Olga Andrini

Mathilde Keck

Rodolfo Briones

Stéphane Lourdel

Rosa Vargas-Poussou

Jacques Teulon
email: jacques.teulon@upmc.fr

ClC-K chloride channels: Emerging pathophysiology of Bartter syndrome type 3

Keywords: Bartter syndrome, CLC family of chloride transporters and channels, chloride channel

teaching and research institutions in France or abroad, or from public or private research centers.

Bartter syndrome

Bartter syndrome is an autosomal recessive salt wasting tubulopathy, characterized by hypokalaemia, metabolic alkalosis and secondary hyperaldosteronism with normal-to-low blood pressure. Clinically, two variants are distinguished: antenatal Bartter syndrome (aBS) and classic Bartter syndrome (cBS) [START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Jeck | Salt handling in the distal nephron: lessons learned from inherited human disorders[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Seyberth | An improved terminology and classification of Bartter-like syndromes[END_REF][START_REF] Seyberth | Bartter-and Gitelman-like syndromes: salt-losing tubulopathies with loop or DCT defects[END_REF][START_REF] Unwin | Bartter's and Gitelman's syndromes: their relationship to the actions of loop and thiazide diuretics[END_REF]. The aBS is a severe form leading to polyhydramnios due to polyuria in utero, and premature birth. It is often complicated by dehydration episodes in the neonatal period and growth retardation. Nearly all patients present with hypercalciuria and medullary nephrocalcinosis. An elevated PGE2 production contributes to aggravate the course of the disease, a fact which prompted the alternate denomination of hyperprostaglandin E syndrome [START_REF] Seyberth | Bartter-and Gitelman-like syndromes: salt-losing tubulopathies with loop or DCT defects[END_REF].

The aBS derives from mutations of the genes encoding the Na + -K + -2Cl -cotransporter and the Kir1.1 K + channel (ROMK), SLC12A1 and KCNJ1, respectively [START_REF] Simon | Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2[END_REF][START_REF] Simon | Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK[END_REF][START_REF] Simon | Mutations in Na(K)Cl transporters in Gitelman's and Bartter's syndromes[END_REF]. In the genetically-based nomenclature they correspond to Bartter syndrome type 1 (OMIM #601678) and 2 (OMIM #241200). The cBS is usually diagnosed in early childhood. Failure to thrive is usually accompanied by low chloremia and severe hypokalemic alkalosis [START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Jeck | Salt handling in the distal nephron: lessons learned from inherited human disorders[END_REF][START_REF] Seyberth | Bartter-and Gitelman-like syndromes: salt-losing tubulopathies with loop or DCT defects[END_REF], polyuria and hypercalciuria being less frequent. The cBS is due to mutations of the CLCNKB gene encoding ClC-Kb Cl -channel [START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF]. This corresponds to Bartter syndrome type 3 in the genetically-based nomenclature (OMIM #607364). There is a large heterogeneity in Bartter syndrome type 3 presentation, extending from aBS to Gitelman syndrome [START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Jeck | Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF]. Gitelman syndrome is classically a defect in distal convoluted tubule (DCT) transport due to mutations in the SLC12A3 encoding the Na + -Cl -cotransporter [START_REF] Jeck | The diuretic-and Bartter-like salt-losing tubulopathies[END_REF][START_REF] Unwin | Bartter's and Gitelman's syndromes: their relationship to the actions of loop and thiazide diuretics[END_REF], but some patients carrying CLCNKB mutations have quite a similar phenotype, exhibiting hypomagnesemia, hypo-or normocalciuria, insensitivity to thiazide administration and absence of polyuria [START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Jeck | Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype[END_REF].

The population of chloride channels in the renal tubule includes two additional players: ClC-Ka, a Cl -channel α-subunit of the same family as ClC-Kb, which is encoded by the CLCNKA gene, and the regulatory β-subunit Barttin encoded by the BSND gene. A fourth variant of the Bartter syndrome, the aBS with sensorineural hearing loss (SNHL) is caused either by BSND mutations [OMIM #602522 [START_REF] Birkenhager | Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure[END_REF](see [START_REF] Fahlke | Physiology and pathophysiology of ClC-K/barttin channels[END_REF] for review)], or by simultaneous mutations in the CLCNKB and CLCNKA genes [OMIM#613090, [START_REF] Nozu | Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness[END_REF][START_REF] Schlingmann | Salt wasting and deafness resulting from mutations in two chloride channels[END_REF]].

