From the * Department of Medicine and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and § Howard Hughes Medical Institute and || Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
Voltage-gated Cl channels belonging to the ClC family appear to function as homomultimers, but
the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl
channels in which one subunit was tagged by a mutation
(D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl
channels
exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an
even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric
WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled
from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.
The ClC family of voltage-gated Cl channels has recently been identified through molecular cloning
(Jentsch et al., 1990
; Jentsch, 1994
), and several distinct
mammalian ClC isoforms have been implicated in various cellular functions (Steinmeyer et al., 1991
; Thiemann et al., 1992
; Uchida et al., 1993
; Adachi et al., 1994
; Fisher et al., 1994
; Kawasaki et al., 1994
; Kieferle
et al., 1994
; van Siegtenhorst et al., 1994
; Malinowska et
al., 1995
). Members of this class of proteins share no
significant structural homology to other known ion
channels, and are therefore likely to have unique properties concerning subunit stoichiometry, gating, and
permeation mechanisms.
Several lines of evidence suggest that the functional
ClC channel unit is composed of multiple identical
components. Based upon detailed analyses of single
Torpedo electroplax Cl channels reconstituted into planar lipid bilayers, Miller and colleagues proposed the
"double-barreled shotgun" model to explain the occurrence of two equally spaced and independently gated
subconductance states (Miller, 1982
; Hanke and Miller,
1983
; Miller and White, 1984
). In this model the Torpedo channel consists of two identical ion conduction
pathways or protochannels which are gated simultaneously by a common slow gate, but each protochannel
is gated independently by a faster process. Examination
of single channel recordings of cloned Torpedo Cl
channels (ClC-0) expressed in Xenopus oocytes show
that these distinct gating and conduction properties
are completely reconstituted in a heterologous system
indicating that a single cDNA is sufficient to code for
this channel behavior (Bauer et al., 1991
). These functional attributes of ClC-0 do not provide direct structural information about the subunit composition of the
channel. However, a recent biochemical study of purified Torpedo Cl
channels demonstrated that the native
configuration of the protein has the sedimentation
properties of a homodimer (Middleton et al., 1994
).
Although it is natural to expect that all ClC channels
will have similar multimeric structures, there is evidence in conflict with the biochemical data on ClC-0
that points toward tetrameric assembly of the skeletal
muscle channel, ClC-1. This information has emerged
from the functional characterization of naturally occurring mutations in a dominant form of congenital myotonia (Thomsen's disease). In this work, co-expression
experiments in Xenopus oocytes revealed that two disease-producing mutants (G230E, P480L) exert negative effects on the functional expression of the wild-type human skeletal muscle Cl channel (hClC-1)
(Steinmeyer et al., 1994
). Based upon RNA titration experiments in which wild-type and mutant transcripts
were co-expressed in oocytes, Steinmeyer and colleagues
proposed that functional channels are composed of
four identical subunits.
The subunit stoichiometry of Shaker and related
mammalian potassium channels has been ascertained
in part by the analysis of artificial multimeric channels
in which subunits have been covalently linked together
(Isacoff et al., 1990; Liman et al., 1992
). This novel and
informative approach requires the "tagging" of at least
one subunit with a mutation that alters a specific functional property such as inactivation or toxin block.
Such a strategy can now be applied to the determination of subunit stoichiometry of the human skeletal
muscle Cl
channel (hClC-1) by assembling multimeric
constructs incorporating a mutation, D136G, that
causes a profound disturbance in voltage-dependent gating (Fahlke et al., 1995
). The distinct gating properties of wild-type (WT)1 and mutant hClC-1 provide the
necessary "tags" to allow recognition of heteromulti-meric channels and to quantify the probable number of subunits required to form a functional channel. In
this paper, we report the successful application of this
method for examining the subunit stoichiometry of
hClC-1, and find strong evidence that the channel is a
functional dimer.
Construction of hClC-1 Dimers
The plasmid pSP64T-hClC-1 (Fahlke et al., 1995) was modified
using recombinant PCR mutagenesis (Higuchi, 1989
) so that the terminal amino acid residue (leu-988) in the open reading frame of hClC-1 is followed by a 20 amino acid linker sequence (SPLHPGLYPYDVPDYAISAV), a new stop codon, and the recognition
sequence for EcoRI. The nucleotide sequence of this linker also
contains an EagI site located 9 bp 5
to the new stop codon. To
construct this modified hClC-1, a 484-bp PCR product was amplified using pSP64T-hClC-1 as a template and the following primers: 2553F, 5
-GACCAGCATGGGGAAGCTCA-3
(nucleotides
[nt] 2553-2572); and the linker-primer, 5
-CCG GAA TTC CTA
AAC GGC CGA AAT TGC ATA GTC AGG TAC GTC ATA AGG
ATA TAG TCC AGG ATG TAG GGG TGA AAG GAT CAG TTC
AT-3
(containing nt 2951-2965 at the 3
end). The amplified
product was then purified with Qiagen PCR Spin columns
(QIAGEN Inc., Chatsworth, CA), digested with Bsu36I (cleaves at
nt 2730 of hClC-1) and EcoRI to give a 307-bp fragment which
was subsequently ligated to Bsu36I/EcoRI digested pSP64T-hClC-1.
