Transcript BMES2006_FW

2+
Ca
Characterization of Different Modes of
Uptake under Different Physiological
Conditions in the Heart Mitochondria
Shivendra G. Tewari1, Ranjan K. Pradhan1,2, Jason N. Bazil1,2, Amadou K.S. Camara3, David F. Stowe2,3, Daniel A. Beard1,2, and Ranjan K. Dash1,2
1Biotechnology
and Bioengineering Center, 2Department of Physiology, and 3Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI
Abstract / Summary
Kinetic Mechanism of
Cardiac mitochondria can act as a significant Ca2+ sink and shape the cytosolic Ca2+ signals
affecting various cellular processes such as energy metabolism and excitation-contraction
coupling. However, mitochondrial Ca2+ uptake mechanisms under different (patho)physiological conditions are not well understood. Characterization of these mechanisms is crucial
in developing a quantitative understanding of Ca2+ signals in the heart. For this purpose, we
performed Ca2+ uptake experiments in isolated guinea pig heart mitochondria under different
experimental concentrations of extra-matrix Mg2+ and Ca2+. The Na+ free respiration buffer
contained 1 mM EGTA with variable levels of Mg2+ (0 mM–2 mM) and CaCl2 (0.0 mM–0.6 mM)
was added as a pulse after mitochondrial energization with substrate was pyruvic acid.
Experimental data were analyzed using an integrated model of mitochondrial bioenergetics
and cation handling. Our model analyses of the data reveal the existence of two Ca2+
uniporters or Ca2+ uptake pathways, namely a fast CU (fCU) and a slow CU (sCU), which
exhibit contrasting differences in [Ca2+]e and [Mg2+]e sensitivities. fCU is a time-dependent
high affinity Ca2+ uniporter, while sCU is a time-independent low affinity Ca2+ uniporter. Both
uniporters are inhibited by extra-matrix Mg2+. The binding affinity of fCU for Mg2+ is higher as
compared to that of sCU. This work was supported by NIH/R01-HL095122.
Model of Mitochondrial Bioenergetics and Ca2+ Handling
H+
H+
H+
C(red)2+
C(ox)3+
H+
H+ H2PO4PIHt
CIV
COQ
CIII
NADH
QH2
ADP3ANT
Fo F1
CI
COQ
ATP4-
C(ox)3+
NAD+
ADP3+ PI2-
ATP4-
TCa
(A)
Ca e2+
2+
e
Ca2+
Ca
2+
e
Influx via
T2Ca
2+
e
Ca2+
Uniporter with
T2Mg
(B)
2+
e
ko
T
KCx,1
ki
2+
x
Ca T
Ca 2+
x
K e  K exp(  e  m ); K x  K exp(  x  m )
0
e
0
x
ki  ki0 exp( 2  e  m ); ko  ko0 exp( 2  x  m )
Mge2+
KMe,2
Ca
KCx,2
Ca 2+
x
Mge2+
TMg
2+
x
2Ca T
2+
e
Mg
Ca
Z Ca F  m
T
 m 
RT
2+
e
KCe,1
 2 KMe,2
Ca e2+
 KMe,1
TCa e2+
Mg
Ca
2+
e
 KMe,1
2