ClC-K chloride channels

ClC-Ka and ClC-Kb (ClC-K1 and ClC-K2 in rat and mouse) chloride channels were cloned in the 90s by the groups of Uchida and Jentsch [START_REF] Kieferle | Two highly homologous members of the ClC chloride channel family in both rat and human kidney[END_REF][START_REF] Takeuchi | Cloning, tissue distribution, and intrarenal localization of ClC chloride channels in human kidney[END_REF][START_REF] Uchida | Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla[END_REF]. They comprise 687 amino acids and are probably present as homodimers at the plasma membrane. Except for the rat ClC-K1 [START_REF] Uchida | Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla[END_REF], functional expression of the ClC-Ks was unsuccessful until the cloning of Barttin in 2001 [START_REF] Birkenhager | Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure[END_REF] when functional studies established that Barttin was a ClC-K regulatory subunit (19, 100). The electrophysiological properties of the ClC-Ks remain imperfectly known. ClC-Ka and rat and mouse ClC-K1 have been studied at the single-channel level and show conductances of 25 to 45 pS [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Fischer | Barttin activates ClC-K channel function by modulating gating[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF][START_REF] Riazuddin | Molecular basis of DFNB73: mutations of BSND can cause nonsyndromic deafness or Bartter syndrome[END_REF] while it has not been possible until now to measure the unit conductance of ClC-Kb / ClC-K2. The only regulatory properties identified so far are the inhibition at acid extracellular pH and the activation at high extracellular calcium (19, 30, 31, 100). In addition, a block at very basic 9-11 pH has also been reported [START_REF] Gradogna | Alkaline pH block of CLC-K kidney chloride channels mediated by a pore lysine residue[END_REF]. The physiological impact of these regulations remains uncertain. On the one hand, ClC-Ka and ClC-Kb are relatively insensitive to calcium between 0.5 and 2 mM [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Gradogna | Molecular Pharmacology of Kidney and Inner Ear CLC-K Chloride Channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF], which makes it unlikely that calcium variations in the physiological range affect channel activity. On the other hand, pH changes such as those experienced during acidosis or alkalosis might affect ClC-Kb (pKa=7.9, (3)) and ClC-Ka (pKa = 7.3, (33)).

At the protein level, the degree of identity between the two isoforms is slightly higher within (about 80-90%) than across (human vs. rodent) species [START_REF] Kieferle | Two highly homologous members of the ClC chloride channel family in both rat and human kidney[END_REF][START_REF] Stauber | Cell biology and physiology of CLC chloride channels and transporters[END_REF][START_REF] Uchida | Function of chloride channels in the kidney[END_REF]. This makes it impossible to determine homologs between species on the basis of sequence comparison. However, the diverse available data have led to the consensus that rodent ClC-K1 and human ClC-Ka, on the one hand, and rodent ClC-K2 and human ClC-Kb, on the other hand, are functional orthologs [START_REF] Stauber | Cell biology and physiology of CLC chloride channels and transporters[END_REF][START_REF] Uchida | Function of chloride channels in the kidney[END_REF].

Collectively, the two ClC-K proteins are present all along the distal nephron, from the thin ascending limb to the collecting duct (in the intercalated cells), but not in the proximal tubule (19). This is confirmed by Barttin antibody staining the same segments of the renal tubule (19). The expression is restricted to the basolateral membrane (19, 89) except for the thin ascending limb (tAL)

where ClC-K is present on both apical and basolateral membranes [START_REF] Uchida | Localization and functional characterization of rat kidney-specific chloride channel, ClC-K1[END_REF]. The differential distribution of the two channels along the renal tubule has been difficult to ascertain in the absence of isoformspecific antibodies. However, immunocytochemistry performed on the kidneys of ClC-K1 -/-mice demonstrated that ClC-K1 was the only isoform present in the tAL [START_REF] Kobayashi | Intrarenal and cellular localization of CLC-K2 protein in the mouse kidney[END_REF] and that ClC-K2 was present in the thick ascending limb (TAL), DCT and the intercalated cells of cortical collecting duct (CCD) [START_REF] Kobayashi | Intrarenal and cellular localization of CLC-K2 protein in the mouse kidney[END_REF].

Patch-clamp experiments have also given some insight into the localization of the two channels: a recent study has demonstrated the correspondence between 45-pS chloride channel in the mouse TAL [START_REF] Paulais | cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney[END_REF] and recombinant mouse ClC-K1 [START_REF] L'hoste | Characterization of the mouse ClC-K1/Barttin chloride channel[END_REF]. Thus, a second 10-pS channel, activated by external calcium and at alkaline pH, which is expressed in the DCT and intercalated cells of the CNT and CCD, probably represents ClC-K2 [START_REF] Lourdel | A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel[END_REF][START_REF] Nissant | Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by singlecell RT-PCR and patch clamp[END_REF][START_REF] Nissant | Similar chloride channels in the connecting tubule and cortical collecting duct of the mouse kidney[END_REF][START_REF] Paulais | cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney[END_REF][START_REF] Teulon | Exploration of the basolateral chloride channels in the renal tubule using[END_REF]. Together with results obtained with RT-PCR on rat renal segments (19, 44-46, 49, 96, 100), a consensus pattern emerges, which places ClC-K1 in the tAL and TAL, and ClC-K2 in the TAL, DCT and the intercalated cells of the CNT and CD (Fig. 1a).