The final construct (designated hClC-DT) was sequenced completely in the region modified by PCR to verify the inserted sequence and to identify recombinants without polymerase errors. Functional expression of this modified hClC-1 in oocytes was performed as previously described (Fahlke et al., 1995
), and the characteristics of the modified channel were indistinguishable from the
original wild-type hClC-1.
The WT-WT hClC-1 dimer construct was assembled in the
mammalian expression vector pRc/CMV by ligating together the
following restriction fragments: WT pRc/CMV-hClC-1, HindIII/
BspHI (containing 76 bp of vector polylinker and nt 1-1395 of
hClC-1); hClC-1-DT, BspHI/EagI (nt 1396-2965, linker); WT-
hClC-1, NotI/XbaI (nt 1-2999); and pRc/CMV, HindIII/XbaI.
Recombinants were screened by PstI digestion, and correct constructs were identified by the presence of a unique 780-bp fragment as well as the appearance of double intensity ethidium bromide-stained fragments derived from the duplicated cDNA sequence. The D136G -D136G dimer construct was assembled in a
similar manner except that the HindIII/BspHI and NotI/XbaI
fragments were derived from D136G (Fahlke et al., 1995). The
presence of the mutant sequence and the absence of WT sequence in the final D136G -D136G construct was verified by Southern
blot hybridization of PstI digested plasmid DNA using allele-specific oligonucleotide probes. The WT-D136G construct was made
by substituting only the NotI/XbaI fragment with the corresponding segment from D136G. Similarly, the D136G -WT construct
was made by substituting the HindIII/BspHI fragment with the
corresponding segment from D136G. Final constructs were shown
to have both WT and D136G sequences in appropriate regions
using allele-specific hybridizations.
Cell Lines and Transient Transfections
HEK-293 cells (ATCC CRL 1573; American Type Culture Collection, Rockville, MD) stably transfected with pRc/CMV-hClC-1
and pRc/CMV-WT-WT were produced as previously described
(Fahlke et al., 1995). Transient transfection of tsA201 (HEK-293
cells stably transformed with the SV40 large T antigen) was performed as described by Chahine et al. (1994)
using 10-15 µg of
plasmid DNA and 10-100 µg of salmon sperm DNA as carrier
(Chahine et al., 1994
). Transfection efficiencies ranged from 20 to 80% as judged by the proportion of cells expressing Cl
currents. For cotransfection experiments, 10 µg of each WT or WT-WT
and D136G or D136G -D136G plasmids were used without carrier DNA. Typically 48 h after transfection, cells were split into 35-mm
culture dishes and investigated at least 3 h later. Cells in which
current amplitude exceeded 10 nA were excluded from analysis.
Electrophysiology
Standard whole-cell recording (Hamill et al., 1981) was performed using an Axopatch 200A amplifier (Axon Instruments,
Foster City, CA). Pipettes were pulled from borosilicate glass and
had resistances of 0.5-0.9 M
. More than 80% of the series resistance was compensated by an analog procedure. The calculated
voltage error due to series resistance was always <5 mV. No digital leakage and capacitive current subtraction were used. Currents were filtered with an internal 4-pole Bessel filter with 1, 2, or 5 kHz (
3 dB) and digitized with sampling rates which are at
least five times the filter frequency using a Digidata AD/DA converter (Axon Instruments). Cells were clamped to 0 mV for at
least 15 s between test sweeps.
The bath solution contained (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES; the pipette solution contained (in mM) 130 CsCl, 2 MgCl2, 5 EGTA, and 10 HEPES. All solutions were adjusted to a pH of 7.4 with CsOH (pipette solutions) or NaOH (bath solutions).
Data Analysis
Data were analyzed by a combination of pClamp (Axon Instruments) and SigmaPlot (Jandel Scientific, San Rafael, CA) programs. All data are shown as means ± SD.
The time course of current activation was fit with an equation
containing either one exponential or a sum of two exponentials and a time-independent value (d) as follows: I(t) = a1exp(t/
1) [+ a2exp(
t/
2)] + d. Activation was analyzed for potentials < 0 mV only. Instantaneous current amplitudes were measured 100 µs after the voltage step. To construct activation curves as shown in Figs. 6 and 7, the instantaneous current amplitude (normalized to its maximum value at a fixed potential of
105 mV) measured after 750 ms prepulses to different voltages (V) was plotted
vs. the preceding potential as described previously (Fahlke et al., 1995
; Fahlke et al., 1996
). This plot yields the voltage dependence of the relative open probability, Popen at the end of the 750-ms pulses. The activation curves obtained in this manner were fit with a single Boltzmann and a voltage-independent value: I(V ) = Amp · [1 + exp([V
V0.5]/kV)]
1 + constant.
