Expression of Human pICln and ClC-6 in Xenopus Oocytes Induces an Identical Endogenous Chloride Conductance

123

ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swellingactivated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl ؊ > I ؊ selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.

Section: Fig 2 Properties Of Endogenous Swelling-activated Currentsmentioning

confidence: 67%

Biophysical Properties of ClC-3 Differentiate It from Swelling-activated Chloride Channels in Chinese Hamster Ovary-K1 Cells

Li¹,

Shimada²,

Showalter³

et al. 2000

123

“…First, the relative anion conductance sequence of pCLC5-injected oocytes was completely different from that of control, water-injected oocytes. Second, the relative anion conductance (Cl Ϫ Ͼ I Ϫ ) and kinetics (rapidly activating, non-inactivating over 500 ms) of the outwardly rectifying current in pCLC5-injected oocytes were very different from that reported in oocytes stimulated by injection with a variety of different cRNA such as pI Cln and CLC6 (43). In the latter case, slowly inactivating currents were observed at positive potentials, with an I Ϫ Ͼ Cl Ϫ conductance preference, and attributed to nonspecific stimulation of an endogenous oocyte channel.…”

Section: Fig 4 Immunodetection Of Clc5 Proteins In Denaturing Polyamentioning

confidence: 87%

Molecular Cloning and Characterization of an Intracellular Chloride Channel in the Proximal Tubule Cell Line, LLC-PK1

Dowland

Luyckx

Enck

et al. 2000

CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate > > Cl ؊ Ϸ Br ؊ > I ؊ > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK a of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.

“…Previous attempts to record plasma membrane currents of ClC-6 have failed (14,29) probably due to lack of insertion into the plasma membrane (20). When we expressed ClC-6 heterologously in mammalian cells, it localized predominantly to punctate intracellular structures and possibly to a very small extent to the plasma membrane (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Neagoe

Stauber

Fidzinski

et al. 2010

112

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...