A major insight in ClC-Ka function was provided by Uchida and associates [START_REF] Akizuki | Impaired solute accumulation in inner medulla of Clcnk1-/-mice kidney[END_REF][START_REF] Liu | Analysis of NaCl transport in thin ascending limb of Henle's loop in CLC-K1 null mice[END_REF][START_REF] Matsumura | Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel[END_REF] who analyzed the consequences of CLCNK1 deletion in the mouse. Matsumura et al [START_REF] Matsumura | Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel[END_REF] observed no hypokalaemic alkalosis in Clcnk1 -/-mice, which would have been suggestive of Bartter syndrome, but polyuria (x5 compared to wild type), associated with low urine osmolality (3-fold lower than WT), was dramatic. Furthermore, Clcnk1 -/-mice failed to concentrate urine after 24-h water deprivation or intraperitoneal injection of dDAVP (∼2500 mOsm/kg H 2 O in WTmice vs. ∼850 mOsm/kg H 2 O in Clcnk1 -/-mice) [START_REF] Matsumura | Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel[END_REF]. A follow-up study showed that the fractional excretion of sodium, chloride and urea, as well as the total osmolar clearance, were not altered in Clcnk1 -/-mice (1). Thus, polyuria is due to water diuresis, not osmotic diuresis. In addition, the urea and NaCl contents in the interstitium of the inner medulla were decreased by a factor 2 in Clcnk1 -/-as compared to Clcnk1 +/+ mice [START_REF] Akizuki | Impaired solute accumulation in inner medulla of Clcnk1-/-mice kidney[END_REF]. Furthermore, no Cl -conductance was detected in microperfused tALs of ClC-K1 -/-mice [START_REF] Liu | Analysis of NaCl transport in thin ascending limb of Henle's loop in CLC-K1 null mice[END_REF][START_REF] Matsumura | Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel[END_REF]. Thus, the loss-of-function of ClC-K1 causes nephrogenic diabetes insipidus by impairing the countercurrent system in the inner medulla [START_REF] Akizuki | Impaired solute accumulation in inner medulla of Clcnk1-/-mice kidney[END_REF]. As pointed out by Matsumura et al [START_REF] Matsumura | Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel[END_REF], it is doubtful that CLCNKA deletion would cause a similar pathology in humans, who have a less developed inner medulla.

The results of Uchida's group confirm that ClC-K1 is mostly present in the thin ascending limb. Thus, although no Clcnk2 -/-mouse line is yet available, we may speculate that ClC-K2 has a predominant role in the other parts of the mouse renal tubule. Ion transport models for the TAL and DCT (Fig 1b,c) involve two distinct Na + -Cl -cotransporters, NKCC2 and NCC, respectively, at the apical membrane. The exit of chloride on the basolateral side is dependent on Cl -channels [START_REF] Eladari | Electroneutral absorption of NaCl by the aldosterone-sensitive distal nephron: implication for normal electrolytes homeostasis and blood pressure regulation[END_REF][START_REF] Jeck | Salt handling in the distal nephron: lessons learned from inherited human disorders[END_REF][START_REF] Sepulveda | Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels[END_REF].

Basolateral Cl -channels are also important for ion transport in the intercalated cells [START_REF] Eladari | Electroneutral absorption of NaCl by the aldosterone-sensitive distal nephron: implication for normal electrolytes homeostasis and blood pressure regulation[END_REF][START_REF] Sepulveda | Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels[END_REF][START_REF] Staruschenko | Regulation of transport in the connecting tubule and cortical collecting duct[END_REF] (Fig1d). Classically, the β intercalated cells participate in bicarbonate secretion but Eladari and Chambrey [START_REF] Chambrey | Renal intercalated cells are rather energized by a proton than a sodium pump[END_REF][START_REF] Eladari | Electroneutral absorption of NaCl by the aldosterone-sensitive distal nephron: implication for normal electrolytes homeostasis and blood pressure regulation[END_REF][START_REF] Leviel | The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice[END_REF] recently discovered a novel, neutral pathway for NaCl absorption in these cells (Fig 1d), which includes a Cl -conductance at the basolateral side [START_REF] Eladari | Electroneutral absorption of NaCl by the aldosterone-sensitive distal nephron: implication for normal electrolytes homeostasis and blood pressure regulation[END_REF][START_REF] Sepulveda | Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels[END_REF][START_REF] Staruschenko | Regulation of transport in the connecting tubule and cortical collecting duct[END_REF]. The α intercalated cells (Fig 1d) participate in acid-base balance by secreting protons via the V-type H + ATPase at the apical membrane and the AE1 HCO 3 -/Cl -exchanger at the basolateral membrane. Basolateral Cl -channels optimize H + secretion by recycling Cl -, thus facilitating the functioning of the AE1 exchanger [START_REF] Staruschenko | Regulation of transport in the connecting tubule and cortical collecting duct[END_REF]. In summary, ClC-K2 is expected to play a key role in Cl -absorption in the DCT, CNT/CCD and to a lower extent in the TAL, where ClC-K1 is present [START_REF] L'hoste | Characterization of the mouse ClC-K1/Barttin chloride channel[END_REF][START_REF] Paulais | cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney[END_REF]. There has been yet no patch-clamp study of ClC-K chloride channels in the α intercalated cells, letting open the possibility that the two channels cooperate at this site.