Simulation of the superposed WT and D136G hClC-1 currents
was done by adding scaled current traces obtained from measurements on HEK 293 cells stably expressing WT or D136G hClC-1
channels. Data shown in Fig. 1, A and C, were used for Fig. 3, C
and D. Scaling factors were chosen to obtain identity of the simulated current amplitudes with the measured current amplitudes
from the WT-D136G hClC-1 transfected cell at two different recording times: either immediately after the voltage step or at the
end of the voltage step. For this purpose a set of two linear equations (IWT-D136G[ti] = a · IWT[ti] + b · ID136G[ti], where ti is either
immediately after or at the end of the voltage step) was solved to
obtain the scaling factors a and b. Simulated data represent the
sum of the WT current trace scaled by the factor a and the
D136G current trace scaled with the factor b. For Fig. 6, current
traces from recordings shown in Fig. 7, A and E, were added in
ratios given in legends.
Expression in Xenopus Oocytes
For expression of WT-D136G and D136G -WT in Xenopus oocytes, coding regions from both were subcloned into the plasmid
vector pSP64T and RNA transcribed in vitro using SP6 RNA polymerase as described previously (Fahlke et al., 1995). Transcripts
were quantified by absorbance measurement at 260 nm and
checked for size and purity by denaturing agarose gel electrophoresis. Co-expression experiments were performed by microinjecting a mixture containing equal quantities (10-20 ng) of
WT-D136G and D136G -WT RNA.
Expression was examined by a two-electrode voltage clamp using
a Warner Instrument Corp. (Hamden, CT) oocyte clamp 7C-725B amplifier. As previously described, WT and D136G hClC-1 channels share a high affinity for 9-anthracene carboxylic acid (9-AC) (Fahlke et al., 1995). To correct for leakage and endogenous currents conducted by channels other than hClC-1, oocytes were
perfused with ND 96 + 0.2 mM 9-AC after each recording. The
blocking process was monitored by repetitive pulses from a holding potential of
30 to
125 mV (0.1 Hz). After reaching steady-state levels, the same pulse protocols were performed, and the
current amplitudes recorded under these conditions were subtracted from the original recording. Only subtracted recordings
were used for analysis. For the calculation of Iss/Ipeak, the peak
current (Ipeak) was measured immediately after settling of the capacitive transient, and the steady-state current (Iss) was measured
at the end of the test pulse (Fig. 8).
Western Blot Analysis
Dishes (100 mm) of tsA201 cells transiently transfected with either WT-D136G or D136G -D136G cDNAs were washed with ice-cold PBS (10 mM Na phosphate, 0.9% NaCl, pH 7.4) and
scraped into 15-ml polypropylene tubes. Cell suspensions were
centrifuged at 2,000 g for 5 min at 4°C, and the resultant cell
were pellets resuspended in 5-10 ml of ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, pH 7.5)
containing freshly added protease inhibitors (5 mM PMSF, 5 mM
N-ethylmaleimide, 1 mM benzamidine), and mixed on a rocking
platform at 4°C for 20 min. The lysates were centrifuged at 5,000 g
for 15 min at 4°C, the pellets discarded, and the supernatants
were used for SDS-PAGE and immunoblotting without further
purification. Plasma membranes from confluent 100-mm dishes
of HEK-293 cells stably expressing either WT-hClC-1 or the WT-
WT dimer were prepared as described by Deal et al. (Deal et al.,
1994). 3-10 µg of protein (protein concentration determined by
a modified Bradford assay, Bio-Rad Corp., Richmond, CA) was
solubilized momentarily at 25°C in SDS sample buffer containing 30 mM DTT before electrophoresis.
Protein samples were fractionated by SDS-PAGE electrophoresis on precast 4-15% polyacrylamide gradient gels (Bio-Rad
Corp.) and electro-transferred to Immobilon PVDF membranes
(Millipore Corp., Bedford MA) at 50 V for 18 h at 4°C. After
transfer, membranes were placed in a blocking solution consisting of 5% nonfat dry milk in TBS-T (50 mM Tris base, 150 mM
NaCl, 0.05% Tween-20, pH 7.5) overnight at 4°C, washed twice
with TBS -T, and probed for 2 h at 25°C with a 1:100 dilution of
an affinity-purified rabbit polyclonal antibody directed against
the carboxyl-terminus of ClC-1 (Gurnett et al., 1995). The membrane was washed twice with TBS-T and incubated for 1 h at 25°C
with a 1:10,000 dilution of goat anti-rabbit IgG conjugated to
horseradish peroxidase (Sigma Chemical Co., St. Louis, MO). After several washes in TBS-T, immunoreactive proteins were detected by enhanced chemiluminescence (ECL; Amersham Corp.,
Arlington Heights, IL).
Sucrose Density Gradient Centrifugation
Plasma membrane protein (250-400 µg) from HEK 293 cells stably transfected with hClC-1 monomer or WT-WT dimer were solubilized in SB buffer (1% Triton X-100, 50 mM Tris, 12.5 mM
MgCl2, 1.5 mM EGTA, 150 mM NaCl, 1.0 mM PMSF, 3.0 mM
benzamidine, 1.0 mM N-ethylmaleimide, pH 7.5) for 1 h at 4 Arlington Heights, ILC. The samples were centrifuged in a Beckman 70.1 ti rotor (Beckman Instruments, Inc., Fullerton, CA) at
100,000 g for 1 h at 4°C. The hClC-1 supernatants and standard
proteins were loaded on separate, continuous 7.5-20% sucrose
gradients prepared with SB buffer containing 0.1% Triton X-100,
then centrifuged in a Beckman SW 40 ti rotor at 100,000 g for 16 h
at 4°C. Individual gradients were fractionated bottom-to-top by
dropwise collection into 32 tubes (8 drops, 370 µl). Aliquots
(24 µl) were fractionated on 4-15% gradients SDS-PAGE gels
and were analyzed either by silver staining (protein standards) or
Western blotting (WT, WT-WT) as described above.
