ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix.
Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface.
The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change – ranging from the cytoplasmic side of the protein to the extracellular side of the protein – is involved in the Ca2+-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca2+. This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.
Citation: Martinez GQ, Maduke M (2008) A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane. PLoS ONE 3(7): e2746. https://doi.org/10.1371/journal.pone.0002746
Editor: Shuguang Zhang, Massachusetts Institute of Technology, United States of America
Received: May 14, 2008; Accepted: June 24, 2008; Published: July 23, 2008
Copyright: © 2008 Martinez, Maduke. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the American Heart Association (0435056N) and the Mathers Foundation.
Competing interests: The authors have declared that no competing interests exist.
Members of the CLC family of chloride channels and transporters are ubiquitously expressed and serve a variety of physiological functions , . Mutations in human CLCs give rise to a number of diseases including Bartter's syndrome (ClC-Kb –), myotonia congenita (ClC-1 –), epilepsy (ClC-2 –), Dent's disease (ClC-5 –), and osteopetrosis (ClC-7 ). ClC-Kb is expressed in the thick ascending limb of the kidney, where it facilitates chloride reabsorption . ClC-Kb, together with the nearly identical homolog ClC-Ka, serves a similar role in chloride homeostasis in the stria vascularis of the inner ear .
Eukaryotic CLC proteins consist of two domains: a membrane domain that forms the chloride-selective permeation pathway and a C-terminal cytoplasmic domain. (Figure 1) , , . In contrast to the relatively much-studied permeation pathway, the functional role of CLC cytoplasmic domains remains less well understood. Even so, their physiological importance is underscored by the large number of truncations and point mutations that result in disease . While disruption of the cytoplasmic domain can interfere with folding and/or trafficking and thus result in a decrease of functional protein at the plasma membrane , –, there are clues that the cytoplasmic domains are also important for electrophysiological function. This has most clearly been shown for the skeletal muscle homologs ClC-0 and ClC-1. In ClC-0, mutations in the cytoplasmic domain affect the “slow” cooperative gating , , . In ClC-1, point mutations in the cytoplasmic domains cause shifts in voltage-dependent gating that are linked to dominantly inherited myotonia , . Disease-causing cytoplasmic domain point mutations are also found in several other homologs; however, in these cases, the electrophysiological effects of the mutations are either controversial (ClC-2) ,  or not yet reported (ClC-6, ClC-7, and ClC-Kb) –.
(A) Homology model of ClC-Kb using ClC-ec1 (1OTS) as a template for the membrane domain and the ClC-Ka C-terminal domain structure (2PFI) as a template for the cytoplasmic domain. Subunit A (grey) has several amino acids removed to show the ion coordination of chloride by Y520 (in spacefill), located on helix-R. R538 is located in the cytoplasmic-domain loop (red) that connects helix-R to CBSD1. (B) Structure of the ClC-Ka C-terminal domain (2PFI) highlighting the different CBSDs. Subunit 1 has CBSD1 in blue and CBSD2 in cyan; subunit 2 has CBSD1 in red and CBSD2 in violet. (C) Structure of TM0935 (1OTS) with the color scheme as in B. All cartoons were generated in PyMol (http://www.pymol.org).
Here we study the point mutation R538P, which occurs in the C-terminal cytoplasmic domain of ClC-Kb and causes Bartter's Syndrome . R538 is located in the linker region between the membrane domain and the cytoplasmic domain (Figure 1A). We show that this mutation changes gating at hyperpolarized membrane potentials, alters selectivity and removes activation induced by extracellular calcium. Thus, the mutation of a single amino acid in the cytoplasmic domain causes a long range conformational change that traverses the membrane-spanning domain of ClC-Kb. In the homologous ClC-Ka (90% identical), the R538P mutation does not yield the same changes in gating or calcium activation. This surprising difference indicates that ClC-Ka and ClC-Kb differ either structurally or in how conformational changes are translated to the extracellular domain.
The C-terminal domains of CLCs contain a pair of conserved protein motifs referred to as cystathionine beta-synthase domains (CBSDs) . CBSDs are present in a diverse array of proteins, where they are independent domains that play roles in the regulation of the core domain's catalytic activity . In cystathionine beta-synthase, mutations in the CBSDs eliminate the protein's ability to be activated by endogenous ligand and lead to the disease homocystinuria, a defect in the methionine metabolism pathway . In AMP-activated protein kinase, mutations in the CBSDs of the gamma subunit result in familial hypertrophic cadiomyopathy, presumably because altered regulation of the other subunits .
