Reconstitution of Functional Voltage-gated Chloride Channels from Complementary Fragments of CLC-1
1997; Elsevier BV; Volume: 272; Issue: 33 Linguagem: Inglês
10.1074/jbc.272.33.20515
ISSN1083-351X
AutoresThomas Schmidt‐Rose, Thomas J. Jentsch,
Tópico(s)Glycogen Storage Diseases and Myoclonus
ResumoWe investigated the effect of truncations on the human muscle chloride channel CLC-1 and studied the functional complementation from partial proteins. Almost complete deletion of the cytoplasmic amino terminus did not affect currents, but truncating the intracellular COOH terminus after Leu720 abolished function. Currents were restored by coexpressing this membrane-embedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine β-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln597 before the first CBS domain, no functional complementation was seen.Complementation was also obtained with channels "split" between transmembrane domains D7 and D8 or domains D8 and D9, but not when split between D10 and D11. Specificity of currents was tested by inserting point mutations in NH2-terminal (G188A and G230E) or COOH-terminal (K585E) fragments. In contrast to G188A and K585E, split channels did not tolerate the D136G mutation, suggesting that it may impede association from nonlinked fragments. Duplication, but not a lack of domain D8 was tolerated in "split" channels. Membrane domains D9–D12 can insert into the membrane without adding a preceding signal peptide to ensure the extracellular amino terminus of D9. Eventually, we succeeded in reconstituting CLC-1 channels from three separate polypeptides: the amino-terminal part up to D8, D9 through CBS1, and the remainder of the cytoplasmic carboxyl terminus.In summary, several regions of CLC channels behave autonomously regarding membrane insertion and folding and mediate protein-protein interactions strong enough to yield functional channels without a direct covalent link. We investigated the effect of truncations on the human muscle chloride channel CLC-1 and studied the functional complementation from partial proteins. Almost complete deletion of the cytoplasmic amino terminus did not affect currents, but truncating the intracellular COOH terminus after Leu720 abolished function. Currents were restored by coexpressing this membrane-embedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine β-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln597 before the first CBS domain, no functional complementation was seen. Complementation was also obtained with channels "split" between transmembrane domains D7 and D8 or domains D8 and D9, but not when split between D10 and D11. Specificity of currents was tested by inserting point mutations in NH2-terminal (G188A and G230E) or COOH-terminal (K585E) fragments. In contrast to G188A and K585E, split channels did not tolerate the D136G mutation, suggesting that it may impede association from nonlinked fragments. Duplication, but not a lack of domain D8 was tolerated in "split" channels. Membrane domains D9–D12 can insert into the membrane without adding a preceding signal peptide to ensure the extracellular amino terminus of D9. Eventually, we succeeded in reconstituting CLC-1 channels from three separate polypeptides: the amino-terminal part up to D8, D9 through CBS1, and the remainder of the cytoplasmic carboxyl terminus. In summary, several regions of CLC channels behave autonomously regarding membrane insertion and folding and mediate protein-protein interactions strong enough to yield functional channels without a direct covalent link. CLC 1The abbreviations used are: CLC, a specific chloride channel family; CLC-X, member X of the CLC chloride channel family; CFTR, cystic fibrosis transmembrane conductance regulator; CBS, cystathionine β-synthase; SP, signal peptide; TMD, transmembrane domain; WT, wild type; PCR, polymerase chain reaction. chloride channels, originally identified by the expression cloning of CLC-0 fromTorpedo electric organ (1Jentsch T.J. Steinmeyer K. Schwarz G. Nature. 1990; 348: 510-514Crossref PubMed Scopus (421) Google Scholar), form a large gene family with at least nine members in mammals and conservation down to organisms likeEscherichia coli and yeast (for review, see Ref. 2Jentsch T.J. Günther W. BioEssays. 1997; 19: 117-126Crossref PubMed Scopus (168) Google Scholar). Their importance is underscored by human inherited diseases; mutations in the muscle channel CLC-1 lead to myotonia (3Steinmeyer K. Ortland C. Jentsch T.J. Nature. 1991; 354: 301-304Crossref PubMed Scopus (363) Google Scholar, 4Koch M.C. Steinmeyer K. Lorenz C. Ricker K. Wolf F. Otto M. Zoll B. Lehmann Horn F. Grzeschik K.H. Jentsch T.J. Science. 