From the Centre for Advanced Biomedical Studies,
University of South Australia, North Terrace, Adelaide, South Australia
5000, Australia and the ¶ Department of Physiology, University of
Adelaide, Adelaide, South Australia 5005, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although hydropathy analysis of the skeletal
muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological
studies have suggested that domain D4 is extracellular and that D13,
conserved in all eukaryotic ClC channels, is located within the
extensive cytoplasmic tail that makes up the carboxyl terminus of the
protein. We have examined the effect of deleting D13 (D13) and the
function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from
rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp
analysis of chloride currents in Sf9 cells showed that only
relatively insubstantial changes could be attributed to the expressed
fs923X, fs895X, and
D13 mutants compared with wild type rat ClC-1.
For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was
demonstrable by polyacrylamide gel electrophoresis, but no chloride
conductance attributable to it could be detected. Overall, our results
indicate that the domain D13 is dispensable, as are the final 100 amino
acids, but not the final 125 amino acids or more, of the carboxyl tail.
Some essential region of unknown significance, therefore, appears to
reside in the 18 amino acids after D13, from Lys877
to Arg894.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mammalian skeletal muscle, the voltage-gated chloride channel, ClC-1, is responsible for the greater proportion of the resting conductance, which acts to stabilize the membrane potential against unwanted perturbations. Its structure, as predicted by hydropathy analysis (1) and in common with other members of the large ClC family, includes 13 relatively hydrophobic domains (termed D1 to D13), most of which probably span the membrane. There is good evidence, however, that D4 and D13 are located entirely extracellularly and intracellularly, respectively (2, 3), with the implication that close to half the amino acids of human ClC-1 (hClC-1)1 must form an extensive cytoplasmic tail (Gln574-Leu988). The D13 domain, which is conserved among all eukaryotic ClC channels (4), is situated in this tail, about two-thirds of the way to the carboxyl terminus (Leu840-Ile870).
Mutations in ClC-1 have been associated with both dominant and recessive forms of myotonia (e.g. Refs. 5 and 6) characterized by abnormal, sustained firing of action potentials that result in prolonged involuntary muscle contractions. In one (R894X) of two myotonic mutations identified in the carboxyl tail (7, 8) the final 95 amino acids of the protein (8) are lost. Functional analysis of this mutant, expressed in Xenopus oocytes, showed a large reduction in chloride currents, which could account for the myotonic symptoms (9). In similar studies (4), chloride currents were totally lost from human ClC-1 truncated at Ser720 (G721X) and at Gln597 (L598X). This suggests that normal function of the ClC-1 protein depends on the integrity of the carboxyl tail and particularly on that portion between Ser720 and Arg894, with some significant role for D13 being implied. Reconstitution experiments (4), in which the nonfunctional mutant S720X was restored to functionality by co-expression with either the complementary or an overlapping carboxyl tail peptide, have reinforced this view. Another hint of the importance of D13 is provided by mutations that truncate the kidney chloride channel, ClC-5, before or within D13, these also being nonfunctional when expressed in Xenopus oocytes (10, 11).
We have investigated the relevance of the cytoplasmic tail by generating truncation and deletion mutants of rat ClC-1, expressing them in the baculovirus Sf9 insect cell system (12), and performing functional analysis by patch clamp.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutagenesis--
Rat ClC-1 cDNA (GenBankTM accession number
P35524) was cloned into the vector pBacPAK 8 (CLONTECH) yielding the plasmid pDA6 (13) in which
the carboxyl terminus truncation mutants Q574X, N596X, and L869X were
generated using the Altered Sites System (Promega) and the mutagenic
primers Q574X (5'-GAGGGAGGGTCACAGACTTTG-3'), N596X
(5'-GCTGAGCTGTCACCAACCAAG-3'), and L869X (5'-TAGCTCTTCTCATGCCAAGAC-3'). Convenient restriction enzyme sites were used to generate additional truncation mutants (fs923X and fs895X). Restriction of pDA6 with SacI yielded fragments of 8.28 and 0.74 kb, the larger of
which was self-ligated to produce the desired mutation (fs923X). In the
process, eight extraneous amino acids (RIPGRPLN) were introduced between Ala922 and the premature termination codon.
