ClC-1 and ClC-2 form hetero-dimeric channels with novel
protopore functions
Gabriel Stölting, Martin Fischer and Christoph Fahlke
Supplemental Text
Control of subunit assembly by hetero-concatameric constructs
Subunit concatenation permits expression of covalently linked multimeric proteins and often
results in the expression of a homogenous populations of heteromultimers [1–4]. However,
concatenated constructs sometimes fail to perfectly control the subunit stoichiometry of
expressed channels, thus giving rise to other channel populations. Synthesis of the desired
protein might be impaired as tandem constructs are not completely transcribed or translated
[5]. Moreover, concatenated subunits might associate to other concatamers forming
multimeric aggregates within which only identical subunits form active conductance
pathways as homo-dimers [1]. This restriction requires careful investigation about the ability
of each concatamer to ensure the intended subunit stoichiometry.
Expression of the ClC-1-ClC-2 concatamer resulted in a homogenous population of
covalently linked dimeric proteins (Fig. 1a). CLC channels are dimeric, and it thus appears
possible that homodimeric ClC-1 and ClC-2 channels might be formed in addition to ClC-1ClC-2 heteroconcatamers. However, in cells expressing the concatamer, no visible current
deactivation occurs upon voltage steps from 0 mV to negative values indicating that the
superposition with homodimeric ClC-1 [6] channels is negligible. Moreover, whole-cell
currents in cells expressing the heteroconcatamer activate at negative voltages without
components that resemble homodimeric ClC-2 and show robust currents at positive voltages
[7]. These results demonstrate that the contribution of homodimeric channels is negligible in
cells expressing the hetero-concatamer and that it is possible to record from a homogenous
population of hetero-dimeric channels.
The linker used in our experiments has been carefully tested in experiments with ClC-1
[4, 8, 9] and ClC-2 homodimers [7]. So far, functional effects of the linker have been neither
observed for ClC-1 nor for ClC-2. However, currents recorded from cells transiently
expressing the ClC-2-ClC-1 concatamer with reverse subunit order exhibited slightly different
functional properties (Supplemental Fig. 1A). In these cases, hyperpolarizing voltage steps
from the holding potential of 0 mV resulted in a small deactivating current component
resembling currents conducted by ClC-1 homodimers. SDS-PAGE of whole cell lysates from
HEK293T cells transiently expressing the ClC-2-ClC-1 concatamer showed a similar ~250
kD band without monomer formation as the concatameric constructs in the ClC-1-ClC-2 order
(data not shown). This result indicates the formation of multimeric aggregates that result in
functional homodimers of two identical subunits with a hanging dysfunctional concatenated
isoform.
Currents from the ClC-2-ClC-1 concatamer could be well described by the addition of
ClC-1-ClC-2 concatamer currents with ClC-1 wild type currents at a ratio of 0.57
(concatamer) to 0.43 (ClC-1) (Supplemental Fig. 1B and C). This superposition was generated
by normalizing representative recordings from ClC-1 and ClC-1-ClC-2 hetero-concatamer
expressing cells and adding these components at the above mentioned ratio so that the first
data point and the last data point at -120 mV superimpose.
Additionally, single channel recordings from cells expressing the ClC-2-ClC-1
heteroconcatamer revealed events that were similar to the inverse heteroconcatamer. Unitary
current amplitudes were not statistically different (Supplemental Fig. 1D and E) (ClC-1-ClC2: at -100 mV: -0.18 ± 0.01 pA, n = 8; at +100 mV: 0.19 ± 0.02 pA, n = 6; ClC-2-ClC-1: at 100 mV: -0.14 ± 0.01 pA, n = 3, P = 0.16; at +100 mV: 0.18 ± 0.05 pA, n = 3, P = 0.882). The
low open probability of heterodimeric channels combined with short open events allows for
the additional comparison of open dwell times even for patches that contain more than one
channel as the probability of finding two simultaneously open channels is low. A comparison
(Supplemental Fig. 1F) demonstrated similar open dwell times for the recorded events (ClC1-ClC-2 at -100 mV: 7.7 ± 1.5 ms, n = 8; ClC-2-ClC-1 at -100 mV: 9.0 ± 2.0 ms, n = 3; P =
0.497 obtained by a Mann-Whitney rank sum test). We thus conclude that expression of ClC2-ClC-1 results in the occurrence of wild-type like homodimeric structure in addition to
heterodimeric channels. We do not know why concatamers of one order are more prone for
this effect than the other one. For analysis of functional properties of a homogeneous
composition of heterodimeric channels, we therefore used the more homogenously expressed
ClC-1-ClC-2 concatameric construct.