1992; 257: 797-800Crossref PubMed Scopus (629) Google Scholar), and those in CLC-5 lead to proteinuria and kidney stones (5Lloyd S.E. Pearce S.H. Fisher S.E. Steinmeyer K. Schwappach B. Scheinman S.J. Harding B. Bolino A. Devoto M. Goodyer P. Rigden S.P. Wrong O. Jentsch T.J. Craig I.W. Thakker R.V. Nature. 1996; 379: 445-449Crossref PubMed Scopus (620) Google Scholar). CLC proteins are structurally unrelated to other channels, including Cl− channels like γ-aminobutyric acid and glycine receptors or the cystic fibrosis transmembrane regulator CFTR. Hydrophobicity analysis indicated 13 hydrophobic domains (D1–D13 (1Jentsch T.J. Steinmeyer K. Schwarz G. Nature. 1990; 348: 510-514Crossref PubMed Scopus (421) Google Scholar)). However, newer experimental findings (6Kieferle S. Fong P. Bens M. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6943-6947Crossref PubMed Scopus (242) Google Scholar, 7Middleton R.E. Pheasant D.J. Miller C. Biochemistry. 1994; 33: 13189-13198Crossref PubMed Scopus (115) Google Scholar, 8Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar) suggest the presence of only 10 (or 12) transmembrane domains (Fig. 1, top). The topology of the D9–D12 region, a long hydrophobic stretch interrupted only once by a short hydrophilic segment, still poses problems. Both the amino- and the carboxyl terminus reside in the cytoplasm, and the loop between D8 and D9 is glycosylated. CLC-0 forms homodimers with one pore per subunit (7Middleton R.E. Pheasant D.J. Miller C. Biochemistry. 1994; 33: 13189-13198Crossref PubMed Scopus (115) Google Scholar, 9Ludewig U. Pusch M. Jentsch T.J. Nature. 1996; 383: 340-343Crossref PubMed Scopus (247) Google Scholar, 10Middleton R.E. Pheasant D.J. Miller C. Nature. 1996; 383: 337-340Crossref PubMed Scopus (221) Google Scholar), and this may also apply for CLC-1 (11Fahlke C. Knittel T. Gurnett C.A. Campbell K.P. George A.L. J. Gen. Physiol. 1997; 109: 93-104Crossref PubMed Scopus (81) Google Scholar). This one-protein-one-pore architecture distinguishes the CLC channels from voltage-gated cation channels. Inshaker K channels, four homologous subunits form a single pore, with equivalent parts of each subunit contributing to it. Although the α-subunit of sodium and calcium channels consists of a single polypeptide chain, it shows the same 4-fold repetition of the K channel subunit motif. Ligand-gated anion channels like the γ-aminobutyric acidA receptor are pentamers, with the second TMD of each monomer contributing to the single common pore. By contrast, and in this respect similar to CLC channels, CFTR also contains 12 TMDs forming one pore per protein. In contrast to CLC channels, it functions as a monomer and consists of two halves that are interrupted by a large cytoplasmic loop. It has two nucleotide binding folds and is a member of the ABC transporter superfamily. In channels with one pore per subunit, several different parts of the polypeptide chain must contribute to the pore, as has been shown both for CFTR (12Anderson M.P. Gregory R.J. Thompson S. Souza D.W. Paul S. Mulligan R.C. Smith A.E. Welsh M.J. Science. 1991; 253: 202-205Crossref PubMed Scopus (893) Google Scholar, 13Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 14McDonough S. Davidson N. Lester H.A. McCarty N.A. Neuron. 1994; 13: 623-634Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 15Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar) and CLC channels (9Ludewig U. Pusch M. Jentsch T.J. Nature. 1996; 383: 340-343Crossref PubMed Scopus (247) Google Scholar, 16Steinmeyer K. Lorenz C. Pusch M. Koch M.C. Jentsch T.J. EMBO J. 1994; 13: 737-743Crossref PubMed Scopus (191) Google Scholar, 17Pusch M. Ludewig U. Rehfeldt A. Jentsch T.J. Nature. 1995; 373: 527-531Crossref PubMed Scopus (302) Google Scholar, 18Fahlke C. Rüdel R. Mitrovic N. Zhou M. George Jr., A.L. Neuron. 1995; 15: 463-472Abstract Full Text PDF PubMed Scopus (104) Google Scholar). These parts must be positioned correctly by intramolecular interactions. In the present work, we show that some of these interactions are strong enough to enable the functional expression of CLC-1 channels from individual polypeptides representing complementary parts of the channel protein. Surprisingly, this not only functions when the "cut" lies between transmembrane spans, but also if an otherwise inactive, COOH-terminally truncated channel is coexpressed with a carboxyl-terminal part lacking any TMD. Here, functional complementation is observed if the cut lies between the first and the second CBS domain (19Bateman A. Trends Biochem. Sci. 1997; 22: 12-13Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 20Ponting C.P. J. Mol. Med. 1997; 75: 160-163Crossref PubMed Scopus (24) Google Scholar) but not if the COOH-terminal part comprises both CBS domains. In addition, we show that "split" channels with a duplicated D8 domain are functional, indicating that D8 lies at the periphery of the channel. Human CLC-1 (GenBankTM accession number Z25884) was cloned betweenNcoI and EcoRI sites of pTLN (21Lorenz C. Pusch M. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13362-13366Crossref PubMed Scopus (215) Google Scholar), yielding pTLNH1. Carboxyl-terminally truncated channels (ΔC390, ΔC451, ΔC520, ΔC597, and ΔC720; numbers indicate last amino acid position) were generated by PCR using 3′-primers that contained two stop codons and an EcoRI restriction site (GCGAATTCTTATCA …) in addition to the template-specific nucleotides. The 5′-primer was chosen upstream of the nearest single-cutter restriction site so that after digestion the PCR product could be ligated into pTLNH1 cut with the same enzymes. Amino-terminal truncations (ΔN110, ΔN369, ΔN413, ΔN509, ΔN598, and ΔN721; number indicates first amino acid position) were constructed using an upstream PCR primer containing the first two codons of CLC-1 (methionine and glutamate) including an NcoI site (GCGATACCATGGAG … ) plus template specific sequences of the new NH2 terminus. The 3′-primer was placed downstream of an appropriate restriction site. Again, PCR products and vector pTLNH1 were cut with the same enzymes and ligated. Since construct ΔN413 starts in an extracellular loop, we constructed ΔN413 + SP in which the signal peptide of the rat nicotinic acetylcholine receptor α-subunit (amino acids 1–47) replaces the first two residues of mutant ΔN413 to direct the amino terminus to the extracellular side. Construct ΔN413 + SPΔC720 is truncated at either end and was obtained by replacing the COOH terminus of mutant ΔN413 + SP with the correspondingNdeI/EcoRI fragment from mutant ΔC720. Point mutations D136G, G188A, K585E, and R496S were introduced into full-length proteins or truncated channels ΔC451 or ΔN413 and ΔN413 + SP by recombinant PCR. All PCR-derived fragments were sequenced. The plasmids were linearized, and capped cRNA was transcribed using SP6 polymerase (mMessage mMachine cRNA synthesis kit, Ambion). cRNA was injected into manually defolliculated Xenopus oocytes, which were kept in modified Barth's solution (90 mm NaCl, 1 mm KCl, 1 mm CaCl2, 0.33 mmCa(NO3)2, 0.82 mmMgSO4, 10 mm Hepes, pH 7.6) at 17 °C for 2–3 days before measurement. In coexpression experiments, we used a molar cRNA ratio of 1:1 for channel fragments containing transmembrane domains and 3:1 for the soluble cytoplasmic carboxyl terminus. Currents were measured at room temperature in ND96 (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm Hepes, pH 7.4) using a two-electrode voltage clamp with a Turbotec amplifier (Npi Instruments) and pCLAMP 5.5 software (Axon Instruments). A typical pulse protocol is depicted in Fig. 2. Currents were recorded in response to 150-ms voltage steps ranging from +80 to −160 mV after a 25-ms prepulse to +80 mV (to open the gate) from the resting potential of the individual oocyte. This was followed by a constant step to −60 mV and a return to the resting potential. For mutant G188A, the voltage was stepped for 4 s from the resting potential to voltages from +20 to −160 mV in steps of −20 mV, followed by a constant pulse at −85 mV. Leakage or capacitive currents were not subtracted, but in the figures capacitive transients were cut off for clarity. Mutants were measured in at least two batches of oocytes (n ≥ 6). Both the amino and carboxyl termini differ in length among CLC family members and are poorly conserved except for few regions in the carboxyl terminus, including domain D13. We asked whether they serve specific functions and at first deleted the amino terminus between glutamate 2 and valine 110 (Fig.1). This leaves only about 10 amino acids before the first TMD. Expression in Xenopus oocytes resulted in currents (Fig. 2, ΔN109) indistinguishable from wild type (WT). CLC-1 WT currents have a chloride > iodide ion selectivity, are inwardly rectifying, and display a rapid, partial deactivation upon hyperpolarization (Ref. 3Steinmeyer K. Ortland C. Jentsch T.J. Nature. 1991; 354: 301-304Crossref PubMed Scopus (363) Google Scholarand Fig. 2). When CLC-1 is truncated behind the conserved domain D13, as in the myotonic mutant R894X, currents expressed inXenopus oocytes are reduced but qualitatively unchanged (22Meyer-Kleine C. Steinmeyer K. Ricker K. Jentsch T.J. Koch M.C. Am. J. Hum. Genet. 