The structures of two human CLC C-terminal cytoplasmic domains, ClC-Ka  and ClC-5 , have been determined. Both of these constructs form dimers, with the intermolecular interface occurring between CBSD2 of each subunit (Figure 1B). This architecture is preserved in solution, as illustrated by cross-linking studies on the ClC-Ka C-terminal domains . In contrast, a cytoplasmic domain construct from the Torpedo homolog ClC-0 crystallizes as a monomer . However, the domain in solution is a dimer, and cross-linking studies on full-length ClC-0 indicates that the dimer interface is similar to that for ClC-Ka and ClC-5 .
Despite the similarities between the CBSD structures described above, there is some plasticity in the oligomeric architecture of CBSDs. In IMPDH, for example, the structure of the full-length protein reveals a monomeric CBSD . In the prokaryotic protein TM0935, a protein comprised entirely of CBSDs, the crystal structure (PDB ID 1O50) reveals a dimeric protein that has a completely different intermolecular interface than that observed in ClC-Ka and ClC-5  (Figure 1C). This interface is also observed in a recombinant protein consisting of two of the CBSDs of the eukaryotic protein AMPK , and in TA0289, a Thermoplasma acidophilum protein containing a C-terminus Zn ribbon domain along with its CBSD .
Because ClC-Kb differs from ClC-Ka in some of the key residues occurring at the dimer interface, we hypothesized that the differences in the effect of R583P on ClC-Ka and ClC-Kb function may reflect differences in the oligomeric architecture of the cytoplasmic domains. On testing this hypothesis, we found that the cytoplasmic domain of ClC-Kb is dimeric and that the dimer interface adopted is similar to that observed in the structure of the C-terminal domain of ClC-Ka.
Characterization of the R538P mutation in ClC-Kb
In ClC-Kb, R538 is located after the last membrane helix (helix-R) immediately preceding CBSD1 (Figure 1). Since helix-R lines the chloride permeation pathway ,  and extends into the cytoplasm, we hypothesized that the R538P mutation could alter helix orientation, thus potentially affecting both gating and permeation. Indeed, we observe alterations in both properties. Wild-type ClC-Kb has a linear steady-state voltage dependence with only slight activation and inactivation at depolarized and hyperpolarized potentials respectively (Figure 2A, C). In contrast, ClC-Kb-R538P is slightly inwardly rectifying and exhibits activation at hyperpolarized potentials (Figure 2B, C). To test whether there are any alterations in the permeation pathway of ClC-Kb-R538P channels, we assessed the relative permeability of different anions. Wild-type ClC-Kb channels show a selectivity sequence of Cl−>Br− = I−, whereas ClC-Kb-R538P shows a slight but significant increase in the relative permeability of bromide (Figure 2D–F).
Two-electrode voltage clamp recordings of (A) wild type and (B) R538P ClC-Kb channels. Currents are in response to a 200-ms prepulse to +60 mV followed by 500-ms test pulses ranging from +60 mV to −140 mV in −20 mV decrements. (C) Steady-state currents at the different voltages were normalized to the current value at +60 mV for wild type (filled squares) and R538P (open squares). Error bars are s.e.m. with n = 8 (WT) and n = 6 (R538P). Error bars smaller than the symbols are not shown. Representative steady state I–V curves for (D) wild type and (E) R538P channels in the presence of chloride (filled squares), bromide (open squares), or iodide (open diamonds). (F) Permeability ratios relative to chloride for wild type (filled bars) and R538P (open bars). The bromide/chloride permeability ratio of R538P is statistically different from that of wild type (p<0.0005, n = 5).
Two characteristic features of ClC-K channels are their activation by extracellular alkalinization and by extracellular Ca2+ , , . No change was observed in the pH dependence of ClC-Kb-R538P relative to wild type channels (data not shown). In contrast, extracellular activation by Ca2+ was abolished: wild type ClC-Kb exhibits a ∼2-fold activation when extracellular Ca2+ is raised from 0.1 to 10 mM calcium, whereas no significant activation is seen with ClC-Kb-R538P under the same conditions (Figure 3).