Supplemental References
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SUPPLEMENTAL FIGURE 1. Expression of ClC-2-ClC-1 concatamers results in
functionally identical channels as expression of ClC-1-ClC-2.
a, Schematic representation of the ClC-2-ClC-1 hetero-dimer, pulse protocol and
representative current traces for ClC-2-ClC-1 hetero-dimeric channels expressed in HEK293
cells. b, Representative current responses to voltage steps between -140 mV and +40 mV
from cells expressing homodimeric ClC-1 (yellow traces) or ClC-1-ClC-2 hetero-dimer (blue
traces). c, Comparison of representative current traces from ClC-2-ClC-1 hetero-dimeric
channels with simulated superposition of currents by ClC-1-ClC-2 heterodimeric and
homodimeric ClC-.1 currents. For this simulation, normalized current traces from cells
expressing ClC-1 (Fig. 1B) and from cells expressing ClC-1-ClC-2 concatamers were scaled
and superimposed so that the peak current amplitude and the current amplitude after 190 ms
are identical to the normalized ClC-2-ClC-1 recording at -120 mV [2]. d, Representative
traces of inside out patches at -100 or +100 mV likely containing more than one ClC-2-ClC-1
hetero-concatameric channel. e, Amplitude histograms from the recordings shown in d. f,
Open dwell time distributions of both variants of the hetero-concatamer. Time constants are
similar in both arrangements (ClC-1-ClC-2: 7.7 ± 1.5 ms, n = 8; ClC-2-ClC-1: 9.0 ± 2.0 ms, n
= 3)
SUPPLEMENTAL FIGURE 2. Variance analysis demonstrates the open channel
rectification of WT ClC-1. a, Non-stationary noise analysis to determine the number of
channels and single channel amplitude at -155 mV. Cells were held at 0 mV and subjected to
+75 mV for 100 ms to open all available channels followed by hyperpolarization at -155 mV
for another 100 ms before clamping the cell to the holding potential for 3 seconds. A
representative mean current of 150 sweeps is shown (top) together with the calculated
variance (middle). Plotting the variance against the isochronal current reveals a parabolic
distribution that is determined by the number of channels and the single channel amplitude. b,
Current recordings from the same cell subjected to voltages from -80 to +100 mV as
indicated. Steady-state current amplitudes and variances were determined for each potential
and used to calculate the single channel amplitude with the number of channels known from
non-stationary noise analysis at -155 mV. c, Plot of unitary current amplitudes from 4 cells
expressing WT ClC-1 revealing a higher conductance at negative (~ 1.5 pS) than at positive
voltages (0.14 pS) delivering the proof of open channel rectification in ClC-1.
SUPPLEMENTAL
FIGURE 3. 9-Anthracenecarboxylic acid blocks ClC-1-ClC-1
homo-concatamers with similar affinity as WT ClC-1. a, Representative current traces to
the protocol shown. Traces were recorded in the presence of 0 µM or 125 µM as indicated. b,
Representative time course of the peak current amplitude upon and after application of 125
µM 9-AC.
A
B
hClC-1
hClC-2
C
+40 mV
+40 mV
0 mV
0 mV
0.0
-0.5
-1.0
0.0
0.1
Time (s)
1 nA
100 ms
0.0
-0.5
-1.0
0.0
0.2
0.4 pA
0.5 s
0.20
0.15
0.10
0.05
Simulation
hClC-2 - hClC-1 concatamer
0.08
+100 mV
-100 mV
0.00
Counts/Total
Normalized Counts
-100 mV
0.2
F
E
+100 mV
0.1
Time (s)
hClC-1
hClC-1 - hClC-2 concatamer
D
-120 mV
0.5
Relative Current
-120 mV
Relative Current
0 mV
-120 mV
0.5
80 mV
0.06
0.04
0.02
0.00
-0.25
0.00
0.25
Amplitude (pA)
100
101
102
Duration (ms)
hClC-1 - hClC-2 concatamer
hClC-2 - hClC-1 concatamer
Supplemental Figure 1
a
b
+75 mV
+100 mV
0 mV
-80 mV
-155 mV
25 ms
2 nA
100 ms
c
500 pA
100
2
0
Variance (pA2)
-200
600
-100
-100
~ 1.5 pS
-200
300
0
-300
0
4
8
Mean Current (nA)
Supplemental Figure 2
~ 0.14 pS
Voltage (mV)
Unitary Amplitude (fA)
2 nA
-120 mV
100
200
200 mV
0 mV
-140 mV
125 µM
0 µM
1 nA
50 ms
b
wash-in
Normalized Current Amplitude
a
1.0
0.5
0.0
0
300
wash-out
Time (s)
600
ClC-1-ClC-1 Homoconcatamer, 125 µM 9-AC
Supplemental Figure 3