of H⁺ are secreted as a result of countertransport with significant Na⁺ reabsorption, chiefly through the apical NHE3. Smaller quantities of H⁺ are transported Na⁺ independently through the proton pump V-ATPase (Laghmani et al. 2002). In the loop of Henle (mainly in the thick ascending limb) and distal convoluted tubule, an additional 5-15% and 5- 9% of the filtrated HCO₃^- is reabsorbed, respectively, with a proposed major role of H⁺/Na⁺ exchange again.

The collecting duct serves as the ﬁnal regulator of the urine pH and H⁺ excretion, affected by several factors like hormones and diet. The two types of intercalated cells

The Molecular Characterization and Expression of New Human SLC26 Anion Transporters