Current recordings for (A) wild type and (B) R538P ClC-Kb channels at 0.1 mM calcium (left) and 10.0 mM calcium (right). (C) Calcium dependent activation of wild type (filled squares) and R538P (open squares) channels. Steady-state current levels at +60 mV plotted as a function of [Ca2+], normalized to the value at 0.1 mM calcium. Each data point is the average of several (n = 4–6) oocytes and error bars show the s.e.m. Only oocytes that showed reversibility were included in the analysis.
Characterization of the R538P mutation in ClC-Ka
Since ClC-Kb shares a high degree of sequence identity with ClC-Ka (∼90% identity), we investigated whether the R538P mutation in ClC-Ka would induce similar changes in voltage dependence, selectivity and calcium activation. Surprisingly, we found only subtle changes: selectivity measurements showed a slight decrease in relative iodide permeation, while voltage- and Ca2+ -activation were unaffected (Figure 4). These results suggest that despite the overall similarity between ClC-Ka and ClC-Kb, the properties imparted to the pore by the cytoplasmic domains are quite different.
Two-electrode voltage clamp current recordings of wild type (A) and R538P (B) expressing channels. Currents are in response to a 100-ms prepulse to +60 mV follow by 500-ms test pulses ranging from +60 mV to −140 mV in −20 mV steps. (C) Relative permeability ratios for ClC-Ka wild type (filled bars) and R538P (open bars) channels. The iodide/chloride permeability ratio of R538P is statistically different from that of wild type (p<0.02, n = 3 (WT) and n = 5 (R538P)). (D) Calcium dependence at +60 mV of wild type (open squares) and R538P (open triangles) normalized to the current at +60 mV in the presence of 0.1 mM calcium, as in Figure 3. Error bars for WT (n = 4) and R538P (n = 3) are the s.e.m.
Analysis of the ClC-Kb cytoplasmic domain
We hypothesized that the differences in the functional effects of the mutation in ClC-Ka and ClC-Kb might be due to a difference between the two C-terminal cytoplasmic domains. The cytoplasmic domain of ClC-Kb is over 90% identical to the cytoplasmic domain of ClC-Ka, for which a crystal structure has been determined , and therefore the structures are likely to be identical. However, when the residues that differ between the two homologs are mapped onto the ClC-Ka crystal structure, it is surprising to note that two of these residues occur at the dimer interface, at the core of the protein. In particular, amino acid 636 is a phenylalanine in ClC-Ka which when mutated removes ClC-Ka cytoplasmic domain dimerization ; in ClC-Kb, amino acid 636 is a non-hydrophobic serine (Figure 5A). This suggests the possibility that the ClC-Kb C-terminal domain may not dimerize or dimerize differently than ClC-Ka. We tested this hypothesis by determining the oligomeric status of a recombinant protein corresponding to the C-terminal domain of ClC-Kb. Gel-filtration chromatography and multi-angle light-scattering both indicate that this protein, like that of the C-terminal domain of ClC-Ka, is a dimer in solution (Figure 5B).
(A) Stereo view of the ClC-Ka cytoplasmic domain structure (2PFI) with amino acid differences between ClC-Ka and ClC-Kb shown in space fill. Amino acid F636 (shown in black) is on the dimer interface in ClC-Ka and when mutated to aspartic acid removes ClC-Ka dimerization. In ClC-Kb this residue is the non-polar residue serine. (B) Gel filtration combined with multi-angle light scattering shows that the C-terminal domain of ClC-Kb forms a dimer in solution. The solid line represents the absorbance at 280 nm as a function of elution volume. Filled circles represent the calculated mass at the different elution volumes. The molar mass was determined to be 37 kDa, consistent with dimerization.
Following this observation, we next sought to compare the dimerization interfaces of the ClC-Ka and Kb cytoplasmic domains. In contrast to the dimerization interface seen in the ClC-Ka crystal structure, a second dimerization interface has been observed in other CBSD-containing proteins, TM0935, TA0289, and AMPK , , . A number of the residues differing between ClC-Ka and ClC-Kb localize to the helices that form this interface (Figure 6A), which led us to question whether the ClC-Kb cytoplasmic domain might form an interface similar to that observed in TM0935 rather than ClC-Ka. To evaluate the ClC-Kb dimer interface, we used a cysteine-crosslinking strategy. In the ClC-Ka C-terminal domain structure, leucine 650 of one subunit is in close proximity (<5Å) to the same residue in the other subunit, and constructs with the L650C mutation spontaneously cross-link in solution . In the ClC-Kb C-terminal domain protein with the L650C mutation, spontaneous cross-linking in solution is also observed (Figure 6D). Thus, the interface seen in the ClC-Ka C-terminal domain crystal structure likely occurs in ClC-Kb as well.