Restriction of pDA6 with XmaI and AccIII yielded
fragments of sizes 6.51, 1.67, and 0.84 kb. The 6.51- and 1.67-kb
fragments were ligated to produce the desired mutation (fs895X) with
three extraneous amino acids (AAA) being introduced between
Arg894 and the premature termination codon. Deletion of the
domain D13 was performed by recombinant PCR (14) using the primers D13F (5'-TGGCCCTTTGTATGAGTCTTGTG-3') and D13R
(5'-CTCATACAAAGGGCCACACCAAA-3') along with Bac 1 and Bac 2 (CLONTECH) flanking primers to amplify the rClC
cDNA sequence prior to D13 (up to and including Thr845)
and following D13 (from and including Lys877),
respectively. The altered cDNA (D13) was cloned into pBacPAK 8 using the restriction enzymes BamHI and KpnI. All
mutant constructs were confirmed by restriction digestion analysis
where possible and, finally, verified by DNA sequencing. Mutation sites
are indicated in Fig. 1.
|
Protein Expression-- Recombinant baculoviruses containing the mutated cDNAs were produced in Sf21 insect cells. Transfer of the relevant DNA insert into the viral genome was confirmed by PCR analysis using the rClC-1-specific primers Se 5 (5'-CCTGGAATCGTTACTTTTGTC-3') and Se 6 (5'-TCCAAAGGCAGCTCCTAGCAC-3'), and viral clones were amplified, titred, and screened for protein production as described elsewhere (12, 13).
Reverse Transcription Polymerase Chain Reaction
(RT-PCR)--
Messenger RNA was extracted from cells infected with the
WT, Q574X, and N596X recombinant baculoviruses. Briefly, 4 × 106 Sf21 insect cells were infected with recombinant
baculovirus with a multiplicity of infection of 20 as described
previously (13). After 48 h of incubation, mRNA was extracted
using the QuickPrep Micro mRNA Purification kit (Pharmacia Biotech
Inc.) and reverse transcribed with the First Strand Synthesis kit
(Pharmacia) to produce cDNA, which was amplified by PCR using the
rClC-1-specific primers Se 3 (5'-GCAGTGATTGGAGCAGCAGCT-3') and Se 4 (5'-CTTGACTGTGGTGGTCTGGAG-3'). The RT-PCR was standardized by
amplification of the cDNAs with -actin-specific primers (15). As
controls, RT-PCR was performed on mRNA extracted from uninfected
cells, and to ensure that mRNA extracted from appropriately
infected cells had no DNA contamination, PCR was also performed on the
mRNA extract without the reverse transcription step. These samples
gave no bands.
Electrophysiology--
Cultured Sf9 cells were infected
with control baculovirus BVDA6.3 containing rClC cDNA or relevant
mutants, incubated for 28-30 h at 28 °C in air, and then seeded
onto glass coverslips and maintained at room temperature as described
previously (13). Whole cell patch clamp experiments were performed
using a List EPC7 patch clamp amplifier and associated standard
equipment. The usual bath solution contained 170 mM NaCl, 2 mM MgSO4, 2 mM CaCl2,
and 10 mM HEPES (adjusted to pH 7.5 with NaOH).
