The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Abstract

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...

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

“…Cultured cells are well established for studying trafficking and function of membrane transport proteins, and a large body of evidence supports the notion that similar motifs might direct targeting in epithelia ClC-3c and neurons (45). However, there are examples of different subcellular targeting of certain proteins in HEK293T cells and in neurons (46). Thus, although our work conclusively demonstrates separate subcellular localizations of ClC-3a, ClC-3b, and ClC-3c, it does not allow conclusions about which organelles ClC-3 splice variants insert into native neurons.…”

Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

“…Constructs for human ClC-5, human ClC-6, and rat ClC-7 in this vector have been described previously (40,41). For the generation of chimeric constructs containing parts of ClC-0 and either ClC-6 or ClC-7, the DNA sequences encoding the N-terminal part (aa 1-48 for ClC-0, aa 1-77 for ClC-6, and aa 1-123 for ClC-7), the transmembrane region (43) from the beginning of helix B until the end of helix R (aa 49 -524 for ClC-0, aa 78 -588 for ClC-6, and aa 124 -614 for ClC-7), and the C-terminal region (aa 525-805 for ClC-0, aa 589 -869 for ClC-6, and aa 615-805 for ClC-7) of the respective CLC were combined by recombinant PCR with overlapping primers and cloned into pcDNA3.…”

Discovery of CLC transport proteins: cloning, structure, function and pathophysiology

“…Before crystal structures were available, site-directed mutagenesis had already revealed the importance of this residue for rectification and gating (Friedrich et al 1999;Waldegger & Jentsch, 2000) and of a Cl − co-ordinating serine (Dutzler et al 2002) for ion selectivity and single-channel conductance (Ludewig et al 1996). Subsequent mutations of the 'gating glutamate' in ClC-0 (Dutzler et al 2003) and CLC exchangers (Leisle et al 2011;Neagoe et al 2010) similarly largely abolished the voltage-dependence of currents. However, channels lacking this glutamate still gate at the single-channel level (Dutzler et al 2003;L'Hoste et al 2013), indicating that CLC gating cannot be explained entirely by the movement of its side chain.…”