(A) Stereo view of TM0935 structure (1O50), a CBSD-containing protein that adopts an alternate dimer interface. Amino acids corresponding to residues that differ between ClC-Ka and ClC-Kb are shown in spacefill. The residue corresponding to 636 in ClC-Kb (see Figure 5A) is shown in black. (B) Residues chosen for cross-linking studies mapped onto the ClC-Ka C-terminal domain structure: L650 (black), 603 (cyan), 604 (green), 644 (olive), 645 (magenta). For this dimer interface, the L650C mutant is expected to form an intersubunit cross-link. (C) Residues chosen for cross-linking studies mapped onto the TM0935 structure; color scheme as in B. For this dimer interface, the 603–645 and the 604–644 cysteine double mutants are expected to form intersubunit cross-links. (D) SDS-PAGE of purified ClC-Kb C-terminal domains with different cysteine mutations in reducing (−) and oxidizing (+) conditions.
While the ClC-Kb-L650C crosslink suggests a dimer interface similar to that observed in ClC-Ka, it does not rule out that in ClC-Kb there is an equilibrium between two dimerization conformations: one corresponding to the ClC-Ka dimer and the other to the TM0935 dimer. Such an equilibrium would suggest a “subunit swapping” mechanism, where the cytoplasmic domain interconverts between conformations with different dimerization interfaces. To assess this possibility, we designed two pairs of cysteine mutations that would be predicted to form intersubunit cysteine cross-links based on the TM0935 structure but not on the ClC-Ka structure (Figure 6B, C). ClC-Kb C-terminal domain constructs containing the double mutants K604C-A644C and L603C-H645C both form monodispersed dimers, as observed with wild-type (data not shown). Unlike the L650C mutant, in neither case was spontaneous cross-linking observed (Figure 6D). Moreover, application of H2O2 to catalyze disulfide-bond formation did not yield cross-links (data not shown). Taken together, these results suggest that ClC-Kb adopts the same dimerization interface as ClC-Ka .
Mutation of residue 538 in the C-terminal cytoplasmic domain of ClC-Kb gives rise to Bartter's syndrome . Although the mechanism by which the disease is caused is unknown, we hypothesized based on this residue's location in the cytoplasmic domain that it may affect function. Indeed, ClC-Kb-R538P exhibits slightly altered bromide permeability relative to wild type, indicating that the mutation induces a change in the permeation pathway. Strikingly, R538P removes extracellular calcium activation. Extracellular calcium activation must result in a conformational change that is transmitted from the calcium binding site to the pore. In order for R538P to abolish the activation, it must prevent calcium from binding or disrupt the calcium induced conformational change being transmitted to the pore.
It is not known whether any of the functional changes observed in ClC-Kb-R538P are related to the Bartter's Syndrome phenotype reported by Konrad et al . ClC-Kb is expressed in the basolateral membrane of kidney epithelia where the membrane potential is around −70 mV. While ClC-Kb-R538P exhibits a greater degree of inward rectification compared to wild type, the current-voltage plots show that deviation from wild type behavior is minimal at −70 mV (Figure 2). At the normal basolateral membrane potential, it is not likely that the change in voltage-dependence observed in ClC-Kb-R538P could alone be responsible for the disease phenotype.
The disruption in Ca2+ dependence may be responsible for the Bartter's syndrome caused by the R538P mutation. Although not definitive, there are several reports in the literature that support this hypothesis. Ca2+ levels are known to affect chloride transport in the thin ascending limb , and in vivo studies of rat kidney have revealed variations in interstitial Ca2+ of about 1 mM , in the range where ClC-Kb activity is affected. Moreover, disruption of basolateral calcium receptors in the kidney results in Bartter's-like symptoms , , which suggests a possible role for calcium fluctuation in the interstitial spaces in the kidney. Together, these results suggest that the loss of Ca2+ activation in the ClC-Kb R538P mutant may be responsible for the disease. Nevertheless, there are other factors that cannot be ruled out. It is possible that ClC-Kb-R538P does not traffic properly to the basolateral membrane or that the single-channel conductance is significantly altered, either of which could lead to altered basolateral chloride conductance. When equal amounts of wild-type and mutant ClC-Kb RNA are injected into the same batch of oocytes, there is only a modest (∼50%) difference between wild-type and mutant currents levels (in low Ca2+). This suggests that expression level, trafficking, and single-channel conductance are similar. However, it is still possible that such a small difference could be responsible for the disease phenotype. Additionally, it is possible that the difference in expression or trafficking is even greater in the native tissue, and so further studies are necessary to determine the cause of the disease.