1995; 57: 1325-1334PubMed Google Scholar). By contrast, truncating the protein before the conserved region D13 (constructs ΔC597 and ΔC720, Fig. 1) abolishes currents (Fig. 2 for ΔC720). The complementary NH2-terminal truncations ΔN598 and ΔN721 did not give any currents either when injected alone (data not shown). Surprisingly, coexpression of the carboxyl-terminal part with the complementary membrane-anchored part of CLC-1 reconstituted robust WT currents for the combination ΔC720 plus ΔN721 but not with ΔC597 plus ΔN598. In another combination (ΔC720 plus ΔN598), we duplicated amino acids 598–720. This resulted in typical CLC-1 currents, although currents were significantly lower than with the complementary pair ΔC720 plus ΔN721 (Fig. 2). We next asked whether similar complementations might be possible if we "cut" the channel protein between TMDs and designed further constructs (Fig. 1). Several loops connecting CLC-1 transmembrane domains are short and highly conserved among CLC-0, CLC-1, and CLC-2 (especially D2-D3, D5-D6, and D6-D7), suggesting that they may be important for function. This has been confirmed by missense mutations found in human myotonia (23Pusch M. Steinmeyer K. Koch M.C. Jentsch T.J. Neuron. 1995; 15: 1-20Abstract Full Text PDF PubMed Scopus (174) Google Scholar) and point mutations introduced in structure-function studies (9Ludewig U. Pusch M. Jentsch T.J. Nature. 1996; 383: 340-343Crossref PubMed Scopus (247) Google Scholar). Therefore, we focused on the large, poorly conserved extracellular loop between D8 and D9 and on the shorter, but also poorly conserved, D7-D8 and D10-D11 stretches. Further, since sequences flanking TMDs may be important for their correct membrane insertion, we decided to use slightly overlapping constructs. To ensure the extracellular position of the amino terminus of construct ΔN413, a signal peptide (SP) from the rat acetylcholine receptor α-subunit was fused in front of it. Expression in Xenopus oocytes of single truncated polypeptides (ΔC390, ΔC451, ΔC520, ΔN369, ΔN413, ΔN413 + SP, ΔN509) did not yield currents different from negative controls (see Fig.3 for ΔN369). When we coexpressed ΔC390 plus ΔN369 and ΔC451 plus ΔN413 + SP, respectively, we observed currents with WT characteristics. The amplitude of ΔC451 plus ΔN413 + SP was 20–30% of WT currents (Fig. 2 and Table I), and only 4% were obtained when coexpressing ΔC390 and ΔN369 (Fig. 3 and Table I). The apparently increased outward current at +80 mV for the latter combination is probably due to endogenous oocyte channels (compare H2O-injected control in Fig. 2) and was still present when CLC-1 currents were specifically blocked with 9-anthracene-carboxylic acid (data not shown). When the boundary was placed in the small hydrophilic loop between hydrophobic domains D10 and D11, we could not detect currents upon coexpression. Obviously, partial CLC-1 proteins can insert correctly into the membrane and form channels by associating with the corresponding counterpart. Voltage-dependent gating, rectification, and ion selectivity are all conserved (ion selectivity data not shown). Thus, they do not need an uninterrupted peptide backbone.Figure 3Functional complementation by coexpression of partial CLC-1 proteins truncated within the membrane-embedded region. A, current trace of ΔN369 showing that this construct is nonfunctional, yielding currents indistinguishable from negative controls. This is representative for all individually expressed truncated constructs shown in thisfigure. B, intracellular "split position" between D7 and D8. C, extracellular boundary between D8 and D9. D, intracellular split between D10 and D11. While typical CLC-1 currents are seen in panels B andC, this combination does not yield chloride currents. Chloride currents were recorded as described for Fig. 2. Current traces are representative examples from at least two batches of oocytes (n ≥ 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Currents expressed from WT CLC-1, truncated channel proteins, and channels split in the carboxyl-terminal cytosolic portion. Top, voltage protocol. Chloride currents were recorded in response to 150-ms voltage steps ranging from +80 to −160 mV after a 25-ms prepulse to +80 mV from the resting potential of the individual oocyte. This prepulse opens the gate of CLC-1, which closes upon hyperpolarization. These test pulses were followed by a constant step to −60 mV and a return to the resting potential. Note the differing scales for individual recordings, as indicated by theangles. Capacitive transients were cut off for clarity infigures. For diagrams of constructs and nomenclature of truncated channels see Fig. 1 and "Experimental Procedures."WT, currents of the full-length channel protein. ΔN109, deletion of the cytoplasmic NH2 terminus. ΔC720, truncation of the COOH terminus at position 720.Bottom, different combinations of amino- and carboxyl-terminal portions. All current traces are representative examples from at least two batches of oocytes (n ≥ 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Schematic diagram of CLC-1 constructs.Hydrophobic domains are termed D1–D13 according to Refs. 1Jentsch T.J. Steinmeyer K. Schwarz G. Nature. 