Borosilicate glass electrodes had a resistance of 1-3 M when filled
with a normal internal solution: 40 mM KCl, 120 mM potassium glutamate, 10 mM Na-EGTA, and 10 mM HEPES (adjusted to pH 7.2 with NaOH). Approximately 90%
of series resistance was compensated. Nominal holding and clamping
potentials reported here must be corrected for a liquid junction
potential of
14 mV estimated to occur between the bath and electrode
solutions (16). Pentobarbitone (0.5 mM) was used to block
native anion channels in Sf9 cells. Experiments were conducted
at room temperatures of 24 ± 1 °C. Data were collected, filtered at 3 kHz, and analyzed on an IBM-compatible PC using pCLAMP
v6.0 software (Axon Instruments). Parameters such as time constants,
apparent open probability, and IC50 were determined as
described previously (16, 17).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of the protein content of Sf21 cells infected with
the appropriate baculovirus construct indicated that the fs923X, fs895X, L869X, and D13 recombinant rClC-1 proteins were being expressed and could readily be detected on Coomassie Blue-stained polyacrylamide gels (see Ref. 13), whereas similar rClC-1-specific bands could not be detected for the N596X and Q574X mutations (data not
shown). Evidence that transcription of the N596X and Q574X constructs
had occurred, however, was provided by RT-PCR analysis of mRNA
extracted from appropriately infected cells, which gave the expected
prominent band in each case (data not shown).
When expressed in Sf9 cells, fs923X, fs895X, and D13
mutations did not change such basic properties of rClC-1 as the
pronounced inward rectification of instantaneous currents, saturation
of outward currents at positive potentials, deactivation of inward currents at negative potentials, and the voltage dependence of apparent
open probability. Kinetics of the inward current deactivation, however,
were slower in the mutants (Fig. 2). Of
the two time constants that can typically be extracted from the
deactivating currents (16), the slow time constant,
2,
was almost doubled (significant by two-way analysis of variance,
p < 0.0001) in the fs895X and
D13 mutants with
respect to WT, whereas the fast time constant,
1, was
not significantly altered. For currents in response to a voltage step
to
120 mV these were: WT
1 = 5.8 ± 0.2 ms, n = 21 and
2 = 26.1 ± 1.4 ms,
n = 20; fs895X
1 = 6.9 ± 0.3 ms, n = 13 and
2 = 45 ± 2.7 ms,
n = 13;
D13
1 = 7.1 ± 1.3 ms,
n = 4 and
2 = 46.1 ± 3.5 ms,
n = 4 (results expressed as mean ± S.E.).
Kinetics of the fs923X mutant were closer to WT. Cells infected with
the L869X, N596X, or Q574X recombinant baculoviruses failed to produce
a chloride conductance attributable to rClC-1 (data not shown).
|
Block of the rClC-1 channel by Cd2+ is sensitive to
mutation, e.g. the R304E mutant (17). When we tested our
present mutants, Cd2+ was as effective on D13 as on the
WT channel (IC50 = 1.1 ± 0.1 mM
(n = 3) for
D13 and 1.0 ± 0.1 mM
(n = 3) for WT), whereas fs895X required higher
concentrations of Cd2+ to achieve the same level of block
(IC50 = 2.7 ± 0.2 mM, (n = 5).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is no doubt from our present study and from previous work (4) that truncations of the carboxyl tail of the protein by 125 or more amino acids eliminate the chloride currents attributable to ClC-1 in Sf9 cell and Xenopus oocyte expression systems. Clearly, without detectable protein products, there seems to be a failure of effective translation of the rClC-1 mutants Q574X and N596X in our expression system. A similar explanation, implying some requirement for the integrity of the mRNA code for the proximal part of the carboxyl tail, might apply to the inability to detect appropriate chloride currents in Xenopus oocytes when the carboxyl tail of hClC-1 had been severely truncated by the mutation L598X (4). Function, in this case, was unable to be restored by co-expression with the complementary carboxyl tail peptide (4). Both more severely and less severely truncated versions of hClC-1, however, such as L391X, P452X, and G721X, must have been expressed and correctly inserted into the membrane because their function was restorable with appropriate complementation (4). By contrast, fully functional, although kinetically slower, versions of rClC-1 are expressed and inserted into the membrane of Sf9 cells, despite lacking the D13 domain or the final 100 amino acids of the carboxyl tail.