Structure of the ClC-K chloride channels

Renal chloride channels belong to the channel subgroup of the ClC family of chloride channels and transporters, which also includes ClC-1 and ClC-2 [START_REF] Fahlke | Physiology and pathophysiology of ClC-K/barttin channels[END_REF][START_REF] Jentsch | CLC chloride channels and transporters: from genes to protein structure, pathology and physiology[END_REF][START_REF] Uchida | In vivo role of CLC chloride channels in the kidney[END_REF]. The structure of this protein family was revealed in 2002 with the crystallization of two bacterial Cl -/H + exchangers from S. typhimurium and the E. coli [START_REF] Dutzler | X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity[END_REF]. Each monomer is defined by 18 α-helices (from A to R) with an antiparallel structure, meaning that two structurally related halves (α-helices A to I and J to R, respectively) span the membrane with an opposite orientation [START_REF] Dutzler | X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity[END_REF]. The ClC proteins are functional dimers constituted by two independent permeation pathways called protopores (see Fig. 2). The protopore, localized between the two structurally related halves of the ClC monomer, is formed by an anion-selective filter between two aqueous vestibules comprising three Cl --binding sites. The binding sites are located close to the extracellular side (S ext ), in the central region of the protein (S cen ) and close to the intracellular side (S in ) [START_REF] Dutzler | Gating the selectivity filter in ClC chloride channels[END_REF]. These are formed by the N-termini of α-helices D and F in the first half of the monomer and the N-termini of α-helices N and R in the second half of the monomer. In ClC-0, ClC-1 and ClC-2 but not ClC-K, the access to each protopore is regulated via one opening / closing mechanism called fast, or "glutamate" gate. The structural determinant of this gating consists of an external glutamate (E148 in E. coli, ec-ClC), the protonation/deprotonation of which is directly implicated in gating [START_REF] Dutzler | Gating the selectivity filter in ClC chloride channels[END_REF][START_REF] Niemeyer | A conserved pore-lining glutamate as a voltage-and chloride-dependent gate in the ClC-2 chloride channel[END_REF]. In addition, the two protopores can be opened / closed simultaneously by the so called common, or slow gate [START_REF] Chen | Structure and function of clc channels[END_REF][START_REF] Pusch | Structural insights into chloride and proton-mediated gating of CLC chloride channels[END_REF]. This second mechanism is particularly important for the renal ClC-Ks that lack the characteristic "glutamate" responsible for protopore gating in the others ClC proteins [START_REF] Dutzler | Gating the selectivity filter in ClC chloride channels[END_REF].

Eukaryotic ClC proteins, including ClC-Ks, have a large cytoplasmic C-terminus domain constituted by a linker sequence connecting α-helix R to two cystathione-β-syntase domains (CBSs)

[see Fig. 2]. The crystal structure of human CBS of ClC-5 [START_REF] Meyer | Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5[END_REF] and ClC-Ka (58) reveals a dimeric interaction between two ClC protein C-terminal domains (59), likely to be conserved among the eukaryotic ClC [START_REF] Meyer | Crystal structure of the cytoplasmic domain of the chloride channel ClC-0[END_REF]. Several studies reported a regulatory role of binding of adenine nucleotides to CBS domains in ClC-1, ClC-2 and ClC-5 [START_REF] Niemeyer | Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies[END_REF][START_REF] Tseng | Binding of ATP to the CBS domains in the C-terminal region of CLC-1[END_REF]105). This part of the protein is known to be involved in the regulation of the common gating [START_REF] Bykova | Large movement in the C terminus of CLC-0 chloride channel during slow gating[END_REF]20). A similar role has yet to be demonstrated for the ClC-Ks.

As noted above, the ClC-K currents recorded in over-expression systems are inhibited at acid pH and activated by an increase of [Ca 2+ ] ext (19,[START_REF] Gradogna | A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels[END_REF][START_REF] Gradogna | Dissecting a regulatory calciumbinding site of CLC-K kidney chloride channels[END_REF]100). A detailed screening of acidic aminoacid residues led to the identification of two critical amino-acids (E261 and D278, localized in the extracellular loop between α-helices I and J), which form the putative extracellular Ca 2+ -binding site [START_REF] Gradogna | Dissecting a regulatory calciumbinding site of CLC-K kidney chloride channels[END_REF]. A histidine residue on position 497 at the beginning of α-helix Q is responsible for the H +induced block [START_REF] Gradogna | A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels[END_REF] [Fig. 2].

CLCNKB mutations in Bartter syndrome

Since the pioneering work of A210V and R351W currents were reduced by 53 and 39%, respectively. Overall, all these mutations (except A210V and R351W) reduced channel currents by more than 60% (19, 102).

Our group has recently investigated 8 novel CLCNKB mutations (L81P, R92W, G120R, V170M, G246R, R351P, G424E and L439P) [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF]. We also selected some published mutations to gain a deeper understanding of the molecular defect behind the development of Bartter syndrome type III (T115P, P124L, L139P and R438H) [START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF]103). In these studies, we co-expressed mutant ClC-Kb proteins with wild-type Barttin in X. laevis oocytes or HEK293 cells. The currents carried by the ClC-Kb mutants were reduced to different extents. For nearly half of the mutants [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF], no current was detected (T115P, G246R, G424E, R438H and L439P). Four mutants showed 50-70% residual activity (R92W, P124L, V170M, R351P), and two had a low activity around 20-40% (L81P, G120R) [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF].

Note that in our hands, P124L generated a noticeable current (40%) while lower currents (0-20%)

were detected in previous studies [START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Simon | Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK[END_REF].