1990; 348: 510-514Crossref PubMed Scopus (421) Google Scholar and 8Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar.Top, wild type CLC-1 with point mutations used in this work. Channel transmembrane topology is shown according to Refs. 8Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar and 42Jentsch T.J. Günther W. Pusch M. Schwappach B. J. Physiol. (Lond.). 1995; 482: 19S-25SCrossref Scopus (203) Google Scholar. The glycosylation site in the extracellular loop connecting D8 and D9 is indicated by branched lines. The CBS domains (19Bateman A. Trends Biochem. Sci. 1997; 22: 12-13Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 20Ponting C.P. J. Mol. Med. 1997; 75: 160-163Crossref PubMed Scopus (24) Google Scholar) in the cytoplasmic carboxyl terminus are shown in black. CBS2 coincides with D13. Bottom, carboxyl-terminal (ΔC) and amino-terminal (ΔN) truncation mutants. Juxtaposed constructs have been coexpressed, but not all combinations tested are included in thisfigure. The hydrophobic domain D8 is drawn ingray in the pair ΔC451 plus ΔN369 to emphasize its duplication.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IComparative summary of currentsConstructConductanceConstructConductanceConstructConductanceμSμSμSWater-injected1.7 ± 0.2ΔC720 + ΔN72163.2 ± 94ΔC390 + ΔN3693.1 ± 0.21-aDespite the low absolute values, these currents were clearly recognized as typically CLC-1 by their characteristic kinetics (see Fig. 5).WT78.2 ± 9.4ΔC597 + ΔN5981.6 ± 0.2ΔC390 + ΔN413 + SP1.5 ± 0.3ΔN10973.9 ± 8.6ΔC720 + ΔN59840.3 ± 5.2ΔC451 + ΔN3695.0 ± 0.81-aDespite the low absolute values, these currents were clearly recognized as typically CLC-1 by their characteristic kinetics (see Fig. 5).ΔC7201.2 ± 0.2ΔC520 + ΔN5091.1 ± 0.1ΔC720 + R496S2.2 ± 0.2ΔN7212.2 ± 0.8ΔC451 + ΔN4135.9 ± 1.21-aDespite the low absolute values, these currents were clearly recognized as typically CLC-1 by their characteristic kinetics (see Fig. 5).ΔC451 + ΔN721 + ΔN413 + SPΔC72010.2 ± 0.9ΔC451 + ΔN413 + SP19.2 ± 1.1Slope conductance at −20 mV for wild type CLC-1, truncation constructs, and coexpressed mutants was calculated. Results ofn ≥ 6 measurements are given in microsiemens (μS) as mean ± S.E. Data of nonfunctional truncated constructs other than ΔC720 and ΔN721 are not listed, but they gave similar conductances. Leak currents were not subtracted, but water-injected controls are included.1-a Despite the low absolute values, these currents were clearly recognized as typically CLC-1 by their characteristic kinetics (see Fig. 5). Open table in a new tab Slope conductance at −20 mV for wild type CLC-1, truncation constructs, and coexpressed mutants was calculated. Results ofn ≥ 6 measurements are given in microsiemens (μS) as mean ± S.E. Data of nonfunctional truncated constructs other than ΔC720 and ΔN721 are not listed, but they gave similar conductances. Leak currents were not subtracted, but water-injected controls are included. Several point mutations characteristically change CLC-1 currents. We wanted to check whether these effects are retained when expressed in "split" CLC-1 proteins. Mutating glycine 188 in D2 to alanine shifts the voltage dependence. Currents activate at positive potentials and exhibit steady state outward rectification (Fig.4). 2M. Pusch and T. J. Jentsch, unpublished observation. This phenotype is retained when the mutation is introduced into ΔC451 and coexpressed with the carboxyl-terminal half ΔN413 + SP. The D136G mutation (in D1) produces strongly inward rectifying currents that slowly activate at voltages more negative than −60 mV (18Fahlke C. Rüdel R. Mitrovic N. Zhou M. George Jr., A.L. Neuron. 1995; 15: 463-472Abstract Full Text PDF PubMed Scopus (104) Google Scholar). In this case, however, no currents were detectable when we coexpressed ΔC451 D136G with ΔN413 + SP (Fig. 4). Replacing lysine 585 at the end of D12 with glutamate leads to channels deactivating more slowly than WT and displaying larger outward currents (24Rychkov G.P. Pusch M. Astill A.S.J. Roberts M.L. Jentsch T.J. Bretag A.H. J. Physiol. (Lond.). 