Supplement “Dynamic buffering of mitochondrial Ca2+ during Ca2+
uptake and Na+-induced Ca2+ release”
Christoph A. Blomeyer1, Jason N. Bazil2, David F. Stowe1,3, Ranjan K. Pradhan2, Ranjan K.
Dash2, Amadou K.S. Camara1
1
Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI
53226, USA
2
Biotechnology and Bioengineering Center and Department of Physiology, Medical College of Wisconsin, 8701
Watertown Plank Road, Milwaukee, WI 53226, USA
3
Research Service, Zablocki Veterans Affairs Medical Center, 5000 W. National Avenue, Milwaukee, WI 53295,
USA
To whom correspondence should be addressed: Amadou K.S. Camara, Department of Anesthesiology, Medical
College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA, Phone: (414) 456-5624
Fax: (414) 456-6507, Email: aksc@mcw.edu
1
Additional data on NADH, membrane potential, matrix pH and matrix volume
Measurements of NADH, membrane potential, matrix pH and matrix volume were conducted as described in detail
in the Methods section. Experimental protocol (Fig. 1) and conditions were identical as used for [Ca 2+]e and [Ca2+]m
measurements. Note that Panels a-c in Figs. S1-S4 depict measurements for a fixed [NaCl] (0, 5 or 20 mM) at
various [CaCl2] (0, 20, 40 µM), whereas Panels d-f show the same data in a re-arranged fashion for a fixed [CaCl2]
(0, 20 or 40 µM) with various [NaCl] (0, 5 or 20 mM).
Fig. S1 NADH. Panels a-f illustrate how NADH (expressed as a percentage) changed over time with addition of
increasing amounts of CaCl2 and NaCl. Note that adding PA induced an abrupt increase in NADH. Furthermore, 40
µM CaCl2 induced a transient oxidation of NADH at t = 120 s. The NADH levels at t = 840 s were not significantly
different. Data were obtained from a mitochondrial protein concentration of 0.5 mg/ml and are presented as mean of
3 individual experiments ± SEM
2
Fig. S2 Membrane potential. Panels a-f show changes in ΔΨm monitored by TMRM fluorescence (expressed as a
ratio) over time with addition of increasing amounts of CaCl2 and NaCl. At the end of each experiment (t = 900 s)
CCCP (4 µM) was added to induce maximal depolarization. Note that 40 µM CaCl2 induced a transient
depolarization of ΔΨm at t = 120 s, which was not observed at lower [CaCl2]. At t = 840 s the levels of ΔΨm were not
significantly different. Data were obtained from a mitochondrial protein concentration of 0.5 mg/ml and are
presented as mean of 3 individual experiments ± SEM
3
Fig. S3 Matrix pH. Panels a-f show changes in pHm over time with addition of increasing amounts of CaCl2 and
NaCl. Note that 40 µM CaCl2 induced a slight increase in pHm at t = 120 s compared to 0 or 20 µM CaCl2. At t =
840 s the levels of pHm were not significantly different. Data were obtained from a mitochondrial protein
concentration of 0.5 mg/ml and are presented as mean of 3 individual experiments ± SEM
4
Fig. S4 Matrix volume. Panels a-f show changes in matrix volume measured by lightscattering over time with
addition of increasing amounts of CaCl2 and NaCl. At the end of each experiment (t = 900 s) valinomycin (VAL, 10
nM), a K+ ionophore, induced maximal matrix swelling. Note that 20 or 40 µM CaCl2 induced a slight swelling
compared to 0 µM CaCl2 (Panels a-c). Furthermore, 5 or 20 mM NaCl at 0 or 20 µM CaCl 2 (Panels d,e) decreased
matrix volume slightly compared to 0 mM NaCl. These differences in matrix volume were not significantly different
at t = 840 s. Data were obtained from a mitochondrial protein concentration of 0.5 mg/ml and are presented as mean
of 3 experiments ± SEM
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NCE stoichiometry
To assess the stoichiometry of the NCE under our conditions, we used equation 4 and the data of [Ca2+]e and [Ca2+]m
for different [CaCl2] and [NaCl] at t = 840 s along with [H+]m = 10-7.38 and [H+]e = 10-7.15. Based on the assumption
that the NCE is an electroneutral exchanger, the Ca2+ gradient across the IMM should be equal to the square of the
H+ gradient (= 0.5892). However, the derived Ca2+ gradients for all experimental groups, shown in Table S1, are
higher than the square of the H+ gradient (~ 0.347) indicating that the NCE stoichiometry is not electroneutral.
[Ca2+]e/[Ca2+]m
0 µM CaCl2
10 µM CaCl2
20 µM CaCl2
30 µM CaCl2
40 µM CaCl2
0 mM NaCl
2.401176
1.712855
2.100135
2.150434
2.408532
1 mM NaCl
1.776672
1.548202
2.006064
2.134651
2.565685
2.5 mM NaCl
1.095368
0.965134
1.649868
1.781208
1.849657
5 mM NaCl
0.614608
0.506378
1.066489
1.128057
1.263521
10 mM NaCl
0.64743
0.473665
0.728307
0.747618
0.7069
20 mM NaCl
0.510285
0.395542
0.505975
0.471393
0.607405
Table S1 NCE stoichiometry. Ca2+ gradients were derived by calculating the ratio of [Ca2+]e to [Ca2+]m and are
separately presented for each combination of added CaCl2 and NaCl
6