CLC C-terminal cytoplasmic domains are required for CLC channel function , –. Numerous missense mutations and truncations in these domains result in disease, with many of the mutations occurring in the CBSDs , , . CBSDs occur in a wide variety of functionally unrelated proteins. As observed in the CLCs, mutations in CBSDs in some of these other proteins lead to disease . In all cases where CBSDs are known to regulate protein function, the CBSDs are structurally distant from the catalytic domain of the proteins (for CLCs, the chloride permeation pathway). Hence, there must be a mechanism for changes in CBSDs to be passed along to the catalytic domain of the proteins to which they are attached.
Surprisingly, we did not observe any changes in calcium activation when R538P was introduced into ClC-Ka. This was surprising, since the ClC-Ka and ClC-Kb channels are very similar (∼90% identical). One possible explanation for the different behaviors is that the structures of the ClC-Ka and ClC-Kb cytoplasmic domains are significantly different, despite their sequence similarity. Supporting this hypothesis is the observation that the majority of amino acids in ClC-Kb that differ from ClC-Ka occur in the residues that would form the two potential dimerization interfaces (the dimerization interface observed in the ClC-Ka C-terminal domain structure and that observed in the prokaryotic CBSD protein TM0935). However, our cross-linking studies indicate that dimer conformation adopted by the ClC-Kb C-terminal domain is that observed in the ClC-Ka structure rather than that of TM0935 and other proteins. Hence the mechanism by which the R538P mutation abolishes Ca2+ activation is not likely to involve changes in the dimer interface.
The location of amino acid 538 suggests possible mechanisms by which such a large conformational change can be transmitted through the membrane domain. Helix-R may extend up to or beyond position 538, and the introduction of a proline may result in helix instability (bend or break) that results in a loss of communication between this helix and the pore/extracellular domain. It is also possible that R538P disrupts a direct interaction with the membrane domain or other amino acids in the cytoplasmic domain (either the N-terminus or C-terminus). Testing these hypotheses will require structural studies on full-length protein.
Materials and Methods
Human ClC-Ka, ClC-Kb and Barttin were subcloned into the HindIII and EcoRI sites of psGEM. psGEM constructs were linearized with NheI and transcribed using mMessage T7 transcription kit (Ambion). ClC-Kb cytoplasmic-domain constructs containing amino acids 538–684 were inserted into the EcorR1 and XbaI sites of the pMAL-c2x vector (NEB) to form a C-terminal fusion protein with maltose-binding protein. All mutations were generated using PCR based site-directed mutagenesis.
Defolliculated Xenopus oocytes were injected with 55.6 nL of a 2∶1 mixture of ClC-Kb∶Barttin-Y98A RNA at 550 ng/µL or 27.5 nL of a 2∶1 mixture of ClC-Ka∶Bartin-Y98A RNA at 5–550 ng/µL. (For these studies, the point mutation Y98A in Barttin, which increases surface expression of ClC-K channels , was used.) Oocytes were incubated at 17°C for 1–7 days before recording using two-electrode voltage clamp. Electrodes were pulled to 0.4–1.6 MΩ and filled with 3 M KCl, 1.6 mM EGTA, 5 mM HEPES, pH 6.5. The standard bath solution was 96 mM NaCl, 1.8 mM CaCl2, 2 mM KCl, 1 mM MgCl2, and 5 mM Tris-Cl at pH 8.4. For selectivity experiments, 80 mM chloride was replaced with the indicated anion. Statistical significance was determined using a student t-test. For calcium experiments, CaCl2 was excluded and calcium acetate was added to reach the desired calcium concentration.