Expression of Tandem hClC-1 Constructs
Initially, to test the feasibility of expressing tandem Cl
channels, we constructed a WT hClC-1 homodimer
(WT-WT) by covalently coupling two complete hClC-1
coding sequences together in a single reading frame
using a short (20 amino acid) hydrophilic linker. This
linker was designed to have minimal predicted secondary structure similar to a linker peptide sequence used
to create heteromultimeric potassium channels (Liman
et al., 1992
). Similarly, we constructed a homodimeric
construct containing two mutant hClC-1 channels having the D136G substitution in the first transmembrane
spanning segment. Both WT-WT and D136G -D136G
constructs were assembled in the mammalian expression plasmid, pRc/CMV, and used to transfect HEK-293 or tsA201 cells. Expression of both homodimers
leads to very high expression levels with many cells exhibiting peak current amplitudes greater than 10 nA.
For data analysis, only cells with current amplitudes
within the range 1-10 nA were used.
Fig. 1 shows the results of whole-cell current recordings made in cells transfected with either monomeric
(WT, D136G) or homodimeric (WT-WT, D136G -
D136G) cDNA constructs. Cells expressing either WT
or WT-WT exhibit rapid deactivation elicited with hyperpolarizing voltage steps from a holding potential of
0 mV that is characteristic of this channel (Steinmeyer
et al., 1991; Pusch et al., 1994
; Fahlke et al., 1996
). Expression of D136G and D136G -D136G constructs resulted in currents which exhibit slow activation upon
hyperpolarization as previously described (Fahlke et
al., 1995
). Current-voltage relationships and steady-state activation curves were identical between corresponding monomeric and dimeric channels (data not shown). These results indicate that the functional phenotypes of both WT and D136G are preserved in the
homodimeric constructs, and that the artificial peptide
linker has no effect on channel function.
Biochemical Characterization of hClC-1 Dimers
To verify that our tandem constructs did indeed encode dimeric proteins, we performed Western blot
analyses on cells transfected with either WT, or one of
the dimeric constructs (Fig. 2 A). Cells transfected with
the monomeric WT channel express a single ~120-130 kD protein detectable by using an anti-ClC-1 antibody
(Gurnett et al., 1995). Cells transfected with WT-WT,
D136G -D136G, and WT-D136G (see below) all express a single protein having a molecular mass of ~240
kD indicating that our cDNA constructs encode
dimeric proteins.
We also characterized the sedimentation properties of monomeric and dimeric hClC-1 by centrifugation through nondenaturing sucrose density gradients to estimate the molecular mass of the native channel complex. Fig. 2 B shows results from a representative experiment in which identical sucrose gradients were loaded with triton X-100 solubilized membranes from HEK-293 cells stably expressing either hClC-1 monomer or the WT-WT dimer. Identical 7.5-20% sucrose density gradients loaded with various purified protein molecular weight standards or HEK cell membranes were centrifuged simultaneously. Individual fractions from each gradient were electrophoresed on SDS-PAGE gels and subjected to either silver staining (molecular weight standards) or Western blotting (WT, WT-WT). The fractions containing the peak silver stained protein standards are plotted on the horizontal axis in Fig. 2 B to provide molecular mass references in the sucrose gradient. The fractions containing either WT hClC-1 monomers or WT-WT dimers were identified by immunodetection using polyclonal anti-ClC-1 antibody and these results are vertically aligned with the fraction number displayed on the horizontal axis. Both WT and WT-WT sediment to a level corresponding to the molecular weight range between aldolase (158 kD) and catalase (240 kD). Peak quantities of immunoreactive protein were observed in fractions 19-23 for the WT monomer, and fractions 19-21 for the WT-WT dimer. The slight difference in sedimentation properties of WT and WT-WT is small in comparison to the separation of the 158 and 240 kD reference proteins, and probably represents minor differences between individual gradients. No immunoreactive protein was detected in either the WT or WT-WT gradients above fraction 25 or below fraction 15. Similar results were obtained in two independent density gradient experiments (data not shown). These results indicate that the native configuration of both WT and WT-WT exhibit similar sedimentation properties in their nondenatured states consistent with formation of homomultimers of hClC-1. Furthermore, the approximate molecular weight of the native complex is within the range expected for a dimeric protein.