Surface expression varied from 0 to 85% when compared to WT ClC-Kb and was proportional to current level, indicating that the primary defect shown by mutants concerns the number of channel units in the membrane [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF]. For mutants generating sufficient current, we evaluated unit conductance (R92W, R351P), anion selectivity (L81P, R92W, V170M and R351P), and pH-and Ca 2+sensitivity (L80P, R92W, R351P, P124L and V170M) [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF]. There was no change in conductance, selectivity or regulation except for two mutants (P124L, V170M) that displayed dramatic alteration in pH-sensitivity (see below). These results go along with the hypothesis that the primary defect induced by mutations is generally an alteration in surface expression of the channel.

Altogether, among the 20 mutations analyzed for the total current, 40% do not show any detectable activity (T115P, G246R, A349D, G424E, G424R, R438C, R438H and L439P), 40% maintain more than 50% activity (R92W, G120R, P124L, V170M, A210V, R351P, R351W and K560M) and 20%

have a smaller activity around 20-30% (L81P, L139P, A204T and R538P). Total protein abundance (considering bands corresponding to monomeric and dimeric proteins) was assayed in HEK293 cells for a minority of mutants [START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF]. Protein abundance was clearly reduced for those mutants that showed no current and no surface expression (G246R, G424E, R438H and L439P). This strongly suggests that the produced transcript is not stable in the expression system. Protein abundance was not reduced for mutants showing more than 50% activity (R92W and R351P) nor for one mutant with 20-30% activity (L81P). This suggests that the stability of the protein (for instance, accelerated retrieval from the membrane) in the membrane could be decreased.

Mutations that alter regulation/conduction appear to be predominant in myotonia caused by mutations in the CLCN1 gene: ClC-1 is a voltage-dependent channel, which is activated by depolarization; many mutations profoundly shift voltage dependence towards more positive voltages [START_REF] Pusch | Myotonia caused by mutations in the muscle chloride channel gene CLCN1[END_REF][START_REF] Pusch | Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 chloride channel[END_REF] or even convert ClC-1 to an inwardly-rectifying channel [START_REF] Fahlke | An aspartic acid residue important for voltage-dependent gating of human muscle chloride channels[END_REF]104). By contrast, mutations altering channel gating are not frequent in Bartter disease type 3. We have evaluated pH-and Ca 2+ -sensitivity for 6 ClC-Kb mutants and found alterations in only 2 [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF], while studies by others detected 2 additional mutants showing some kind of altered regulation. Maduke et al [START_REF] Martinez | A cytoplasmic domain mutation in ClC-Kb affects long-distance communication across the membrane[END_REF] reported that R538P mutation totally abolished sensitivity to Ca 2+ . This is associated with a clear reduction in current (19). According to the authors, the arginine 538 being located in the cytoplasm after helix R, the lack of sensitivity to Ca 2+ implies a long distance effect from the cytoplasmic side of the protein to its extracellular side [START_REF] Martinez | A cytoplasmic domain mutation in ClC-Kb affects long-distance communication across the membrane[END_REF]. Similar Ca 2+ -independence was reported by Yu et al (102) for R351W mutation (at the outer aspect of the protein in K-L linker), which was also associated with 60% decrease in current.

In our hands, two mutations (V170M and P124L) dramatically altered pH and Ca 2+ sensitivity

(3). The curve of H + -dependent inhibition shifted by about 1.5 pH units towards more acid values for V170M and P124L mutants as compared to WT ClC-Kb (pK H : 6.0 for V170M and 6.1 for P124L vs. 7.6

for WT). Our results suggest that these mutations do not alter directly the binding-site of H + but disrupt the gating function of the channel, thus indirectly affecting the sensitivity to pH ext [START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF]. As a direct consequence, the mutant ClC-Kb channels are maximally active at pH 7.4 while the activity of WT ClC-Kb at this pH is only 20% of its maximal activity. In addition, we observed that the mutants were completely independent of Ca 2+ at pH 7.4, perhaps because they are maximally active at this pH (3). In parallel, there was a clear reduction in current and surface expression in the two mutants (3).

Therefore, these mutations result in hyperactive channels, which tend to offset the functional impact of reduced expression at the membrane. The opposite mechanism might operate in the case of T481S ClC-Kb, a CLCNKB single-nucleotide polymorphism that has been linked to essential hypertension [START_REF] Jeck | Activating mutation of the renal epithelial chloride channel ClC-Kb predisposing to hypertension[END_REF][START_REF] Sile | CLCNKB-T481S and essential hypertension in a Ghanaian population[END_REF]. Jeck et al [START_REF] Jeck | A common sequence variation of the CLCNKB gene strongly activates ClC-Kb chloride channel activity[END_REF] showed that the 5-20-fold increase in ClC-Kb current induced by T481S mutation was associated with higher sensitivity to pH (lower activity at pH 7.4) and an increase in membrane channel expression, a mirror image of what is observed in Bartter syndrome for ClC-Kb mutants.

