(Received for publication, April 21, 1997, and in revised form, May 28, 1997)
From the Center for Molecular Neurobiology Hamburg (ZMNH), Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany
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 -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.
CLC1 chloride channels, originally identified by the expression cloning of CLC-0 from Torpedo electric organ (1), form a large gene family with at least nine members in mammals and conservation down to organisms like Escherichia coli and yeast (for review, see Ref. 2). Their importance is underscored by human inherited diseases; mutations in the muscle channel CLC-1 lead to myotonia (3, 4), and those in CLC-5 lead to proteinuria and kidney stones (5).
CLC proteins are structurally unrelated to other channels, including
Cl channels like
-aminobutyric acid and glycine
receptors or the cystic fibrosis transmembrane regulator CFTR.
Hydrophobicity analysis indicated 13 hydrophobic domains (D1-D13 (1)).
However, newer experimental findings (6-8) 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 (7, 9, 10), and this
may also apply for CLC-1 (11). This one-protein-one-pore architecture
distinguishes the CLC channels from voltage-gated cation channels. In
shaker K channels, four homologous subunits form a single
pore, with equivalent parts of each subunit contributing to it.
Although the -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
-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
(12-15) and CLC channels (9, 16-18). 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 (19, 20) 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 between
NcoI and EcoRI sites of pTLN (21), yielding
pTLNH1. Carboxyl-terminally truncated channels (C390,
C451,
C520,
C597, and
C720; 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 (
N110,
N369,
N413,
N509,
N598, and
N721; 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
N413 starts in an
extracellular loop, we constructed
N413 + SP in which
the signal peptide of the rat nicotinic acetylcholine receptor
-subunit (amino acids 1-47) replaces the first two residues of
mutant
N413 to direct the amino terminus to the
extracellular side. Construct
N413 + SP
C720 is truncated at either
end and was obtained by replacing the COOH terminus of mutant
N413 + SP with the corresponding
NdeI/EcoRI fragment from mutant
C720.
Point mutations D136G, G188A, K585E, and R496S were introduced into
full-length proteins or truncated channels C451 or
N413 and
N413 + 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 mM
Ca(NO3)2, 0.82 mM
MgSO4, 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, N109)
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. 3
and Fig. 2). When CLC-1 is truncated behind the conserved domain D13,
as in the myotonic mutant R894X, currents expressed in
Xenopus oocytes are reduced but qualitatively unchanged
(22). By contrast, truncating the protein before the conserved region
D13 (constructs
C597 and
C720, Fig. 1)
abolishes currents (Fig. 2 for
C720). The complementary
NH2-terminal truncations
N598 and
N721 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
C720 plus
N721 but not with
C597 plus
N598. In another combination
(
C720 plus
N598), we duplicated amino
acids 598-720. This resulted in typical CLC-1 currents, although
currents were significantly lower than with the complementary pair
C720 plus
N721 (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 (23) and point mutations introduced in structure-function studies (9). 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 N413, a signal peptide (SP) from the rat
acetylcholine receptor
-subunit was fused in front of it.
Expression in Xenopus oocytes of single truncated
polypeptides (C390,
C451,
C520,
N369,
N413,
N413 + SP,
N509) did not yield currents
different from negative controls (see Fig.
3 for
N369). When we
coexpressed
C390 plus
N369 and
C451 plus
N413 + SP, respectively, we
observed currents with WT characteristics. The amplitude of
C451 plus
N413 + SP was 20-30% of WT
currents (Fig. 2 and Table I), and only
4% were obtained when coexpressing
C390 and
N369 (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.
|
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).2
This phenotype is retained when the mutation is introduced into C451 and coexpressed with the carboxyl-terminal half
N413 + SP. The D136G mutation (in D1) produces
strongly inward rectifying currents that slowly activate at voltages
more negative than
60 mV (18). In this case, however, no currents
were detectable when we coexpressed
C451 D136G with
N413 + 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 (24). When inserted into
N413 + SP and coexpressed with
C451,
the same properties were found (Fig. 4).
D8 Is Essential for Channel Function, but an Additional D8 Is Tolerated
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 (C390 +
N413 + SP) or contain it twice (
C451 +
N369). 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. 5B for
C451 +
N369, currents were lower but did not differ
qualitatively from WT.
When expressing C451 together with
N413, which lacks the signal peptide added in construct
N413 + SP, one may expect nonfunctional proteins
because D9 may have inserted with an inverted orientation. However, we
again observed WT currents (Fig. 5C). Their size was reduced
as compared with
N413 + SP (Fig. 3C). Thus,
a significant proportion of
N413 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 N369 or
N413 + SP. This mutation was identified in patients
with recessive myotonia. It abolishes chloride currents in the
physiological voltage range (25). 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
N369 or
N413 + SP in molar ratios of
1:2 to 1:6 did not yield currents. Coexpression of WT
C520 with
N413 + 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 C451,
N413 + SP
C720, and
N721
(see Fig. 1, bottom), and again observed typical CLC-1
currents (Fig. 5D). Current amplitudes were lower than for
the pair
C451 +
N413 + 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 (26, 27) as well as eukaryotic
systems. For example, the human 2-adrenergic receptor could be functionally expressed in oocytes from two separate
polypeptides (28). Similar results were obtained for M2/M3 muscarinic
acetylcholine receptors (29) and the anion exchanger AE1 (30). With
CFTR, surprising results by Sheppard et al. (31) 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 (32). 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 -helical membrane proteins is proposed to be a two-stage
process (33). 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 (34).
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 (17, 23). 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 N413. Its intrinsic topogenic activity
cannot be easily explained by the "positive-inside" rule (35-37),
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
(8).
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 (38). 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 (39). D13 was originally identified as a region of intermediate hydrophobicity (1) and is now known to be cytoplasmic (8, 40). 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 (5, 41). A myotonic mutation (R894X) that truncates CLC-1 shortly after D13 leads to reduced but qualitatively unchanged currents (22).
Recently, Bateman (19) and Ponting (20) 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
-synthase) domain
probably consists of two short
-helical and three
-strand
stretches in the order
. 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
C720 +
N721, which contain CBS1 and CBS2, respectively, could
associate to form functional channels. No currents could be seen when
C597 was coexpressed with
N598, which
contains both CBS domains. The successful complementation of
C720 with
N721 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
C720 and
N598
(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
N598 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 (25), together with
C720 (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
C720-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
N369 (D8-D13) or
N413 + SP (D9-D13) or coexpressing
C520 (D1-D10) with
N413 + 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 (11), as unambiguously shown for the CLC-0 chloride channel (9, 10).
We thank Michael Pusch for the G188A mutant and useful discussions and Klaus Steinmeyer for critical reading of the manuscript.