Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Zifarelli, Giovanni; Pusch, Michael

doi:10.1038/emboj.2008.284

Cited by 97 publications

(142 citation statements)

References 37 publications

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“…They increased ϳ3-fold in the presence of extracellular NO 3 Ϫ (Fig. 5E), coinciding with the recent parallel work by Zifarelli and Pusch (38). Whereas currents of WT ClC-5 are also larger with NO 3 Ϫ than with Cl Ϫ (Fig.…”

Section: Resultssupporting

confidence: 76%

“…ϩ exchange both in the present study as well as in parallel work by Zifarelli and Pusch (38). A novel method (43) to measure proton transport allowed these authors to show that ClC-5(S168P) had gained an NO 3 Ϫ /H ϩ coupling ratio of ϳ2 at voltages between ϩ40 and ϩ60 mV, indistinguishable from that for Cl Ϫ /H ϩ exchange (38).…”

Section: /Hsupporting

confidence: 49%

“…A novel method (43) to measure proton transport allowed these authors to show that ClC-5(S168P) had gained an NO 3 Ϫ /H ϩ coupling ratio of ϳ2 at voltages between ϩ40 and ϩ60 mV, indistinguishable from that for Cl Ϫ /H ϩ exchange (38). Our data on other ClC-5 mutants at this position (Fig.…”

Section: /Hmentioning

confidence: 70%

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Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Bergsdorf

Zdebik

Jentsch

2009

Journal of Biological Chemistry

108

127

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CLC proteins are found in all phyla from bacteria to humans and either mediate electrogenic anion/proton exchange or function as chloride channels (1). In mammals, the roles of plasma membrane CLC Cl Ϫ channels include transepithelial transport (2-5) and control of muscle excitability (6), whereas vesicular CLC exchangers may facilitate endocytosis (7) and lysosomal function (8 -10) by electrically shunting vesicular proton pump currents (11). In the plant Arabidopsis thaliana, there are seven CLC isoforms (AtClC-a-AtClC-g) 2 (12-15), which may mostly reside in intracellular membranes. AtClC-a uses the pH gradient across the vacuolar membrane to transport the nutrient nitrate into that organelle (16). This secondary active transport requires a tightly coupled NO 3 Ϫ /H ϩ exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1 (one of the two CLC isoforms in Escherichia coli) display tightly coupled Cl Ϫ /H ϩ exchange, but anion flux is largely uncoupled from H ϩ when NO 3 Ϫ is transported (17-21). The lack of appropriate expression systems for plant CLC transporters (12) has so far impeded structure-function analysis that may shed light on the ability of AtClC-a to perform efficient NO 3 Ϫ /H ϩ exchange. This dearth of data contrasts with the extensive mutagenesis work performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues (22, 23) and the investigation of mutants (17, 19 -21, 24 -29) have yielded important insights into their structure and function. CLC proteins form dimers with two largely independent permeation pathways (22,25,30,31). Each of the monomers displays two anion binding sites (22). A third binding site is observed when a certain key glutamate residue, which is located halfway in the permeation pathway of almost all CLC proteins, is mutated to alanine (23). Mutating this gating glutamate in CLC Cl Ϫ channels strongly affects or even completely suppresses single pore gating (23), whereas CLC exchangers are transformed by such mutations into pure anion conductances that are not coupled to proton transport (17,19,20). Another key glutamate, located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark of CLC anion/proton exchangers. Mutating this proton glutamate to nontitratable amino acids uncouples anion transport from protons in the bacterial EcClC-1 protein (27) but seems to abolish transport altogether in mammalian . In those latter proteins, anion transport could be restored by additionally introducing an uncoupling mutation at the gating glutamate (21).The functional complementation by 32) 2 The abbreviations used are: AtCIC-n, member n of the CLC family of Cl Ϫ channels and transporters in the plant Arabidopsis thaliana; CIC-n, member n of the CLC family of chloride channel and transporters (in animals); pH i , intracellular pH; pH 0 , extracellular pH; I(NO 3 Ϫ ) and I(Cl Ϫ ), current in the presence of NO 3 Ϫ and Cl Ϫ , respectively, which in CLC exchangers also involves an H ϩ component; WT, wild-type...