1996; 497: 423-435Crossref Scopus (142) Google Scholar). When inserted into ΔN413 + SP and coexpressed with ΔC451, the same properties were found (Fig.4). We also co-injected some of the "half-channels" in different combinations. As shown in Fig. 1, this produces channels that either lack domain D8 (ΔC390 + ΔN413 + SP) or contain it twice (ΔC451 + ΔN369). While no currents were obtained when D8 was lacking (data not shown), a duplication of this TMD was tolerated. As can be seen in Fig. 5 B for ΔC451 + ΔN369, currents were lower but did not differ qualitatively from WT. When expressing ΔC451 together with ΔN413, which lacks the signal peptide added in construct ΔN413 + SP, one may expect nonfunctional proteins because D9 may have inserted with an inverted orientation. However, we again observed WT currents (Fig. 5 C). Their size was reduced as compared with ΔN413 + SP (Fig. 3 C). Thus, a significant proportion of ΔN413 proteins have inserted correctly even without an additional signal peptide. As shown above, an additional transmembrane domain D8 was tolerated in split channels. This suggests that either the one in the NH2- or the COOH-terminal fragment is displaced from its normal position. We wondered whether a carboxyl-terminal half-channel can replace its equivalent from the full-length channel after coexpression, which would imply that the channel structure is flexible enough to allow such a replacement after the assembly of the complete channel from the full-length protein. We expressed the CLC-1 mutant R496S (in the D9/D10 block) together with constructs ΔN369 or ΔN413 + SP. This mutation was identified in patients with recessive myotonia. It abolishes chloride currents in the physiological voltage range (25Lorenz C. Meyer Kleine C. Steinmeyer K. Koch M.C. Jentsch T.J. Hum. Mol. Genet. 1994; 3: 941-946Crossref PubMed Scopus (109) Google Scholar). If this part of the protein can be displaced by the nonmutated equivalent of the carboxyl-terminal "half-channel," one should be able to detect CLC-1 currents. However, coexpression of full-length CLC-1 R496S with constructs ΔN369 or ΔN413 + SP in molar ratios of 1:2 to 1:6 did not yield currents. Coexpression of WT ΔC520 with ΔN413 + SP, which duplicates the D9-D10 stretch, did not yield functional channels either (data not shown). We could obtain functional channels when "splitting" CLC-1 between TMDs D8 and D9 or after residue 720 in the cytoplasmic carboxyl terminus. This suggests that also the stretch extending from D9 to the end of CBS1 may fold correctly to an independent "module." We therefore co-injected cRNA for the three constructs ΔC451, ΔN413 + SPΔC720, and ΔN721(see Fig. 1, bottom), and again observed typical CLC-1 currents (Fig. 5 D). Current amplitudes were lower than for the pair ΔC451 + ΔN413 + SP. This is probably due to decreasing efficiency of assembly with an increasing number of fragments. Reconstitution of membrane proteins from artificial fragments has been demonstrated in prokaryotic (26Bibi E. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4325-4329Crossref PubMed Scopus (166) Google Scholar, 27Zen K.H. McKenna E. Bibi E. Hardy D. Kaback H.R. Biochemistry. 1994; 33: 8198-8206Crossref PubMed Scopus (80) Google Scholar) as well as eukaryotic systems. For example, the human β2-adrenergic receptor could be functionally expressed in oocytes from two separate polypeptides (28Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (609) Google Scholar). Similar results were obtained for M2/M3 muscarinic acetylcholine receptors (29Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar) and the anion exchanger AE1 (30Groves J.D. Tanner M.J.A. J. Biol. Chem. 1995; 270: 9097-9105Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). With CFTR, surprising results by Sheppard et al. (31Sheppard D.N. Ostedgaard L.S. Rich D.P. Welsh M.J. Cell. 1994; 76: 1091-1098Abstract Full Text PDF PubMed Scopus (101) Google Scholar) show that its NH2-terminal half can dimerize with itself and form Cl− channels without the COOH-terminal half. Similar truncated CFTR proteins have been described in kidney (32Morales M.M. Carroll T.P. Morita T. Schwiebert E.M. Devuyst O. Wilson P.D. Lopes A.G. Stanton B.A. Dietz H.C. Cutting G.R. Guggino W.B. Am. J. Physiol. 1996; 270: F1038-F1048Crossref PubMed Google Scholar). This probably reflects the symmetry inherent to ABC transporters, although, with the exception of the nucleotide binding folds, the first and the second part of the protein are not highly homologous. In contrast, and not surprising given its structure, none of the CLC-1 proteins lacking TMDs could form functional channels when expressed by itself. In many transmembrane proteins, internal topogenic sequences ensure a correct integration even if the normal translation start is absent. The folding of α-helical membrane proteins is proposed to be a two-stage process (33Popot J.L. Engelman D.M. Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (821) Google Scholar). First, hydrophobic domains are established in the lipid bilayer, which then interact to form the final three-dimensional structure. Thus, there seems to be no principal difference between inter- and intramolecular assembly of transmembrane domains, apart from the facilitated encounter of helices in a single polypeptide chain. Expression of truncated proteins lacking one or more transmembrane domains may, however, result in misfolding if a certain succession of the amino acid sequence is required to achieve the correct insertion of the nascent polypeptide chain. Moreover, newly synthesized TMDs may need previously translated TMDs for "guided" folding and assembly. The successful assembly of functional proteins from individual parts suggests that these form structurally independent subdomains or "modules" whose tertiary structure does not differ too much from the one found within the native protein. Further, the intermolecular interactions between these different modules is strong enough to allow their association in the absence of a covalent link. Examples of modular structures of membrane channels and transporters are provided by some gene superfamilies: voltage-gated sodium and calcium channels have four modules covalently linked in a single polypeptide, whereas they are encoded separately in K channels; ABC transporters can be encoded by one, two, or four genes, whose individual products will assemble and form the complete transporter (34Higgins C.F. Annu. Rev. Cell Biol. 1992; 113: 67-113Crossref Scopus (3375) Google Scholar). In the CLC-1 chloride channel, at least three regions are structurally autonomous: an amino-terminal one comprising D1–D8, a central one containing the D9–D12 block and part of the cytoplasmic carboxyl terminus, and the rest of the hydrophilic tail, which includes D13. A cut between D10 and D11, however, was not tolerated. This suggests that either the NH2- or the COOH-terminal portion is misfolded (or both) or that interactions between these different parts are too weak to allow for their association even if folded correctly. The D9–D12 region is a broad hydrophobic region that is important for permeation and gating (17Pusch M. Ludewig U. Rehfeldt A. Jentsch T.J. Nature. 1995; 373: 527-531Crossref PubMed Scopus (302) Google Scholar, 23Pusch M. Steinmeyer K. Koch M.C. Jentsch T.J. Neuron. 1995; 15: 1-20Abstract Full Text PDF PubMed Scopus (174) Google Scholar). In the absence of clearly separated hydrophobic domains, it seems possible that it folds correctly only if translated as a continuous polypeptide. This view is strengthened by the fact that no cleavable signal peptide was necessary to correctly express fragment ΔN413. Its intrinsic topogenic activity cannot be easily explained by the "positive-inside" rule (35von Heijne G. Nature. 1989; 341: 456-458Crossref PubMed Scopus (431) Google Scholar, 36Sipos L. von Heijne G. Eur. J. Biochem. 1993; 213: 1333-1340Crossref PubMed Scopus (253) Google Scholar, 37Spiess M. FEBS Lett. 1995; 369: 76-79Crossref PubMed Scopus (105) Google Scholar), since there is no conspicuous charge asymmetry in this region. Exact topology is still unknown here except for the fact that the region before D9 is extracellular and the region after D12 is intracellular (8Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar). The CLC-1 protein could also be split between D7 and D8, but expression was less efficient than with the D8-D9 cut. The D7-D8 linker is shorter than the one connecting D8 and D9 and rather poorly conserved. However, it is always highly positively charged, and mutagenesis showed that it is important for CLC-2 gating (38Jordt S.E. Jentsch T.J. EMBO J. 1997; 16: 1582-1592Crossref PubMed Scopus (205) Google Scholar). D8 is probably located at the channel periphery, since split channels functionally tolerated an extra D8 copy. Thus, the channel structure must allow for an extrusion of D8 domain into the lipid bilayer. Channels deleted for D13 could be functionally complemented by coexpressing the lacking hydrophilic COOH-terminal portion. Similar results were recently reported for the Torpedo channel CLC-0 (39Maduke M. Miller C. Biophys. J. 1997; 72: A5Abstract Full Text PDF PubMed Scopus (60) Google Scholar). D13 was originally identified as a region of intermediate hydrophobicity (1Jentsch T.J. Steinmeyer K. Schwarz G. Nature. 1990; 348: 510-514Crossref PubMed Scopus (421) Google Scholar) and is now known to be cytoplasmic (8Schmidt-Rose T. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7633-7638Crossref PubMed Scopus (101) Google Scholar, 40Gründer S. Thiemann A. Pusch M. Jentsch T.J. Nature. 1992; 360: 759-762Crossref PubMed Scopus (362) Google Scholar). It is conserved in all eukaryotic CLC proteins, suggesting some important, as yet unknown function. By contrast, it is absent from the two E. coli CLC proteins. Mutations in human disease already hinted at the importance of D13: point mutations truncating CLC-5 before or within D13 lead to Dent's disease, and these truncated proteins were nonfunctional in Xenopus oocytes (5Lloyd S.E. Pearce S.H. Fisher S.E. Steinmeyer K. Schwappach B. Scheinman S.J. Harding B. Bolino A. Devoto M. Goodyer P. Rigden S.P. Wrong O. Jentsch T.J. Craig I.W. Thakker R.V. Nature. 