Expression of Heterodimeric WT-D136G Channels
We combined WT and D136G together in a single reading frame as a tool to explore the functional subunit
stoichiometry of hClC-1. Cells transiently transfected
with the WT-D136G construct expressed large Cl currents and synthesize a protein of molecular mass appropriate for an hClC-1 dimer (Fig. 2 A). Fig. 3 A shows
representative whole-cell recordings made from cells
transfected with WT-D136G. In contrast to D136G but
similar to WT channels, WT-D136G cells express currents
exhibiting rapid deactivation upon hyperpolarization. However, WT-D136G currents deactivate to an extent
much less than WT channels. In WT-D136G -expressing
cells, current measured 300 ms after onset of a
165 mV
voltage-step ("steady-state" current) is approximately
fivefold larger than it is for WT; the fractional steady-state currents were 0.47 and 0.1 for WT-D136G and
WT, respectively. At
165 mV, WT-D136G currents
also exhibit a very small (<5% of peak current) slowly
activating component which is never seen in WT channels. The voltage dependence of the instantaneous as well as that of the current amplitude at the end of the
test pulse displays inward rectification (data not
shown). Thus, expression of WT-D136G gives rise to
Cl
currents with gating properties distinct from either
WT or D136G. We also considered that subunit order
could be a factor in the genesis of the novel gating phenotype observed in WT-D136G expressing cells as was
found in studies of tandem voltage-gated K+ channels
(McCormack et al., 1992
), and therefore constructed a
heterotandem construct with the reversed order of subunits (designated as D136G -WT). In cells transiently
transfected with D136G -WT, we observed an identical
gating phenotype as seen in WT-D136G expressing
cells (Fig. 3 B).
To evaluate whether the current recordings made in
WT-D136G expressing cells could result from a simple
superimposition of the individual current components
of WT and D136G, we compared these data with simulations of currents that would result from the addition
of the two individual channels. The results of this simulation done at two voltages (165 and
115 mV) were
then compared to current recordings of the monomeric and heterodimeric channels. The simulated currents exhibit rapid deactivation followed by a large
slowly activating component. In a qualitative manner, actual WT-D136G current recordings are obviously distinct from the simple sum of the two separate channels
(Fig. 3, C and D). These data indicate that channels encoded by WT-D136G and D136G -WT are gated by a
mechanism resulting from an interaction between the
two covalently coupled subunits and are consistent with
the formation of heteromultimeric channels. Furthermore, this subunit-subunit interaction is independent
of the subunit order.
A quantitative analysis of the gating properties of Cl
currents in WT-D136G transfected cells reveal that
there is a homogenous population of channels present.
Evidence for channel homogeneity comes from studies
of the time course of activation. The time course of current activation elicited by depolarizing test potentials following a prepotential of
100 mV is shown for WT-
D136G (Fig. 4 A), WT channels (Fig. 4 B), and D136G
(Fig. 4 C). The time course for WT-D136G activation is
well fit by a single exponential function, whereas WT
channel activation is biexponential and consists of fast
and slow components. The activation time constants
for WT-D136G and WT are not voltage-dependent in
the negative potential range, and the mean value of the
activation time constant for WT-D136G (
= 7.7 ± 1.4 ms, n = 4) is not statistically different from the fast activation time constant for WT channels (
fast = 7.4 ± 1.1 ms, n = 4). The absence of a second exponential component in WT-D136G activation indicates that the contribution of homomultimeric WT channels (and by inference, homomultimeric D136G channels) is negligible.
Homogeneity of the expressed current phenotype suggests that functional channel complexes are formed by
an even number of subunits. If the channel complex
were formed by an odd number of subunits, we would
expect a mixed current phenotype because of unequal
incorporations of mutant and WT subunits. These data
also imply that a single mechanism simultaneously gates
the ion pore or pores of hClC-1, although it is not possible to know the exact pore stoichiometry from our results. Based upon our results from the heterotandem expression experiments, we conclude that a cooperative
interaction between an even number of at least two subunits is required to form functional hClC-1 channels.
Co-Expression of WT-WT and D136G-D136G Homodimers
We considered the various subunit configurations
which might exist for channel complexes comprised of
either two or four subunits and this is illustrated in Fig.
5. Because we cannot functionally distinguish between
dimeric channels consisting of one or two identical
pores (with a common gate), we have chosen to draw
the ion pore as a shared structure in the two subunit
configurations. For tetrameric channel assemblies, we
have considered both one and two pore architectures.
To help distinguish among these possible subunit configurations, we performed co-expression studies in which both WT-WT and D136G -D136G homodimers
were introduced into the same cell. This was accomplished by cotransfecting tsA201 cells with equal quantities of each cDNA construct and then examining the
cells for transient channel expression. We predicted
that if the functional channel unit is composed of only
two subunits, then co-expression of the two homodimers
should result in a simple summation of the two current
phenotypes of WT and D136G. If the channel is tetrameric, then WT homotetramers, D136G homotetramers, and WT-D136G heterotetramers should co-exist.
The distinct gating properties of WT, D136G, and WT-
D136G provide a tool to identify the presence of these
three different channel configurations.
Fig. 6 shows representative current recordings made from three different cells cotransfected with WT-WT and D136G -D136G (Fig. 6, A, C, and E). In all cells, the currents exhibit rapid deactivation followed by a large slowly activating component, but there is heterogeneity among the cells with respect to the balance of the two current components. We explain the multiple current phenotypes by cell-to-cell variability in the relative expression levels of WT-WT and D136G-D136G. These three different current patterns strongly resemble the simple summation of WT and D136G phenotypes in varying proportions. Simulations reveal similar patterns of channel gating behavior with 5:1, 2:1, and 1:2 ratios of WT:D136G currents (Fig. 6, B, D, and F ). These data provide qualitative evidence that WT-WT and D136G - D136G express independently, and that there is no formation of heteromultimeric channels in these experiments.