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Section: Resultssupporting

confidence: 76%

Section: /Hsupporting

confidence: 49%

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Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Bergsdorf

Zdebik

Jentsch

2009

Journal of Biological Chemistry

108

127

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“…It is expressed in different cell types in vesicles of the endocytotic-lysosomal pathway; in osteoclasts, ClC-7 is localized also in the ruffled border and is involved in the acidification of the resorption lacuna. Recently it has been shown that, as previously suggested by work on bacterial ClC homologues, the ClC-7 protein and other members of the ClC family are not passive chloride ion channels, but rather secondary active chloride/proton antiporters (Accardi & Miller, 2004;Picollo & Pusch, 2005;Scheel et al, 2005;Graves et al, 2008;Zifarelli & Pusch, 2009).…”

Section: Introductionmentioning

confidence: 82%

Molecular and clinical heterogeneity in CLCN7-dependent osteopetrosis: report of 20 novel mutations

et al. 2010

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ABSTRACT:The "Osteopetroses" are genetic diseases whose clinical picture is caused by a defect in bone resorption by osteoclasts. Three main forms can be distinguished on the basis of severity, age of onset and means of inheritance: the dominant benign, the intermediate and the recessive severe form. While several genes have been involved in the pathogenesis of the different types of osteopetroses, the CLCN7 gene has drawn the attention of many researchers, as mutations within this gene are associated with very different phenotypes. We report here the characterization of 25 unpublished patients which has resulted in the identification of 20 novel mutations, including 11 missense mutations, 6 causing premature termination, 1 small deletion and 2 putative splice site defects. Careful analysis of clinical and molecular data led us to several conclusions. First, intermediate osteopetrosis is not homogeneous, since it can comprise both severe dominant forms with an early onset and recessive ones without central nervous system involvement. Second, the appropriateness of haematopoietic stem cell transplantation in CLCN7-dependent ARO patients has to be carefully evaluated and exhaustive CNS examination is strongly suggested, as transplantation can almost completely cure the disease in situations where no primary neurological symptoms are present. Finally, the analysis of this largest cohort of CLCN7-dependent ARO patients together with some ADO II families allowed us to draw preliminary genotype-phenotype correlations suggesting that haploinsufficiency is not the mechanism causing ADO II. The availability of biochemical assays to characterize ClC-7 function will help to confirm this hypothesis.

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“…Residue S107 in this sequence appears to be an intracellular gate to the Cl − -permeation pathway (3,10). Mutations at 107 unvaryingly disrupt anion transport, with mixed effects on H + transport (21,25,(42)(43)(44). The surgical precision with which I109F retards H + transport demonstrates that different regions of the GSGIPE signature sequence can have separable effects on H + and Cl − transport, despite the close juxtaposition of the H + /Cl − pathways at this region of the protein.…”

Section: Discussionmentioning

confidence: 99%

Water access points and hydration pathways in CLC H ⁺ /Cl ⁻ transporters

Han

Cheng

Maduke

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl − has been established, the pathway of H + translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H + -translocation. An extended (0.4 μs) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, sitespecific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-Å hydrophobic region between the two known H + transport sites [E148 (Glu ex ) and E203 (Glu in )]. Our finding that the formation of these water wires requires the presence of Cl − explains the previously mystifying fact that Cl − occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H + transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H + and Cl − ions in CLC transporters. membrane transporters | membrane proteins | membrane exchangers | antiporters | coupling mechanism T he chloride channel (CLC) family (1, 2) includes both passive Cl − channels and secondary active H + -coupled Cl − transporters (3-8). The latter, also known as H + /Cl − exchangers, drive uphill movement of H + by coupling the process to downhill movement of Cl − or vice versa, thereby exchanging the two types of ions across the membrane at fixed stoichiometry (9). ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3)(4)(5)(6)(7)(8)(12)(13)(14)(15)(16)(17). Detailed structural and functional studies of 11,[18][19][20][21][22][23][24][25][26][27] have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl − permeation pathway and its lining residues (10,18,25) and established the role of E148, also known as Glu ex , as the extracellular gate for the Cl − pathway (9, 11).Although much less is known about the H + translocation pathway (and mechanism), experimental studies have provided key information ...

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Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Cited by 97 publications

References 37 publications

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Molecular and clinical heterogeneity in CLCN7-dependent osteopetrosis: report of 20 novel mutations

Water access points and hydration pathways in CLC H ⁺ /Cl ⁻ transporters

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Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Cited by 97 publications

References 37 publications

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Molecular and clinical heterogeneity in CLCN7-dependent osteopetrosis: report of 20 novel mutations

Water access points and hydration pathways in CLC H + /Cl − transporters

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Water access points and hydration pathways in CLC H ⁺ /Cl ⁻ transporters