Many proteins of the CLC gene family are Cl ؊ channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl ؊ /H ؉ exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl ؊ and H ؉ transport. Surface expression was unchanged by these mutations. Uncoupled Cl ؊ transport could be restored in the ClC-4 E281A and ClC-5 E268A proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H ؉ donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl ؊ /H ؉ exchange independently from the transport activity of the neighboring subunit. CLC 6 transport proteins are encoded by a large gene family with members in all phyla (1, 2). Because the founding member of this gene family, ClC-0, from the electric organ of Torpedo (3), is a chloride channel, all CLC genes were believed to encode anion channels. This is undoubtedly true for mammalian ClC-1, -2, and -K, which belong to the same homology branch as ClC-0. However, the bacterial CLC protein ecClC-1 turned out to be an electrogenic Cl Ϫ /H ϩ exchanger (4). Our studies then revealed that endosomal ClC-4 and -5, which reach the plasma membrane to some degree, are Cl Ϫ /H ϩ exchangers as well (5, 6). The fact that several members of the gene family function as ion channels, whereas others carry out stoichiometrically coupled ion exchange, provides unprecedented opportunities to elucidate the structural basis for these different transport modes.The linear I/V relationship of ecClC-1 allowed the estimation of a 2:1 stoichiometry of transport from reversal potentials (4). Unlike ecClC-1, ClC-4 and -5 mediate strongly outwardly rectifying currents (7), precluding a precise determination of their coupling ratio from reversal potentials. Comparing Cl Ϫ and H ϩ transport rates yielded estimates for the Cl Ϫ /H ϩ stoichiometry between 1 and 5 (5, 6). The biological consequences of endosomal CLCs being Cl Ϫ /H ϩ antiporters rather than Cl Ϫ channels are intriguing (8). These proteins are thought to facilitate endosomal/lysosomal acidification by neutralizing proton pump currents, a process important for endocytotic trafficking and lysosomal function. Indeed, ClC-5 is crucial for renal endocytosis and is mutated in a human disorder associated with proteinuria and kidney stones (9).It remains unclear whether Cl Ϫ /H ϩ exchange depends on the dimeric structure of CLC proteins. It is known that both pores of the double-barreled ClC-0 Cl Ϫ channel can be shut closed simultaneously by a "common gate" that depends on both subunits (10 -13). Similarly, it may be that Cl Ϫ /H ϩ flux coupling ...

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...

Members of the CLC protein family of ClMutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the "anion-coordinating" tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO 3 ؊ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl ؊ /H ؉ antiporter.The CLC gene family, originally thought to encode exclusively chloride channels, is now recognized to comprise both channels and anion-proton antiporters (1). Following the discovery that the bacterial EcClC-1 (one of the two CLC isoforms in Escherichia coli) functions as a 2ClϪ /H ϩ exchanger (2), mammalian endosomal ClC-4 and -5 were shown to mediate anion/proton exchange as well (3, 4). These endosomal electrogenic exchangers may facilitate endosomal acidification by shunting currents of the V-type ATPase and have a role in luminal Cl Ϫ accumulation (5, 6). The plant AtClC-a functions physiologically as an NO 3 Ϫ /H ϩ exchanger that uses the pH gradient over the vacuole membrane to accumulate the nutrient NO 3 Ϫ into that organelle (7). With the notable exception of renal ClC-K channels (8), both channel-and exchanger-type CLC proteins share a glutamate in the permeation pathway that is involved in gating (in CLC channels) and in coupling chloride to proton countertransport (in CLC exchangers), respectively. Mutations in this gating glutamate profoundly affect CLC channel gating and uncouple anion from proton countertransport in CLC exchangers. All confirmed CLC antiporters display another glutamate (the proton glutamate) at their cytoplasmic surface that probably transfers protons to the central exchange site given by the gating glutamate (9 -11). Because this proton glutamate is not found in confirmed CLC channels, its presence might indicate that the respective CLC is an exchanger.Based on this hypothesis, ClC-3-7 should function as Cl Ϫ /H ϩ exchangers, but this remains to be shown for ClC-3, -6, and -7 by heterologous expression. Contrasting with ClC-4 and -5, which reach the plasma membrane to a degree that allows for detailed biophysical studies, currents mediated by ClC-3 were too low to determine whether it transports protons (3, 12). On the other hand, ClC-3 is ϳ80% identical in sequence to the established exchangers ClC-4 and ClC-5, with which it shares current properties that are similarly affected by mutations in the gating glutamate (12, 13). Hence, it is very likely that ClC-3 also functions as an exchanger. So far, it has been impossible to functionally express ClC-6 and ClC-7, which form a distinct branch of the CLC family (14), in the plasma membrane. This may be a consequence of their efficient targeting to late endosomes and lysosomes, respectively. ClC-7 is the only member of the CLC family significantly expressed on lysosomes (15-17). Lysosomes display 2Cl Ϫ /H ϩ exchange activity (18,19), which was strongly reduced by small interfering RNA in culture (18) and in ClC-7 knock-out (KO) 2 mic...