These results are consistent with previous observations of the
naturally occurring myotonic mutant of human ClC-1, R894X (8), and of
ClC-5 mutants associated with Dent's disease (10, 11). The conclusions
from these earlier studies have, however, implicated D13 as an
essential functional region (4). This view has received support from
the recent identification of a structural motif, conserved across
archaea, bacteria, and eukarya, the so-called CBS (cystathionine
-synthase) domain (18). This motif is present in tandem (denoted
CBS1 and CBS2) in all eukaryotic ClC proteins (19) with CBS1 located
between D12 and D13 and CBS2 overlying D13, extending from
Cys825 to Lys877 in rClC-1 (Fig.
3). A high level of conservation
frequently indicates physiological importance, but our present results
are inconsistent with any obvious function for D13 or the CBS2 domain
in which it lies. In our quite functional
D13 mutant, all of D13
and, simultaneously, the final two-thirds of CBS2 have been
eliminated.
|
Although the absence of any evidence of function in ClC-1 truncated by
125 or more amino acids could be fortuitous, a more likely
interpretation that encompasses both the earlier observations of others
and our new results with the D13 mutant is that there is some
essential region of presently unknown significance contained within the
immediate post-D13 region. An argument that the region of functional
significance might lie prior to D13 but after Ser717 could
only be entertained if our L869X mutant, which is expressed, failed to
be correctly inserted into the membrane. We have no proof of correct
insertion, but note that wherever there has been evidence of expression
of ClC-1, there has been concurrent evidence of correct insertion for
both shorter and longer truncation mutants (Ref. 4 and present
results).
Coincident with the diminished status of D13 and CBS2, then, it is the region immediately beyond these domains (from Lys877 to Arg894) that takes on an unexpected prominence. Of these 18 amino acids, the final 7 (PPLASFR) are identical in ClC-0, ClC-1, and ClC-2, and mutations around Arg894 (Arg888 in hClC-1) are known to modify ClC-1 function modestly, as in our fs895X mutant, or substantially, as in the myotonic goat (corresponding to A885P in hClC-1) (7) and human R894X (8) mutants. The seeming discrepancy between our fs895X mutant and the more severe effects of the human R894X might be explained by dramatic differences in the reactivity of their terminal sequences, RAAA and RNTTST (RNTTSI in rClC-1; Fig. 3), respectively. Lack of homology with the post-D13 area of ClC-5 makes it unlikely that the D13 truncation mutants of ClC-5 lose function in the same way as similar mutants of ClC-1 (Fig. 3).
Finally, our results with Cd2+ block, which occurs only when Cd2+ is applied to the extracellular side of the channel (17), suggest that the immediate post-D13 region is structurally in close proximity to the channel pore or that very long range conformational changes originating in this distant region can influence the extracellular site of Cd2+ binding.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Professor T. J. Jentsch of the Center for Molecular Neurobiology, Hamburg, for advice on recombinant PCR and to Dr. D. St. J. Astill for suggestions relating to the frameshift mutations.
![]() |
FOOTNOTES |
---|
* This work was supported by the Neuromuscular Research Foundation of the Muscular Dystrophy Association of South Australia, the Australian Research Council, and the Research Committee of the University of South Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Ross Stuart Postgraduate Scholarship of the Muscular Dystrophy Association of South Australia.
To whom correspondence should be addressed: Centre for
Advanced Biomedical Studies, School of Pharmacy and Medical Sciences, University of South Australia, North Terrace, Adelaide, SA 5000, Australia. Tel.: 61-8-8302-2398; Fax: 61-8-8302-2389; E-mail: a.bretag{at}unisa.edu.au.
1
The abbreviations used are: hClC-1, human ClC-1;
rClC-1, rat ClC-1; CBS, cystathionine -synthase; PCR, polymerase
chain reaction; RT, reverse transcription; kb, kilobase pair(s); WT,
wild type.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|