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...

ClC-3 is an intracellular chloride transport protein known to reside on endosomes and synaptic vesicles. The endogenous protein has been notoriously difficult to detect in immunohistological experiments because of the lack of reliable antibodies. Using newly generated antibodies, we now examine its expression pattern at the cellular and subcellular level. In all tissues examined, immunostaining indicated that ClC-3 is a vesicular protein, with a prominent expression in endocrine cells like adrenal chromaffin cells and pancreatic islet cells. In line with a possible function of ClC-3 in regulating vesicle trafficking or exocytosis in those secretory cells, capacitance measurements and amperometry indicated that exocytosis of large dense-core vesicles (LDCVs) was decreased in chromaffin cells from ClC-3 knock-out mice. However, immunohistochemistry complemented with subcellular fractionation showed that ClC-3 is not detectable on LDCVs of endocrine cells, but localizes to endosomes and synaptic-like microvesicles in both adrenal chromaffin and pancreatic ␤ cells. This observation points to an indirect influence of ClC-3 on LDCV exocytosis in chromaffin cells, possibly by affecting an intracellular trafficking step.

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Arginine (Arg)-based endoplasmic reticulum (ER)-localization signals are involved in the quality control of different heteromultimeric membrane protein complexes. ATP-sensitive potassium (KATP) channels are unique because each subunit in the heterooctamer contains an Arg-based ER-localization signal. We have dissected the inactivation events that override the ER-localization activity of the eight peptide-sorting motifs. Employing a 14-3-3-scavenger construct to lower the availability of 14-3-3 proteins, we found that 14-3-3 proteins promote the cell-surface expression of heterologously expressed and native KATP channels. 14-3-3 proteins were detected in physical association with KATP channels in a pancreatic β-cell line. Our results suggest that the Arg-based signal present in Kir6.2 is sterically masked by the SUR1 subunit. By contrast, 14-3-3 proteins functionally antagonized the Arg-based signal present in SUR1. The last ten amino acids were required for efficient 14-3-3 recruitment to multimeric forms of the Kir6.2 C-terminus. Channels containing a pore-forming subunit lacking these residues reached the cell surface inefficiently but were functionally indistinguishable from channels formed by the full-length subunits. In conclusion, 14-3-3 proteins promote the cell-surface transport of correctly assembled complexes but do not regulate the activity of KATP channels at the cell surface.