Expression and purification of ClC-Kb cytoplasmic domains
ClC-Kb cytoplasmic domains were expressed as C-terminal maltose-binding protein fusions (MBP-KbC). E. coli transformed with MBP-KbC were grown at 37°C in LB media to OD600 of ∼0.5, transferred to 25°C, induced with 0.3 mM IPTG, and grown for 5 additional hours. Cells were harvested by centrifugation, and pellets were suspended in 50 mL storage buffer (200 mM NaCl, 20 mM Tris-Cl pH 7.4, 1 mM EDTA, 20% glycerol) and frozen overnight or stored at −20°C. Cells were thawed and lysed by sonication after application of protease inhibitor cocktail. Cell lysates were centrifuged for 60′ @ 96,000×g. The supernatant was loaded onto an amylose column (NEB), equilibrated with column buffer (200 mM NaCl, 20 mM Tris-Cl pH 7.4, 1 mM EDTA), and protein was eluted with 10 mM maltose in column buffer. MBP-KbC fusion was digested with Factor Xa overnight at 4°C (8–10 hours). The digestion reaction was diluted 10× into cation-exchange buffer A (6 mM NaCl, 20 mM sodium phosphate, pH 6.0) and loaded onto a POROS strong cation-exchange column (Applied Biosystems). After washing away MBP with cation-exchange buffer B (25 mM NaCl, 20 mM sodium phosphate pH 6.0), purified ClC-Kb C-terminal domain (KbC) was eluted with a 25 mM to 500 mM NaCl gradient (in sodium phosphate, pH 6.0).
For cysteine cross-linking experiments, purified KbC was buffer exchanged (using gel-filtration chromatography) into TBS7.5 (200 mM NaCl, 10 mM Tris pH 7.5, 5 mM ß-mercaptoethanol). Samples were reduced with 250 mM ß-mercaptoethanol.
Multiangle light scattering
A Shodex PROTEIN KW-802.5 size exclusion chromatography column, equilibrated with running buffer (200 mM NaCl, 20 mM Tris-Cl, pH 7.5) was coupled with in-line DAWN EOS multiangle light scattering, refractive index (Wyatt Technology Corporation) and UV (Jasco Corporation) detectors (SEC-MALLS). Monomeric bovine serum albumin at 1 mg/mL was used to calibrate light scattering detectors. Protein at 1 mg/mL was run through the size exclusion column at room temperature (23°C). The molecular weight of the eluted species was calculated using the ASTRA 4.90.08 software provided by Wyatt Technology Corporation.
We thank Dr. Thomas Jentsch for providing ClC-Ka, ClC-Kb, and Barttin DNA and Shelley Elvington for careful reading of this manuscript.
Conceived and designed the experiments: GQM MM. Performed the experiments: GQM. Analyzed the data: GQM. Wrote the paper: GQM MM.
- 1. Zifarelli G, Pusch M (2007) CLC chloride channels and transporters: a biophysical and physiological perspective. Rev Physiol Biochem Pharmacol 158: 23–76.
- 2. Jentsch TJ (2008) CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit Rev Biochem Mol Biol 43: 3–36.
- 3. Rodriguez-Soriano J, Vallo A, Perez de Nanclares G, Bilbao JR, Castano L (2005) A founder mutation in the CLCNKB gene causes Bartter syndrome type III in Spain. Pediatr Nephrol 20: 891–896.
- 4. Fukuyama S, Hiramatsu M, Akagi M, Higa M, Ohta T (2004) Novel mutations of the chloride channel Kb gene in two Japanese patients clinically diagnosed as Bartter syndrome with hypocalciuria. J Clin Endocrinol Metab 89: 5847–5850.
- 5. Hebert SC (2003) Bartter syndrome. Curr Opin Nephrol Hypertens 12: 527–532.
- 6. Zelikovic I, Szargel R, Hawash A, Labay V, Hatib I, et al. (2003) A novel mutation in the chloride channel gene, CLCNKB, as a cause of Gitelman and Bartter syndromes. Kidney Int 63: 24–32.
- 7. Konrad M, Vollmer M, Lemmink HH, van den Heuvel LP, Jeck N, et al. (2000) Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome. J Am Soc Nephrol 11: 1449–1459.
- 8. Pusch M (2002) Myotonia caused by mutations in the muscle chloride channel gene CLCN1. Hum Mutat 19: 423–434.
- 9. Jentsch TJ, Lorenz C, Pusch M, Steinmeyer K (1995) Myotonias due to CLC-1 chloride channel mutations. Soc Gen Physiol Ser 50: 149–159.