We exploited the distinct gating properties of WT,
WT-D136G, and D136G as a tool to evaluate the subunit composition of the expressed channels using a
more quantitative analysis. This was accomplished by
subtracting the "pure" D136G component from the currents observed in co-transfection experiments, and
determining if the residual current components resemble the pure WT phenotype or a mixture of WT and
heteromultimeric channels. To accomplish this, we examined peak instantaneous and late currents resulting from a test pulse of 105 mV that is preceded by various prepotentials (Fig. 7). This is a similar pulse protocol used in Fig. 1 except our analyses were restricted to
the "tail" portion of the records. Fig. 7 illustrates the results obtained from cells expressing WT, WT-D136G,
or D136G alone to determine the essential characteristics of each channel with this pulse protocol (Fig. 7, A,
C, and E). For both WT and WT-D136G, the current
amplitudes measured at the end of the
105 mV test
potential (Iss) were the same for all prepotentials (i.e.,
are voltage independent, Fig. 7, B and D), whereas the
D136G currents decrease with more depolarized prepotentials (Fig. 7 F ). Therefore, a decrease of Iss at the
end of the
105 mV test potential can be used as a
marker of pure D136G current. Furthermore, WT can
be distinguished from WT-D136G by the ratio Iss/Ipeak
determined at the most negative prepotential (WT: Iss/ Ipeak = 0.11 ± 0.03, n = 5; WT-D136G: Iss/Ipeak = 0.48 ± 0.06, n = 5). Moreover, the voltage dependence of the
normalized Ipeak differs greatly between WT and WT-
D136G; the voltage dependence of Ipeak can be well fit
with a single Boltzmann function for WT alone, but not
for WT-D136G.
In Fig. 8, A, B, and C, we show the analysis of a representative cell cotransfected with both WT-WT and
D136G -D136G. These data show a clear decrease in Iss
(open squares) between 165 and
85 mV. The slope of
a straight line fit to the first four data points in Fig. 8 B
was divided by the slope of a similar line fit to the data
in Fig. 7 F. The ratio of these two slopes was used as a
scaling factor to estimate the proportion of steady-state current in Fig. 8 B due to pure D136G channels. This
value was obtained by multiplying the normalized current values in Fig. 7 F by the derived scaling factor and
then subtracting these values at each prepotential from
the data shown in Fig. 8 B. This subtraction gives the
normalized current voltage relationship for the residual current component. Inspection of this residual
component reveals its close similarity to the WT current-voltage relationship shown in Fig. 8 C, and the dependence of the instantaneous current amplitude on
the prepulse potential can be fit with a single Boltzmann function and a constant term. The parameters of
this fit in the WT-WT:D136G -D136G cotransfected
cell are indistinguishable from values obtained from
WT expressing cells (V0.5 =
53.9 ± 7.6 vs.
51.1 ± 5.9 mV for WT, n = 7, P > 0.1; kV =
21.9 ± 4.1 vs.
18.9 ± 0.9 mV for WT, n = 7, P > 0.1). Furthermore, the Iss/Ipeak
ratio determined at the most negative prepotential for
the residual current component in WT-WT:D136G -
D136G cotransfected cells was 0.13 ± 0.09 (n = 9) and
is not significantly different from WT channels (0.11 ± 0.03, n = 5). It is therefore unlikely that this residual
current has a component due to the formation of heteromultimeric channels. This analysis should be sufficiently sensitive to detect heteromultimeric channel
phenotypes in the context of a tetrameric channel assembly. If the channel were a tetramer, then WT, heteromultimer, and D136G phenotypes would exist in proportions consistent with a binomial distribution. Even
in the case of threefold lower expression levels of the
D136G homodimer, a current component resulting
from formation of heterotetramers should represent
37.5% of the total current (calculation based on a standard binomial distribution in which current phenotypes would exist in the ratio of a2:2ab:b2, where a = WT-WT density, b = D136G-D136G density, a2= probability of forming WT homotetramers, b2 = probability
of forming D136G homotetramers, 2ab = probability of
heterotetramer formation).
This quantitative analysis was able to detect the presence of heteromultimeric channels in an experiment where WT and D136G monomer constructs were cotransfected into tsA201 cells. Fig. 8, D, E, and F, show analysis of a representative WT:D136G co-expressing cell. In this cell, subtraction of pure D136G steady-state current leaves a residual component with an Iss/Ipeak ratio of 0.39. This Iss/Ipeak value is significantly larger than observed for WT alone or what was observed in the homodimer co-expression experiment and is intermediate between values observed for WT and WT-D136G. Furthermore, the voltage dependence of the subtracted Ipeak cannot be fit with a single Boltzmann function consistent with more than one current component. Similar evidence for heteromultimeric channel formation was observed in all cells examined (n = 7). This experiment demonstrates the ability of this method to detect heteromultimeric current components and also helps exclude the possibility that WT and D136G subunits do not co-assemble unless covalently linked.