Alteration of functional motifs as a cause of Bartter syndrome

Several of the mutations that have been functionally analyzed are located around the selectivity filter. Three mutations in α-helix N (R438C, R438H, L439P) and one close to it (G424E) are associated with reduction in total protein abundance, absence of expression at the membrane and absence of current. Two mutations (P124L and V170M) alter channel regulation. These results strongly suggest that the selectivity filter is of paramount importance for structure stability and gating. The hyperactivity of P124L and V170M is puzzling when considered in the context of disease pathophysiology. As already mentioned, mutations of the CLCN1 gene causing myotonia often result in gating alterations. However, these alterations always render the channel less active [START_REF] Pusch | Myotonia caused by mutations in the muscle chloride channel gene CLCN1[END_REF]. Thus, it is currently difficult to appreciate whether the uncoupling of channel activity from pH-and Ca 2+dependent gating is a co-incidental side-effect of the mutations or whether these mutations alter yet unidentified regulators of channel activity.

By contrast, the available data provides little information about the functional effects of CLCNKB mutations in two other regions: α-helices B and J (8 mutations), which interact with Barttin, and CBS domains (7 mutations). The CBS domains might be particularly critical for channel function.

They participate in ClC common gating (20,[START_REF] Fong | Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata[END_REF], likely by sensing intracellular metabolites [START_REF] Ignoul | CBS domains: structure, function, and pathology in human proteins[END_REF]. They also participate in channel trafficking and targeting to the plasma membrane. The partial deletion of the cytoplasmic domain leads to a retention of ClC-0 and ClC-1 proteins into the ER (20,[START_REF] Hebeisen | The role of the carboxyl terminus in ClC chloride channel function[END_REF][START_REF] Maduke | Formation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains[END_REF][START_REF] Schmidt-Rose | Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1[END_REF].

The only CBS1-located mutation that has been functionally characterized in over-expression systems (K560M) reduces ClC-Kb current to 25% of control (19).

The interface between the two monomers, mainly composed of α-helices H, I, P and Q, is potentially another critical region. In the case of Dent's disease, an X-linked hereditary disease due to mutations in the CLCN5 gene encoding the Cl -/H + exchanger ClC-5, about 60% of the mutations are located at the dimer interface [START_REF] Lourdel | ClC-5 mutations associated with Dent's disease: a major role of the dimer interface[END_REF]. These mutations either result in ER retention or channel dysfunction [START_REF] Lourdel | ClC-5 mutations associated with Dent's disease: a major role of the dimer interface[END_REF]. Mutations at the same location are also frequent in CLCN7 gene causing osteopetrosis, or in CLCN1 gene, where they modify channel gating [START_REF] Pusch | Myotonia caused by mutations in the muscle chloride channel gene CLCN1[END_REF]. Unexpectedly, only 3 mutations have been found at the dimer interface in Bartter disease: P216L (51) and A242E (5), which have not been functionally investigated and G246R which shows no current, no surface expression and reduced protein abundance [START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF]. The latter result suggests a destabilization of the protein.

Obviously, further experiments focusing on surface channel expression and sub-cellular distribution would be necessary in the future to investigate the functional consequences of mutations in the CBS region, in the α-helices interacting with Barttin and at the dimer interface.

Phenotype-genotype relationship

The first papers reporting mutations in the CLCNKB gene causing Bartter syndrome type 3 already mentioned that the severity of the disease was highly variable [START_REF] Jeck | Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF]. In particular, whole gene deletion, which is frequent in this disease, may cause aBS or cBS [START_REF] Jeck | Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF]. This is also the case for A204T and R438H mutations [START_REF] Castano | Genetics of type III Bartter syndrome in Spain, proposed diagnostic algorithm[END_REF][START_REF] Rodriguez-Soriano | A founder mutation in the CLCNKB gene causes Bartter syndrome type III in Spain[END_REF]103). R438H mutation results in a non-functioning channel [START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF], A204T reduces current to 0-25% of control [START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF]. In particular, Zelikovic et al (103) showed that patients carrying the mutation R438H within one same large family could present with mild or severe symptoms. Especially in the case of CLCNKB gene deletion, the variable severity of the disease suggests that ClC-Ka Cl -channel, K + -Cl -cotransporter (8) or additional Cl -channels (60) might be expressed in the TAL in a variable fashion and compensate for the loss of function of ClC-Kb in some patients. In the case of point mutations, one alternative possibility would be that the defect being due to conformational destabilization has variable impact depending on the individual.

The 8 patients homozygous for V170M, or compound heterozygous with another mutation (whole gene deletion or mutations resulting in the production of unstable mRNAs or truncated proteins) have a mild phenotype (3). It is likely that these patients mainly express V170M homodimers of this hyperactive channel with reduced expression at the membrane. This is also the case for one patient homozygous for P124L, but not for 2 other patients compound heterozygous for P124L and G465R (3). It will be interesting to observe whether more mutations of this type are detected in the future and whether they are associated with a mild presentation.

Conclusion

Overall, currently available data suggests that CLCNKB mutations might be arranged in two main classes: (i) mutations causing fully impaired (about 40% of the mutations) or reduced (about 60% of the mutations) channel expression at the membrane; (ii) mutations combining reduced channel surface expression with altered channel properties (about 20% of the total number of mutations analyzed).