- 10. Kubisch C, Schmidt-Rose T, Fontaine B, Bretag AH, Jentsch TJ (1998) ClC-1 chloride channel mutations in myotonia congenita: variable penetrance of mutations shifting the voltage dependence. Hum Mol Genet 7: 1753–1760.
- 11. Koch MC, Steinmeyer K, Lorenz C, Ricker K, Wolf F, et al. (1992) The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 257: 797–800.
- 12. Haug K, Warnstedt M, Alekov AK, Sander T, Ramirez A, et al. (2003) Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies. Nat Genet 33: 527–532.
- 13. Stogmann E, Lichtner P, Baumgartner C, Schmied M, Hotzy C, et al. (2006) Mutations in the CLCN2 gene are a rare cause of idiopathic generalized epilepsy syndromes. Neurogenetics 7: 265–268.
- 14. Heils A (2005) CLCN2 and idiopathic generalized epilepsy. Adv Neurol 95: 265–271.
- 15. Gunther W, Piwon N, Jentsch TJ (2003) The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease. Pflugers Arch 445: 456–462.
- 16. Wang SS, Devuyst O, Courtoy PJ, Wang XT, Wang H, et al. (2000) Mice lacking renal chloride channel, CLC-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis. Hum Mol Genet 9: 2937–2945.
- 17. Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ (2000) ClC-5 Cl−-channel disruption impairs endocytosis in a mouse model for Dent's disease. Nature 408: 369–373.
- 18. Cox JP, Yamamoto K, Christie PT, Wooding C, Feest T, et al. (1999) Renal chloride channel, CLCN5, mutations in Dent's disease. J Bone Miner Res 14: 1536–1542.
- 19. Lloyd SE, Pearce SH, Fisher SE, Steinmeyer K, Schwappach B, et al. (1996) A common molecular basis for three inherited kidney stone diseases. Nature 379: 445–449.
- 20. Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, et al. (2001) Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104: 205–215.
- 21. Estevez R, Boettger T, Stein V, Birkenhager R, Otto E, et al. (2001) Barttin is a Cl− channel beta-subunit crucial for renal Cl− reabsorption and inner ear K+ secretion. Nature 414: 558–561.
- 22. Nozu K, Inagaki T, Fu XJ, Nozu Y, Kaito H, et al. (2008) Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness. J Med Genet 45: 182–186.
- 23. Matulef K, Maduke M (2007) The CLC ‘chloride channel’ family: revelations from prokaryotes. Mol Membr Biol 24: 342–350.
- 24. Estevez R, Pusch M, Ferrer-Costa C, Orozco M, Jentsch TJ (2004) Functional and structural conservation of CBS domains from CLC chloride channels. J Physiol 557: 363–378.
- 25. Schwappach B, Stobrawa S, Hechenberger M, Steinmeyer K, Jentsch TJ (1998) Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p. J Biol Chem 273: 15110–15118.
- 26. Schmidt-Rose T, Jentsch TJ (1997) Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1. J Biol Chem 272: 20515–20521.
- 27. Mo L, Xiong W, Qian T, Sun H, Wills NK (2004) Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation. Am J Physiol Cell Physiol 286: C79–89.
- 28. Maduke M, Williams C, Miller C (1998) Formation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains. Biochemistry 37: 1315–1321.
- 29. Jentsch TJ, Poet M, Fuhrmann JC, Zdebik AA (2005) Physiological functions of CLC Cl− channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67: 779–807.
- 30. Uchida S, Sasaki S (2005) Function of chloride channels in the kidney. Annu Rev Physiol 67: 759–778.
- 31. Carr G, Simmons N, Sayer J (2003) A role for CBS domain 2 in trafficking of chloride channel CLC-5. Biochem Biophys Res Commun 310: 600–605.
- 32. Fong P, Rehfeldt A, Jentsch TJ (1998) Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata. Am J Physiol 274: C966–973.
- 33. Beck CL, Fahlke C, George AL Jr (1996) Molecular basis for decreased muscle chloride conductance in the myotonic goat. Proc Natl Acad Sci USA 93: 11248–11252.
- 34. Niemeyer MI, Yusef YR, Cornejo I, Flores CA, Sepulveda FV, et al. (2004) Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies. Physiol Genomics 19: 74–83.
