Mouse bulbus olfactorius under the microscope

Jentsch Lab

Physiology and Pathology of Ion Transport

CLC Cl - channels and transporters

Research Project 1

We discovered the CLC gene family in 1990

By expression cloning of the voltage-gated Cl- channel from the electric organ of the marine ray Torpedo [1], we discovered the CLC gene family. This channel, which we baptized ClC-0, is the founding member of the CLC gene family which is present from bacteria to men. In humans, it comprises 9 members (Figure 1) (for review, see [2]). Originally thought to only represent plasma membrane Cl- channels, many of these proteins rather function as electrogenic Cl-/H+-exchangers in intracellular membranes. Mutations of CLC genes underlie several human genetic diseases. We have shown that some CLC proteins associate with other, structurally unrelated proteins, mutations in which cause pathologies that overlap with those observed with the loss of their respective CLC partners. 

Figure 1. The CLC gene family of Cl- channels and Cl-/H+-exchangers. Associated structurally unrelated β-subunits include the cell adhesion molecule GlialCAM (mutations in which underlie a distinct form of leukodystrophy) for ClC-2, the 2-membrane span membrane protein barttin (mutations in which lead to Bartter syndrome with deafness) for both ClC-K isoforms, and for ClC-7 Ostm1, with disruption of either gene leading to osteopetrosis associated with neurodegeneration.

Important new insights from CLC-gene disruption in mice

Unexpectedly, our disruption of the chloride transporter ClC-7 led to osteopetrosis [3], a hypercalcification of bone. This phenotype is due to a dysfunction of osteoclasts, the cells that are responsible for bone degradation. ClC-7 currents may balance the electrogenic transport of the H+-ATPase that acidifies the resorption lacuna (Figure 2).

Figure 2. Role of ClC-7 in osteopetrosis. a, X-ray of bones from wild-type (WT) and ClC-7 KO mice reveals hypercalcification in the KO. b, model for the acidification of the osteoclast resorption lacuna.

Motivated by these findings, we identified mutations in either the ClC-7 chloride channel [3], or in the a3 subunit of the H+-ATPase [4], in human patients with severe juvenile osteopetrosis. ClC-7 is normally present in a lysosomal compartment, but is inserted into the plasma membrane of osteoclasts. Similarly, our results show that ClC-3 through ClC-6 reside normally in membranes of intracellular compartments, where they may contribute to their acidification by electrical shunting of the proton ATPase. ClC-5, a Cl-/H+ exchanger mutated in the kidney stone disorder Dent’s disease [5], is present in renal endosomes [6]. Our ClC-5 knock-out mouse model [7] revealed that an impaired endosomal acidification led to a broad defect in proximal tubular endocytosis. This entails secondary changes in calciotropic hormones, eventually resulting in the hyperphosphaturia and hypercalciuria (and kidney stones) in Dent’s disease. The related ClC-3 putative Cl-/H+ exchanger is also present in endosomes, as well as in synaptic vesicles [8]. As the uptake of neurotransmitters into synaptic vesicles is coupled to the proton gradient, this may have interesting consequences for synaptic transmission. Surprisingly, the knock-out of ClC-3 led to nearly complete degeneration of the hippocampus [8].

Vesicular acidification

In the classical model of vesicular acidification (Figure 3a), the electrical current of the vesicular H+-ATPase is neutralized by Cl- influx through a Cl- channel.  The discovery that vesicular CLCs are 2Cl-/H+-exchangers rather than Cl- channels [9, 10] therefore came as a surprise because a role in acidification rather seemed counterintuitive.

Figure 3. We generated ClC-5 and ClC-7 knock-in mice in which we converted the WT 2Cl-/H+-exchangers ClC-5 and ClC-7 (b) with point mutations into mere Cl- conductances (c) to conform to the classical model (a) of vesicular acidification.

To elucidate whether vesicular CLCs just serve to neutralize proton pump currents, we converted ClC-5 and ClC-7 in mice to pure Cl- conductances [11, 12]. Any phenotype apparent in those mice cannot be ascribed to a defect in vesicular acidification. Surprisingly, these mice displayed grossly the same phenotype as the corresponding KO mice, suggesting an important role of vesicular Cl- accumulation. Another recent ClC-7 knock-in mouse [13] revealed that not only ion transport activity, but also (so far unknown) protein-protein interactions account for ClC-7 functions. Several current projects of our lab focus on the exciting roles of vesicular CLCs in basic cellular processes and pathological states.
ClC-2 is a widely expressed plasma membrane Cl- channel that is slowly activated by inside-negative voltage or cell swelling [14, 15]. We showed previously that disruption of ClC-2 in mice leads to testicular and retinal degeneration [16], as well as leukodystrophy [17]. Recently van der Knaap and colleagues showed that human ClC-2 mutations also lead to leukodystrophy, a disease associated with vacuolization of the white matter (glia) of the central nervous system.  Together with Raúl Estévez we have recently shown that ClC-2 associates with GlialCAM, an Ig-like cell adhesion cell surface molecule, and that this interaction profoundly changes ClC-2 gating [18]. Importantly, GlialCAM mutations also lead to human leukodystrophy, as do mutations in MLC1, a protein interaction partner of GlialCAM that displays several transmembrane domains. We recently generated GlialCAM mouse models and compared their CNS pathology to those of ClC-2 and MLC1 KO mice [19]. Unexpectedly, the localization, abundance and biophysical properties of ClC-2 depended on both GlialCAM and MLC1, suggesting changes in ClC-2 as a common pathogenic factor in the respective diseases. Several current projects focus on ClC-2 and its role in the CNS.