It is clear that further investigation is needed to fully characterize the pathogenesis of Bartter disease type 3, which has been hampered by technical difficulties in the expression of this particular chloride channel. However, Bartter disease type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing retention in the endoplasmic reticulum and accelerated channel degradation. Accordingly, a classical approach to treatment would be to test the efficacy of adapted chaperons for enhancing ClC-Kb surface expression. An approach of this type has been recently performed with some success on W610X ClC-Kb mutants in MDCK cells using an aminoglycoside derivative [START_REF] Cho | Translational read-through of a nonsense mutation causing Bartter syndrome[END_REF]. This long-term strategy might be helpful for those patients who present with a severe form of Bartter syndrome type 3.

At the end of this review dedicated to the pathophysiology of Bartter syndrome, it is worth reminding that one CLCNKB single-nucleotide polymorphism (T481S) has been linked to essential hypertension [START_REF] Jeck | Activating mutation of the renal epithelial chloride channel ClC-Kb predisposing to hypertension[END_REF][START_REF] Sile | CLCNKB-T481S and essential hypertension in a Ghanaian population[END_REF]. Although the association with hypertension was not replicated by some studies [START_REF] Fava | The functional variant of the CLC-Kb channel T481S is not associated with blood pressure or hypertension in Swedes[END_REF][START_REF] Kokubo | Association analysis between hypertension and CYBA, CLCNKB, and KCNMB1 functional polymorphisms in the Japanese population--the Suita Study[END_REF][START_REF] Speirs | No association with hypertension of CLCNKB and TNFRSF1B polymorphisms at a hypertension locus on chromosome 1p36[END_REF], the idea that ClC-Kb might be functionally important for long-term control of blood pressure remains attractive, and keeps in line with recent findings pointing out the importance of chloride in blood pressure regulation [START_REF] Eladari | Electroneutral absorption of NaCl by the aldosterone-sensitive distal nephron: implication for normal electrolytes homeostasis and blood pressure regulation[END_REF]. In addition, several CLCNKA single-nucleotide polymorphisms have been associated with salt-sensitive hypertension (4) or heart failure [START_REF] Cappola | Loss-of-function DNA sequence variant in the CLCNKA chloride channel implicates the cardio-renal axis in interindividual heart failure risk variation[END_REF].

Altogether, these various reports suggest that the pathophysiological impact of ClC-Kb and ClC-Ka on NaCl balance might be not restricted to Bartter syndrome. ClCs using using Multalin software for multiple sequence alignments [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF], plus manual adjustements.

The location of the binding sites slightly depends on the homology model, especially for the Ca 2+ binding site. As a result, the number of spheres is larger than the number of involved amino acids.

Pymol software (The PyMOL Molecular Graphics System, Version 1.7, Schrödinger, LLC) was used to render  ic

 ic

FIGURE LEGEND Figure 1 .

 1 FIGURE LEGENDFigure1. Localization of the two ClC-K channels along the rodent renal tubule and ion transport systems in various parts of the distal nephron. A) Localization of ClC-K1 (blue) and ClC-K2 (orange) along the nephron. The localization is based on RTPCR on rat isolated renal segments[START_REF] Kramer | Mechanisms of Disease: the kidneyspecific chloride channels ClCKA and ClCKB, the Barttin subunit, and their clinical relevance[END_REF][START_REF] Nissant | Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by singlecell RT-PCR and patch clamp[END_REF] 100), immunochemistry(19,[START_REF] Kobayashi | Intrarenal and cellular localization of CLC-K2 protein in the mouse kidney[END_REF] and patch-clamp studies on the mouse renal tubule[START_REF] L'hoste | Characterization of the mouse ClC-K1/Barttin chloride channel[END_REF][START_REF] Lourdel | A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel[END_REF][START_REF] Nissant | Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by singlecell RT-PCR and patch clamp[END_REF][START_REF] Nissant | Similar chloride channels in the connecting tubule and cortical collecting duct of the mouse kidney[END_REF][START_REF] Paulais | cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney[END_REF].The distribution pattern remains incomplete: 1) in the absence of a dedicated patch-clamp study, it is

Figure 2 .

 2 Figure 2. Homology model of the ClC-Kb Cl -channel based on the Eukaryotic ClC of

Figure 3 .

 3 Figure 3. Mutations in the ClC-Kb Cl -channel associated with Bartter disease. The location of mutations is illustrated on a topological model of ClC-Kb where rectangles represent α-helices. The plasma membrane is not shown; the lower part of the model is intracellular, the upper part extracellular. The letters indicate the corresponding α-helices and CBS1 and CBS2 the two cystathione-β-syntase domains. The α-helices participating in the selectivity filter, those interactingwith Barttin and those located at the dimer interface are colored in green, yellow and purple, respectively. Each symbol gives the position and the type of a single mutation, the number referring to mutations in Table1.

