Supplementary Material
Supplementary Methods
Molecular biology and oocyte expression
Human ClC-5 was cloned into the pTLN vector and expressed in oocytes obtained by
partial ovarectomy of anesthesized Xenopus laevis females, followed by collagenase
treatment. Mutations were introduced by recombinant PCR and confirmed by
sequencing. cRNA was synthesized by SP6 mMessage mMachine kit (Ambion).
Oocytes were kept in a solution containing (in mM) 90 NaCl, 10 Hepes, 2 KCl, 1
MgCl2, 1 CaCl2, pH 7.5 for three to six days at 18oC.
Voltage-clamp measurements
Two-electrode voltage clamp experiments were performed with a Turbotec 03
amplifier (npi, Tamm, Germany) and a custom acquisition program (GePulse, written
in Visual C++, Microsoft) at room temperature (20-25 oC) as described earlier
(Zdebik et al. 2008). The standard bath solution contained (in mM): 100 NaCl, 4
MgSO4, 10 HEPES, pH 7.3. The voltage protocol consisted of voltage steps of 50 ms
from 100 to –80 mV in decrements of 20 mV. For measurements with the mutants
E211A, E211A-Y617A and E211A-D727A that conduct also in the negative voltage
range, steps were from 100 to –120 mV. Holding potential was the resting potential of
the oocytes.
Non-stationary noise analysis
Data for non-stationary noise analysis were obtained by repeatedly applying a voltage
step to 140 mV. The mean current, I, and the variance, σ2, were calculated from these
traces as described (Zdebik et al. 2008), and the variance was plotted as a function of
the mean current and fitted to the following equation
I2
σ = iI −
N
2
where i is the apparent single channel current and N the number of “channels”.
Extracellular pH measurements
Acidification of the extracellular solution due to proton transport was measured with a
pH-sensitive microelectrode placed close to the vitelline membrane of the oocyte
(Picollo and Pusch 2005). The microelectrode consisted of a silanized glass pipette
tip-filled with a proton ionophore (Cocktail B, Fluka), backfilled with a solution
containing a phosphate-buffered saline, and connected to a custom high-impedance
amplifier. The electrodes were routinely checked and responded consistently with a
slope of 57-61 mV/pH unit. The oocyte was simultaneously voltage-clamped and
acidification was induced by applying a train of voltage-clamp pulses to 100 mV.
Solutions contained 0.5 mM Hepes and were at pH 7.3.
Legend Supplementary Figures
Supplementary Figure 1. Additive effects of ATP and ADP. A, representative
recordings from an inside-out patch on WT ClC-5 perfused with control solution (no
nucleotide added) and with solutions containing either 1 mM ADP, 1 mM ATP or the
combination of 1 mM ADP and 1 mM ATP simultaneously. For clarity, data were
filtered at 3 kHz. B, mean values of the normalized currents obtained in experiments
similar to the one described in A. Normalization was performed with the current in
control solution at 160 mV. Error bars indicate SEM (n>= 3).
Supplementary Figure 2. Effect of NAD on ClC-5. A, representative recordings
from an inside-out patch on WT ClC-5 perfused with control solution and with
solutions containing the indicated NAD concentration.
Supplementary Figure 3. Adenine does not compete with ATP. A, representative
recordings from an inside-out patch on ClC-5 perfused with control solution and with
solutions containing 1 mM ATP and 1 mM ATP in combination with 3 mM adenine
simultaneously. For clarity, data were filtered at 3 kHz. B, mean values of the
normalized currents obtained in experiments similar to the one described in A (n = 6).
Normalization was performed with the current in control solution at 160 mV.
Supplementary Figure 4. Influence of the oxidation state on the ATP effect. A,
representative recordings from an inside-out patch on WT ClC-5 perfused with
control solution and with solutions containing either 1 mM ATP, 1 mM DTT or the
combination of 1 mM DTT and 1 mM ATP simultaneously. For clarity, data were
filtered at 3 kHz. B, mean values of the normalized currents obtained in experiments
similar to the one described in A (n>= 3). Normalization was performed with the
current in control solution at 160 mV.
Supplementary Figure 5. Analysis of mutants Y617A and D727A by voltage-clamp
and extracellular pH recording. A, representative recordings from voltage-clamp
experiments on WT ClC-5, mutant Y617A and D727A. B, representative
measurements of the extracellular acidification produced by the activation of Y617A
and D727A with repetitive voltage pulses to 100 mV. The stimulation period is
indicated by the bar. C, mean values of the currents at 100 mV obtained in
experiments similar to the one described in A for WT ClC-5, Y617A and D727A (n
>= 34).
Supplementary Figure 6. Analysis of the double mutant E211A-Y617A by voltageclamp. A, representative recordings from voltage-clamp experiments on the mutant
E211A and the double mutant E211A-Y617A. B, mean values of the normalized
currents obtained in experiments similar to the one described in A for E211A (filled
rectangles) (n=6) and E211A-Y617A (open circles) (n=7). Normalization was
performed for each data set with the current at 100 mV of the corresponding mutant.
Error bars indicate SEM.
References for Supplementary Material
Picollo A, Pusch M (2005) Chloride/proton antiporter activity of mammalian CLC
proteins ClC-4 and ClC-5. Nature 436: 420-423
Zdebik AA, Zifarelli G, Bergsdorf EY, Soliani P, Scheel O, Jentsch TJ, Pusch M
(2008) Determinants of anion-proton coupling in mammalian endosomal CLC
proteins. J Biol Chem 283: 4219-4227
Supplementary Figure 1
A
1 mM ADP
1 mM ATP
1 mM ATP
1 mM ADP
5 pA
control
10 ms
B
2.0
Inorm
1.5
1.0
0.5
0.0
ADP
1mM
ATP
1 mM
ATP 1 mM
ADP 1 mM
wash
Supplementary Figure 2
5 pA
control
5 ms
1 mM NAD
10 mM NAD
wash
Supplementary Figure 3
A
2 pA
control
1 mM ATP
1 mM ATP
3 mM Adenine
5 ms
B
2.0
Inorm
1.5
1.0
0.5
0.0
ATP 1 mM
ATP 1 mM
Adenine 3 mM
wash
Supplementary Figure 4
A
1 mM ATP
2 pA
control
1 mM DTT
5 ms
B
2.0
Inorm
1.5
1.0
0.5
0.0
DTT
1 mM
DTT 1 mM
ATP 1 mM
ATP
1 mM
1 mM DTT
1 mM ATP
Supplementary Figure 5
A
Y617A
D727A
2 µA
WT
10 ms
B
D727A
5 mV
V
10 mV
V
Y617A
20 s
20 s
C
I (µA)
6
4
2
0
WT
Y617A
D727A
Supplementary Figure 6
A
2 µA
E211A
E211A-Y617A
10 ms
B
E211A
E211A+Y617A
1.0
Inorm
0.5
0.0
-0.5
-100
-50
0
50
Voltage (mV)
100