Cited own publications:
[1]  Jentsch TJ, Steinmeyer K, Schwarz G (1990) Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348: 510-514
[2]  Stauber T, Weinert S, Jentsch TJ (2012) Cell biology and physiology of CLC chloride channels and transporters. Compr Physiol 2: 1701-1744
[3]  Kornak U, Kasper D, Bösl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ (2001) Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104: 205-215
[4]  Kornak U, Schulz A, Friedrich W, Uhlhaas S, Kremens B, Voit T, Hasan C, Bode U, Jentsch TJ, Kubisch C (2000) Mutations in the a3 subunit of the vacuolar H+-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet 9: 2059-2063
[5]  Lloyd SE, Pearce SH, Fisher SE, Steinmeyer K, Schwappach B, Scheinman SJ, Harding B, Bolino A, Devoto M, Goodyer P, Rigden SP, Wrong O, Jentsch TJ, Craig IW, Thakker RV (1996) A common molecular basis for three inherited kidney stone diseases. Nature 379: 445-449
[6]  Günther W, Luchow A, Cluzeaud F, Vandewalle A, Jentsch TJ (1998) ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc Natl Acad Sci U S A 95: 8075-8080
[7]  Piwon N, Günther W, Schwake M, Bösl MR, Jentsch TJ (2000) ClC-5 Cl--channel disruption impairs endocytosis in a mouse model for Dent's disease. Nature 408: 369-373
[8]  Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bösl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ (2001) Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29: 185-196
[9]  Scheel O, Zdebik A, Lourdel S, Jentsch TJ (2005) Voltage-dependent electrogenic chloride proton exchange by endosomal CLC proteins. Nature 436: 424-427
[10] Leisle L, Ludwig CF, Wagner FA, Jentsch TJ, Stauber T (2011) ClC-7 is a slowly voltage-gated 2Cl-/1H+-exchanger and requires Ostm1 for transport activity. EMBO J 30: 2140-2152
[11] Novarino G, Weinert S, Rickheit G, Jentsch TJ (2010) Endosomal chloride-proton exchange rather than chloride conductance is crucial for renal endocytosis. Science 328: 1398-1401
[12] Weinert S, Jabs S, Supanchart C, Schweizer M, Gimber N, Richter M, Rademann J, Stauber T, Kornak U, Jentsch TJ (2010) Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation Science 328: 1401-1403
[13] Weinert S, Jabs S, Hohensee S, Chan WL, Kornak U, Jentsch TJ (2014) Transport activity and presence of ClC-7/Ostm1 complex account for different cellular functions. EMBO Rep, 10.15252/embr.201438553
[14] Gründer S, Thiemann A, Pusch M, Jentsch TJ (1992) Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. Nature 360: 759-762
[15] Thiemann A, Gründer S, Pusch M, Jentsch TJ (1992) A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356: 57-60
[16] Bösl MR, Stein V, Hübner C, Zdebik AA, Jordt SE, Mukhophadhyay AK, Davidoff MS, Holstein AF, Jentsch TJ (2001) Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl--channel disruption. EMBO J 20: 1289-1299
[17] Blanz J, Schweizer M, Auberson M, Maier H, Muenscher A, Hübner CA, Jentsch TJ (2007) Leukoencephalopathy upon disruption of the chloride channel ClC-2. J Neurosci 27: 6581-6589
[18] Jeworutzki E, López-Hernández T, Capdevila-Nortes X, Sirisi S, Bengtsson L, Montolio M, Zifarelli G, Arnedo T, Müller CS, Schulte U, Nunes V, Martínez A, Jentsch TJ, Gasull X, Pusch M, Estévez R (2012) GlialCAM, a protein defective in a leukodystrophy, serves as a ClC-2 Cl- channel auxiliary subunit. Neuron 73: 951-961
[19] Hoegg-Beiler MB, Sirisi S, Orozco IJ, Ferrer I, Hohensee S, Auberson M, Godde K, Vilches C, de Heredia ML, Nunes V, Estevez R, Jentsch TJ (2014) Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction. Nat Commun 5: 3475

Prof. Dr. Thomas Jentsch
Prof. Thomas J. Jentsch
Group Leader
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) & Max-Delbrück-Centrum für Molekulare Medizin (MDC)
Robert-Rössle-Str. 10
13092 Berlin, Germany
Research Lab Coordinator
Dr. Norma Nitschke
+49 30 9406-2974