- 35. Poet M, Kornak U, Schweizer M, Zdebik AA, Scheel O, et al. (2006) Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proc Natl Acad Sci U S A 103: 13854–13859.
- 36. Cleiren E, Benichou O, Van Hul E, Gram J, Bollerslev J, et al. (2001) Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the CLCN7 chloride channel gene. Hum Mol Genet 10: 2861–2867.
- 37. Jeck N, Konrad M, Peters M, Weber S, Bonzel KE, et al. (2000) Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype. Pediatr Res 48: 754–758.
- 38. Bateman A (1997) The structure of a domain common to archaebacteria and the homocystinuria disease protein. Trends Biochem Sci 22: 12–13.
- 39. Ignoul S, Eggermont J (2005) CBS domains: structure, function, and pathology in human proteins. Am J Physiol Cell Physiol 289: C1369–1378.
- 40. Janosik M, Kery V, Gaustadnes M, Maclean KN, Kraus JP (2001) Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region. Biochemistry 40: 10625–10633.
- 41. Daniel T, Carling D (2002) Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White syndrome. J Biol Chem 277: 51017–51024.
- 42. Markovic S, Dutzler R (2007) The structure of the cytoplasmic domain of the chloride channel ClC-Ka reveals a conserved interaction interface. Structure 15: 715–725.
- 43. Meyer S, Savaresi S, Forster IC, Dutzler R (2007) Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5. Nat Struct Mol Biol 14: 60–67.
- 44. Meyer S, Dutzler R (2006) Crystal structure of the cytoplasmic domain of the chloride channel ClC-0. Structure 14: 299–307.
- 45. Zhang R, Evans G, Rotella FJ, Westbrook EM, Beno D, et al. (1999) Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase. Biochemistry 38: 4691–4700.
- 46. Day P, Sharff A, Parra L, Cleasby A, Williams M, et al. (2007) Structure of a CBS-domain pair from the regulatory gamma1 subunit of human AMPK in complex with AMP and ZMP. Acta Crystallogr D Biol Crystallogr 63: 587–596.
- 47. Proudfoot M, Sanders SA, Singer A, Zhang R, Brown G, et al. (2008) Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain. J Mol Biol 375: 301–315.
- 48. Lin CW, Chen TY (2003) Probing the pore of ClC-0 by substituted cysteine accessibility method using methane thiosulfonate reagents. J Gen Physiol 122: 147–159.
- 49. Engh AM, Maduke M (2005) Cysteine accessibility in ClC-0 supports conservation of the CLC intracellular vestibule. J Gen Physiol 125: 601–617.
- 50. Waldegger S, Jentsch TJ (2000) Functional and structural analysis of ClC-K chloride channels involved in renal disease. J Biol Chem 275: 24527–24533.
- 51. Uchida S, Sasaki S, Nitta K, Uchida K, Horita S, et al. (1995) Localization and functional characterization of rat kidney-specific chloride channel, ClC-K1. J Clin Invest 95: 104–113.
- 52. Miller MD, Schwarzenbacher R, von Delft F, Abdubek P, Ambing E, et al. (2004) Crystal structure of a tandem cystathionine-beta-synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 A resolution. Proteins 57: 213–217.
- 53. Kondo Y, Yoshitomi K, Imai M (1988) Effect of Ca2+ on Cl− transport in thin ascending limb of Henle's loop. Am J Physiol 254: F232–239.
- 54. Mupanomunda MM, Tian B, Ishioka N, Bukoski RD (2000) Renal interstitial Ca2+. Am J Physiol Renal Physiol 278: F644–649.
- 55. Watanabe S, Fukumoto S, Chang H, Takeuchi Y, Hasegawa Y, et al. (2002) Association between activating mutations of calcium-sensing receptor and Bartter's syndrome. Lancet 360: 692–694.
- 56. Vargas-Poussou R, Huang C, Hulin P, Houillier P, Jeunemaitre X, et al. (2002) Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. J Am Soc Nephrol 13: 2259–2266.
- 57. Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503–568.
- 58. Estevez R, Jentsch TJ (2002) CLC chloride channels: correlating structure with function. Curr Opin Struct Biol 12: 531–539.
- 59. Hayama A, Rai T, Sasaki S, Uchida S (2003) Molecular mechanisms of Bartter syndrome caused by mutations in the BSND gene. Histochem Cell Biol 119: 485–493.