1996; 379: 445-449Crossref PubMed Scopus (620) Google Scholar, 41Lloyd S.E. Pearce S.H.S. Günther W. Kawaguchi H. Igarashi T. Jentsch T.J. Thakker R.V. J. Clin. Invest. 1997; 99: 967-974Crossref PubMed Scopus (136) Google Scholar). A myotonic mutation (R894X) that truncates CLC-1 shortly after D13 leads to reduced but qualitatively unchanged currents (22Meyer-Kleine C. Steinmeyer K. Ricker K. Jentsch T.J. Koch M.C. Am. J. Hum. Genet. 1995; 57: 1325-1334PubMed Google Scholar). Recently, Bateman (19Bateman A. Trends Biochem. Sci. 1997; 22: 12-13Abstract Full Text PDF PubMed Scopus (442) Google Scholar) and Ponting (20Ponting C.P. J. Mol. Med. 1997; 75: 160-163Crossref PubMed Scopus (24) Google Scholar) identified a more general structural motif by data base screening to which the D13 domain conforms. Based on the crystal structure of inosine-5′-monophosphate-dehydrogenase, this so-called CBS (cystathionine β-synthase) domain probably consists of two short α-helical and three β-strand stretches in the order βαββα. It is found in various other proteins like a certain protein kinase subunit and ABC transporters, but its functional role is unknown. Interestingly, the CBS domain is found twice in every eukaryotic CLC protein. The second CBS domain (CBS2) roughly coincides with D13, while the other one (CBS1) lies between D12 and D13. Strikingly, only constructs ΔC720 + ΔN721, which contain CBS1 and CBS2, respectively, could associate to form functional channels. No currents could be seen when ΔC597 was coexpressed with ΔN598, which contains both CBS domains. The successful complementation of ΔC720 with ΔN721 suggests that CBS2 forms a correctly folded module, which can bind somewhere to the protein truncated behind CBS1. It is presently unclear whether this is by direct interaction between CBS1 and CBS2. When we coexpressed the overlapping constructs ΔC720 and ΔN598(which contains CBS1 and CBS2), we again observed functional complementation, which, however, was less efficient. Thus, if CBS1-CBS2 binding is necessary, their intramolecular interaction in construct ΔN598 is weak enough to allow intermolecular competition with CBS1 from the channel backbone. On the other hand, coexpressing a nonconducting full-length channel, CLC-1 R496S (25Lorenz C. Meyer Kleine C. Steinmeyer K. Koch M.C. Jentsch T.J. Hum. Mol. Genet. 1994; 3: 941-946Crossref PubMed Scopus (109) Google Scholar), together with ΔC720 (lacking CBS2) did not yield currents. This could mean that CBS2 interacts too strongly with the rest of the CLC-1 R496S channel protein and therefore does not swing over to a nearby ΔC720-protein in a heteromultimer and does not complement this truncated channel. A displacement of larger intramembranous parts of the channel also failed when coexpressing CLC-1 R496S (D1–D13) with constructs ΔN369 (D8–D13) or ΔN413 + SP(D9–D13) or coexpressing ΔC520 (D1–D10) with ΔN413 + SP (D9–D13). Channel properties like gating kinetics and rectification were preserved in functional split channels, and two of the point mutations introduced into partial channels retained their characteristic effects. However, mutation D136G prevented functional reconstitution from "half-channels." Possibly, neutralization of the negative charge in D1 at position 136 affects the contact surfaces between the two parts, thereby disturbing their association. In the full-length protein, tight spatial proximity is ensured by covalent links, leading to a functional pore whose gating, however, is drastically changed. In summary, we could show that functional CLC-1 chloride channels can be formed from fragments, indicating the presence of at least three modular domains that can insert and fold independently and then associate. This includes the cytoplasmic domain D13, which conforms to the newly identified CBS motif. The subunits formed by the association of these partial proteins are then likely to further associate to form a homodimeric channel (11Fahlke C. Knittel T. Gurnett C.A. Campbell K.P. George A.L. J. Gen. Physiol. 1997; 109: 93-104Crossref PubMed Scopus (81) Google Scholar), as unambiguously shown for the CLC-0 chloride channel (9Ludewig U. Pusch M. Jentsch T.J. Nature. 1996; 383: 340-343Crossref PubMed Scopus (247) Google Scholar, 10Middleton R.E. Pheasant D.J. Miller C. Nature. 1996; 383: 337-340Crossref PubMed Scopus (221) Google Scholar). We thank Michael Pusch for the G188A mutant and useful discussions and Klaus Steinmeyer for critical reading of the manuscript.
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