Co-Expression of WT-D136G and D136G -WT Heterodimers
The absence of a heteromultimeric current component
in the co-transfection experiments with WT-WT and
D136G -D136G excludes a tetrameric channel assembly
with two pores (Fig. 5 B). In considering the various
channel architectures shown in Fig. 5, we recognized the remote possibility that subunit arrangement in a
single pore tetramer could be a factor in determining
the gating phenotype. For example, assembly of one
WT-WT dimer with one D136G -D136G dimer into a
single pore tetramer having the identical subunits in
adjacent positions gives rise to currents indistinguishable from either WT alone, D136G alone, or the linear
sum of WT and D136G. However, we can evaluate this
possibility by co-expressing WT-D136G with D136G -
WT. If the channel is a single pore tetramer, then one
would expect formation of two complexes in which the identical subunits are diagonally arranged (similar to
the situation with WT-D136G alone) and one complex
with the identical subunits in adjacent positions (Fig. 5
C). If the latter complex gives rise to WT, D136G, or
summed current phenotypes, then we should observe
one of these possibilities in addition to heteromulti-meric channels. To test his idea, we expressed WT-
D136G and D136G -WT simultaneously in oocytes and
measured current with the two-electrode voltage
clamp. Oocytes were used in this experiment to better control the stoichiometry of channel expression. Expression of both WT-D136G and D136G -WT alone or
in combination give rise to identical current phenotypes (Fig. 9, A, B, and C), with indistinguishable peak
current amplitudes (instantaneous current measured
at 145 mV [mean ± SEM, n = 7]: WT-D136G, 5.4 ± 1.0 µA; D136G -WT, 6.7 ± 1.2 µA; WT-D136G + D136G -WT, 6.6 ± 1.7 µA).
To quantitatively test for possible contributions of
pure WT or pure D136G components in oocytes co-
injected with WT-D136G and D136G -WT RNA, we examined the voltage dependence of Iss/Ipeak in oocytes
expressing the three different channel populations (WT-D136G, WT-D136G + D136G -WT, and D136G -
WT) (Fig. 9 D). In these experiments, Iss/Ipeak was measured during a series of test potentials from a holding
potential of 30 mV. Expression of both heterotandems alone or in combination exhibit identical voltage dependencies of Iss/Ipeak that are distinct from that observed for pure WT and D136G currents. This observation rules out any contribution from diagonal vs. adjacent subunit arrangements making it highly unlikely
that a tetrameric assembly of one WT-D136G and one
D136G -WT give rises to current phenotypes as observed in the cotransfection studies with WT-WT and
D136-D136G. Therefore our co-expression studies provide strong evidence that hClC-1 forms functional dimers.
Discerning the oligomeric structure of voltage-gated
ion channels continues to be an important but challenging area of investigation. Because of the difficulties
applying structural approaches such as x-ray diffraction
or electron microscopy to the study of ion channels, a
variety of functional approaches have been developed. MacKinnon (1991) described a method in which co-
expression of wild-type and charybdotoxin-insensitive
mutant Shaker potassium channels in Xenopus oocytes
helped to deduce the number of subunits per channel
by means of a binomial analysis of the blocking action of the toxin. To avoid uncertainties regarding the expression of heterogenous RNA mixtures in oocytes,
other investigators have chosen to construct cDNAs encoding artificial heteromultimeric potassium channels
with fixed subunit stoichiometries to investigate oligomeric structure (Isacoff et al., 1990
; Liman et al., 1992
). It is now possible to apply a similar approach toward
defining the subunit composition of mammalian voltage-gated ClC-type chloride channels. This is due to
the availability of a mutant hClC-1 (D136G) having a
well characterized functional phenotype that differs substantially from the WT channel (Fahlke et al., 1995
).
Our data demonstrates that the heterodimeric WT- D136G construct encodes a homogenous population of channels with novel gating properties that cannot be explained by simple addition of the two separate WT and D136G phenotypes (Fig. 3). The functional homogeneity of the WT-D136G channel population is supported by the demonstration of monoexponential time course of current activation (Fig. 4, A and B). These findings are not consistent with the expression of a mixture of homomultimeric and heteromultimeric channels, and therefore rule out the possibility that homodimeric channels are formed in these experiments by misassembly of WT-D136G (channels formed by subunits contributed by more than one WT-D136G molecule). However, these data alone do not rule out that the channel complex is a tetramer.
Our cotransfection experiments using WT-WT and
D136G-D136G help to rule out the possibility that the
number of subunits per channel is greater than two.
This is best appreciated by considering simple possible
configurations of the two homodimeric channels as
shown in Fig. 5 B. In the case of heterotetramer formation, we would expect three Cl channel phenotypes
(WT, D136G, WT-D136G) to co-exist. What we observed fits best with a simple superimposition of the two
WT and D136G current phenotypes (Fig. 7). We cannot completely exclude the possibility that WT-WT and
D136G-D136G only interact to form homotetrameric
pores or that heterotetramer formation is unstable in
this experiment. However, preferential assembly of homomultimeric channels seems very unlikely in view of
the highly efficient expression of both heterotandem
constructs, and the lack of evidence for homomulti-meric channels in WT-D136G transfected cells. Similarly, trimeric channels resulting from the assembly of
one dimer molecule with a single subunit of a separate
dimer molecule seems unlikely because of the homogeneity of channel expression with WT-D136G alone,
and the absence of heteromultimeric channels in the
homodimer co-transfection experiment.