  Figure 1

Figure 3

 3 Figure 3

  Simon et al. identifying mutations in the ClCNKB gene as a cause of Bartter syndrome type 3[START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF], a number of publications have reported pathogenic mutations[START_REF] Andrini | CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca dependence of ClC-Kb channels[END_REF][START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Fukuyama | Novel mutations of the chloride channel Kb gene in two Japanese patients clinically diagnosed as Bartter syndrome with hypocalciuria[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Lee | Genetic basis of Bartter syndrome in Korea[END_REF][START_REF] Rodriguez-Soriano | A founder mutation in the CLCNKB gene causes Bartter syndrome type III in Spain[END_REF][START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF] 102, 103). As a whole, according to the Human Gene Mutation Database (free access HGMD database, Biobase International, www.hgmd.cf.ac.uk), more than 54 mutations have been described, including complex rearrangements, large and small deletions, small insertions, nonsense or missense mutations and splice site mutations. One characteristic feature of the disease is the frequent occurrence of total CLCNKB gene deletion[START_REF] Brochard | Phenotype-genotype correlation in antenatal and neonatal variants of Bartter syndrome[END_REF][START_REF] Castano | Genetics of type III Bartter syndrome in Spain, proposed diagnostic algorithm[END_REF][START_REF] Jeck | Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype[END_REF][START_REF] Konrad | Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome[END_REF][START_REF] Simon | Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III[END_REF]. aggregates so that the band corresponding to the monomeric protein is often hardly visible [(10, 43, 100, 101) but see[START_REF] Janssen | Diseasecausing dysfunctions of barttin in Bartter syndrome type IV[END_REF]]. Obviously, a limit of this interesting study lies in the use of chimeric channels, as it is difficult to anticipate how the presence of rat ClC-K1 stretches in the ClC-Kb protein might influence the observed effects. Soon afterwards,Estevez et al. (19) recorded for the first time ClC-K/Barttin complexes in X. laevis oocytes. They tested several of the mutations reported by Simon et al and Konrad et al (48, 83) by co-injecting mRNA for several ClC-Kb mutants with Y98A-mutant Barttin in

	Before the discovery of Barttin, Waldegger et al. (101) succeeded in recording chloride
	currents from a chimeric human ClC-Kb/rat ClC-K1 construct [ClC-Kb (c)] and analyzed a selection of
	mutations reported by Simon et al. (83) in X. laevis oocytes using two-electrode voltage-clamp:
	P124L, A204T, A349D and R438C abolished ClC-Kb (c) current while Y432H dramatically reduced it, by
	at least 4 fold.
	been found in patients (A77P, L81P, T115P, P124L, G164C, V170M, A204T, G246R, S297R, R351W,
	H357Q, G424E, R438H, K560M, R595X, W610X). A few mutations such as A204T (29, 76, 83), P124L
	(3, 5, 43, 48, 83), R438H (3, 5, 43, 48, 83) and W610X (14, 51, 68) have been reported independently

The 50 point mutations (missense and nonsense) illustrated in Fig.

1

and summarized in Table

1

are distributed all along the protein with no clear hotspots. Several mutations are located close to several parts of the selectivity filter, in C-D linker and α-helix D (3 mutations), E-F linker and α-helix F (2mutations) and α-helix N (8 mutations), respectively. By contrast, no mutations were reported in the vicinity of H + and Ca 2+ binding sites. Altogether, 60% mutations are located on α-helices, 20% in helix linkers and 20% in the cytoplasmic part of the protein. About 16 homozygous mutations have by several authors. In particular, A204T, a founder mutation in Spain, is the prevalent mutation in this country

[START_REF] Garcia-Nieto | Mutation G47R in the BSND gene causes Bartter syndrome with deafness in two Spanish families[END_REF]

. W610X is the most common mutations in Korea

[START_REF] Lee | Genetic basis of Bartter syndrome in Korea[END_REF]

.

Functional consequences of CLCNKB mutations

Only 20 CLCNKB mutations have been functionally analyzed. This low figure is mainly due to technical problems regarding ClC-Kb functional expression. Current recording of ClC-Kb in X. laevis oocytes or cultured cells has remained difficult even after the discovery of the Barttin regulatory subunit: ClC-Kb levels of current are 5-fold lower than those elicited by ClC-Ka

[START_REF] Janssen | Diseasecausing dysfunctions of barttin in Bartter syndrome type IV[END_REF][START_REF] Keck | Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression[END_REF]

. An additional, specific problem concerns Western blotting of the ClC-K protein: over-expressed ClC-K proteins tend to form stable order to increase surface expression of the channel complex. Overall A349D and R438C mutations totally abolished currents, as in the abovementioned study, while P124L, A204T, R538P and K560M mutations decreased the currents by 60-80%. More recently, Yu et al (102) analyzed R30X, A210V and R351W mutations by the same methods. As expected, R30X mutant generated no current;

Table 1 . 695 Table 1 . CLCNKB mutations and corresponding changes in amino acid sequence of the ClC-Kb chloride channel. 696 697

 16951

	Mutat		Amino
	ion numb	Nucleotide change	acid change	Protein domain	Remaining current	Sensitivity to H + and Ca 2+	Surface expression	Total ClC-Kb protein
	er

Acknowledgments

We thank the patients, their families, and the nephrologists from the French tubulopathy network. The English text was edited by Charlotte Sumida. The present address of Mathilde Keck is UMR INSERM 1166 ICAN, 91 boulevard de l'Hôpital, 75013 PARIS, France

Grants

This work was supported by grants from French Research National Agency (ANR BLAN 2010-111201, ANR-14-CE12-0013-02), French Kidney Foundation and ECOS-sud (C14-S01). The work of R. Briones was supported by a grant from DFG (SFB803).

Conflict of interest

None