Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters

Abstract

Several members of the CLC family of Cl- channels and transporters are expressed in vesicles of the endocytotic-lysosomal pathway, all of which are acidified by V-type proton pumps. These CLC proteins are thought to facilitate vesicular acidification by neutralizing the electric current of the H+-ATPase. Indeed, the disruption of ClC-5 impaired the acidification of endosomes, and the knock-out (KO) of ClC-3 that of endosomes and synaptic vesicles. KO mice are available for all vesicular CLCs (ClC-3 to ClC-7), and ClC-5 and ClC-7, as well as its beta-subunit Ostm1, are mutated in human disease. The associated mouse and human pathologies, ranging from impaired endocytosis and nephrolithiasis (ClC-5) to neurodegeneration (ClC-3), lysosomal storage disease (ClC-6, ClC-7/Ostm1) and osteopetrosis (ClC-7/Ostm1), were crucial in identifying the physiological roles of vesicular CLCs. Whereas the intracellular localization of ClC-6 and ClC-7/Ostm1 precluded biophysical studies, the partial expression of ClC-4 and -5 at the cell surface allowed the detection of strongly outwardly rectifying currents that depended on anions and pH. Surprisingly, ClC-4 and ClC-5 (and probably ClC-3) do not function as Cl- channels, but rather as electrogenic Cl--H+ exchangers. This hints at an important role for luminal chloride in the endosomal-lysosomal system.

Figure 1

Subcellular localization of vesicular CLC proteins A, proposed localization of different CLC isoforms along the endosomal–lysosomal pathway. Vesicles are progressively acidified from the neutral extracellular pH (∼7.4) to the acidic pH (∼4.5) of lysosomes. Acidification is performed by an ATP-driven proton pump that needs a net influx of negative charge for electroneutrality. The neutralizing current is thought to be mediated by CLC isoforms. ClC-4 and -5 mediate nCl⁻–H⁺ exchange (with an imprecisely known stoichiometry n, which might be n = 2 as in ClC-e1) and this very likely applies for ClC-3 as well. ClC-6 and ClC-7/Ostm1 are also shown as antiporters, although this remains to be demonstrated. The localization of ClC-4 is rather uncertain, but may also be endosomal (Suzuki et al. 2006). B, in osteoclasts attached to bone, ClC-7/Ostm1 is co-inserted with the proton pump into the highly infolded ‘ruffled border’ that acidifies the underlying resorption lacuna.

Figure 1

Subcellular localization of vesicular CLC proteins A, proposed localization of different CLC isoforms along the endosomal–lysosomal pathway. Vesicles are progressively acidified from the neutral extracellular pH (∼7.4) to the acidic pH (∼4.5) of lysosomes. Acidification is performed by an ATP-driven proton pump that needs a net influx of negative charge for electroneutrality. The neutralizing current is thought to be mediated by CLC isoforms. ClC-4 and -5 mediate nCl⁻–H⁺ exchange (with an imprecisely known stoichiometry n, which might be n = 2 as in ClC-e1) and this very likely applies for ClC-3 as well. ClC-6 and ClC-7/Ostm1 are also shown as antiporters, although this remains to be demonstrated. The localization of ClC-4 is rather uncertain, but may also be endosomal (Suzuki et al. 2006). B, in osteoclasts attached to bone, ClC-7/Ostm1 is co-inserted with the proton pump into the highly infolded ‘ruffled border’ that acidifies the underlying resorption lacuna.

Figure 2

Model for renal pathology in Dent's disease (due to a loss of ClC-5) The small peptide PTH (parathyroid hormone) and various forms of vitamin D (VitD; bound to their binding protein) pass the glomerular filter into the early proximal tubule (A). PTH and VitD-binding protein complexes are normally endocytosed by proximal tubular cells after binding to megalin. PTH is degraded in lysosomes, whereas 1,25-VitD reaches VitD receptors that regulate the transcription of nuclear target genes (not shown) and 25-VitD is metabolized by mitochondrial enzymes: 1α-hydroxylase activates the precursor to the active hormone 1,25(OH)₂-vitaminD₃ (1,25-VitD), whereas the 24-hydroxylase inactivates VitD. The endocytotic uptake of PTH and VitD is severely impaired in the absence of ClC-5 (A and B; indicated by red minus symbols). As a consequence, their concentration increases in the lumen of later nephron segments compared to wild-type (A–C). This leads to an enhanced stimulation of apical PTH receptors (B; green plus symbols), causing the endocytosis and degradation of the sodium–phosphate cotransporter NaPi-2a (leading to phosphaturia) and increasing α-hydroxylase while decreasing 24-hydroxylase levels. Similar changes in hydroxylase levels also result from the impaired apical endocytosis of VitD, which acts on the transcription of the respective genes (not shown). The altered hydroxylase activities tend to produce more active hormone (1,25-VitD). Such an increase, however, is counteracted by a decrease in precursor availability due to its defective endocytotic uptake. Thus, depending on other factors, the amount of 1,25-VitD released into the blood may be decreased or increased (B). C, in contrast to proximal tubules, in which VitD is taken up primarily by apical endocytosis of its binding protein and is hence decreased in ClC-5 KO cells, it enters distal tubular cells mainly by diffusion of the free hormone. The increased luminal concentration of 1,25-VitD in distal segments of Clcn5⁻ nephrons enhances the transcription of distal VitD target genes like that of the ion channel TRPV5 (Maritzen et al. 2006).

Figure 3

Lysosomal storage in neurons of mice lacking ClC-7 or ClC-6 Electron-dense lysosomal storage material (indicated by arrows) is present throughout the cytoplasm of Clcn7^−/− neurons (A), whereas it accumulates exclusively in initial axon segments of Clcn6^−/− neurons (B). Scale bars, 1.5 μm in A and 0.4 μm in B. The plasma membrane of the neuron in B is highlighted by a dashed line. nuc, nucleus. Modified images from Kasper et al. (2005) and Poët et al. (2006).

Figure 4

ClC-5 is a Cl⁻–H⁺ exchanger, whereas ClC-0 is a Cl⁻ channel HEK cells transfected with ClC-5 (A) or ClC-0 (B) were clamped to different voltages (lower traces) using the gramicidin-perforated patch clamp technique to minimize the equilibration of their internal pH (pH_i) with the patch pipette. The middle traces shows clamp currents, while the upper panels show pH_i (measured using a ratiometric pH-sensitive dye). When ClC-5-transfected cells were clamped to voltages more positive than +30 mV, pH_i increased, indicating an exit of H⁺ in exchange for Cl⁻ entry. Consistent with the steep outward rectification of ClC-5 currents (Friedrich et al. 1999), the rate of intracellular alkalinization increased steeply with inside-positive voltage. By contrast, a similar voltage-clamp protocol did not change the pH_i of cells expressing the Torpedo channel ClC-0 (B). Panels taken from Scheel et al. (2005).