Finally, we have excluded that a tetrameric assembly could be responsible for the expression of summed WT and D136G phenotypes uniquely in the homodimer co-expression experiments due to the adjacent arrangement of the subunits. To do this we examined the heterotandem constructs WT-D136G and D136G -WT together in Xenopus oocytes. This experiment was performed to rule out that adjacent (vs. diagonal) arrangement of hClC-1 subunits in a tetrameric complex might lead to a summed phenotype. As illustrated in Fig. 5 C, co-assembly of the two heterotandem channels will produce diagonal and adjacent complexes in a 2:1 ratio. If the adjacent configuration gives rise to the summed phenotype while the diagonal configurations result in channels that exhibit a mixed gating phenotype resembling WT-D136G alone, we should have observed a more complex gating behavior in cells co-expressing both heterotandems. The absence of such a complex gating phenotype and the identity of these results with those obtained with either heterotandem channel alone rules out cross-talk within the context of a tetrameric channel complex and indicates that only dimeric channels are functional.
In support of a dimeric structure for hClC-1, we have also presented biochemical evidence that the recombinant channel forms native complexes consistent with a two subunit structure. Sucrose density gradient centri-fugation of triton X-100 solubilized membranes from hClC-1 expressing HEK-293 cells indicates that the molecular mass of the native channel (~158-240 kD) is close to twice the predicted mass of a single subunit (~120-130 kD). Furthermore, the sedimentation properties of hClC-1 are the same for proteins encoded by both monomeric and tandem cDNA constructs. Our results indicate a dimeric structure for hClC-1 when it is expressed heterologously. The size of the channel complex in native skeletal muscle should be similar unless additional non-identical subunits or cytoskeletal elements unique to muscle are incorporated.
Our conclusion that hClC-1 is a dimer conflicts with
the previously published study by Steinmeyer et al. that
infers a tetrameric structure of this channel from RNA
titration experiments using two nonfunctional hClC-1
mutants (Steinmeyer et al., 1994). There are several issues that can be raised about this previous study that
could explain this discrepancy. First, these experiments were performed in Xenopus oocytes and current recordings are subject to contamination with an endogenous
calcium-activated Cl
channel. Second, the authors may
have underestimated the extent of competition for expression by comparing mixtures of two different hClC-1
alleles with mixtures of hClC-1 with the cystic fibrosis transmembrane conductance regulator (CFTR). Because
competition for expression is expected to depend upon
the number of molecules competing for ribosomal engagement, use of CFTR (twice the molecular weight of
hClC-1) contributes ~50% less on an equal weight basis than would another hClC-1 allele (higher molar quantity). Finally, these experiments were performed with
non-functional mutants and therefore these investigators are limited in their ability to evaluate the true proportion of expressed WT vs mutant channel proteins.
This limitation raises some uncertainty as to the validity of their binomial analysis for determining subunit stoichiometry of hClC-1.
Middleton et al. (1994) discussed two fundamentally
different quaternary architectures for the formation of
two identical, but independently gated pores in the
dimeric ClC-0 protein: either each ion pore is formed
completely by one subunit (one pore/one subunit), or
each subunit contributes to the formation of both protochannels (shared pore concept). The observation
that WT-D136G forms a homogenous population of
Cl
channels with novel gating properties raises interesting possibilities for the function of hClC-1. If each
hClC-1 subunit encodes a complete ion pore, then
there must be a single mechanism that gates both pores
in the dimeric channel in an identical fashion. This is analogous to the slow gate of ClC-0, but in hClC-1 this
gating mechanism has fast kinetics. Our results are not
consistent with a separate mechanism that gates each
protochannel separately. In other words, independent
gating of two separate ion pores seems improbable
based upon the results we obtained with WT-D136G.
Additional studies, possibly exploiting the heterotandem strategy with a pore altering mutation, will be
needed in the future to determine the true stoichiometry of the hClC-1 pore.
Original version received 18 June 1996 and accepted version received 10 September 1996.
Address correspondence to Alfred L. George, Jr., S-3223 MCN, Vanderbilt University Medical Center, 21st Avenue South at Garland, Nashville, TN 37232-2372. Fax: 615-343-7156; E-mail: ageorge{at}mbio.mc.vanderbilt.edu
1 Abbreviations used in this paper: nt, nucleotide; WT, wild-type.This work was supported by grants from the Muscular Dystrophy Association and the Lucille P. Markey Charitable Trust. C. Fahlke is supported by the Deutsche Forschungsgemeinschaf (DFG, Fa301/1-1), C.A. Gurnett is supported by a predoctoral fellowship from the American Heart Association (Iowa Affiliate), K.P. Campbell is an investigator of the Howard Hughes Medical Institute, and A.L. George, Jr. is a Lucille P. Markey Scholar.