2
2  ni 1
4
n
[
Ca
]
[
B
]
i
Ca
,
i
 1  
n
ni
2  ni

i K i
1

[
Ca
]
/
K
Ca ,i
Ca ,i

Ca e2+
KCe,2




2


Table 1. Buffering Model Parameters
T2Cae2+
The uniporter T is assumed to have two binding sites for both Ca2+ on either side of the inner mitochondrial
membrane (IMM) and two distinct binding sites for Mg2+ on the cytoplasmic side of the IMM. (A) Two Ca2+ ions
from the cytoplasmic side cooperatively bind to the uniporter in two steps to form the complex T2Ca2+e which
then undergoes conformal changes to form the complex 2Ca2+xT. This complex goes through the reverse
process where it dissociates in two steps to form the unbound uniporter T and two Ca2+ ions in the matrix
side. (B) A linear mixed-type scheme for Mg2+ inhibition of Ca2+ influx via the uniporter. The Mg2+ interacts with
the Ca2+ only in the cytoplasmic side of the IMM. There is no Mg2+ binding to uniporter T from the matrix side
of the IMM. But in the matrix side, Ca2+ binding is symmetrical to that of the cytoplasmic side as shown in this
figure. The transport of Ca2+ via the uniporter is limited by the rate constants ki and ko which are dependent on
m, and modulated by extra-matrix free [Ca2+], matrix free [Ca2+] and extra-matrix free [Mg2+].
The figure is reproduced from Pradhan et al., Biophysical J 101(9):2071-2081, 2011.
ADP3-
For highly buffered conditions, βCa is large. Mathematically:
TMge2+ 2Ca e2+
 KCe,2
2+
e
Ca2+ Buffering Power (βCa) is a manifestation of the Ca2+ sequestration system. For every Ca2+ ion entering (or
leaving) the mitochondria, only a fraction remain free. This can be written as:
Ca  [TCa]x / [Ca ]x
Mge2+
TMge2+Ca e2+
2+
e
Mg
KMe,1
T2Mge2+ 2Ca e2+
2
 KMe,2
 KCe,1
2+
e
Ca e2+
Ca2+ Sequestration System in Mitochondrial Matrix
Inhibition
 2 KCe,2
 2 KCe,1
KCe,2
KCe,1
Ca e2+
T2Mge2+Ca e2+
Mg2+
Type
[BCa,1]
KCa,1
n1
[BCa,2]
KCa,2
n2
[BCa,3]
KCa,3
n3
[BCa,4]
KCa,4
n4
Definition
BP1
Total protein concentration
Ca2+ binding constant
Number of binding sites
BP2
Total protein concentration
Ca2+ binding constant
Number of binding sites
BP3
Total protein concentration
Ca2+ binding constant
Number of binding sites
BP4
Total protein concentration
Ca2+ binding constant
Number of binding sites
Value
Units
30.0
6.00
1
mM
µM
-
35.0
5.9
2
mM
µM
-
40.0
5.8
3
mM
µM
-
45.0
5.7
4
mM
µM
-
NCE
CHE
Ca2+
Na+
BP1
Ca2+
BP2
H+
Ca2+
NHE
Na+
H+
KHE
K+
H+
H+ leak
BP4
BP3
Ca2+ sequestration systems
H+
Ca2+
Ca2+
Ca2+
matrix space
inner mitochondrial membrane
 m 
Matrix and Extra-matrix Free Ca2+ Dynamics with Mg2+ Inhibition
Model Simulations of Fluxes through Two Types of Uniporter: sCU and fCU
(A)
(A)
(B)
(B)
sCU
fCU
Mg2+
RaM
Extra-matrix space
Mg2+
The model is developed from the Beard’s model of mitochondrial respiratory system and oxidative
phosphorylation (1) to account for the dynamics of mitochondrial Na+-Ca2+ cycle and interaction
between extra-matrix Mg2+ and Ca2 (2, 3), and additional pathway (fCU) for mitochondrial Ca2+ uptake.
The Ca2+ sequestration model consists of four types of buffering proteins : BP1, BP2, BP3 and BP4 in
the matrix. The integrated model includes the reactions at complex I, III, IV, and F0F1 ATPase of the
electron transport system; the substrate transporters (ANT and PHT), cation transporters (sCU, fCU,
NCE, NHE, CHE, and KHE), and passive K+ and H+ permeations across the mitochondrial inner
membrane; and the passive substrate transport fluxes of adenines and phosphates across the
mitochondrial outer membrane. Both uniporters sCU and fCU are inhibited by extra-matrix Mg2+. The
flux through the TCA cycle producing NADH is expressed in terms of a phenomenological
dehydrogenase flux. ANT: adenine nucleotide translocase, PHT: phosphate-hydrogen cotransporter,
sCU: slow Ca2+ uniporter, fCU: fast Ca2+ uniporter, NCE: 3Na+/1Ca2+ exchanger, NHE: Na+/H+
exchanger, CHE: Ca2+/H+ exchanger, and KHE: K+/H+ exchanger.
(1): Beard, PLoS Comput Biol 1(4): e36, 2005; (2): Dash and Beard, J Physiol 586(13): 3267-3285, 2008;
(3): Pradhan et al., Biophysical J 101(9):2071-2081, 2011.
(D)
(E)
(A,B,C): Dynamics of slow,
low affinity uniporter fluxes
(sCU) in response to added
CaCl2 (0, 0.25, 0.5, 0.6 mM),
in the absence (A) and
presence of 0.5 mM MgCl2
(B) and 1 mM MgCl2 (C)
added to the buffer medium.
(D,E,F): Dynamics of fast,
high affinity uniporter fluxes
(sCU) in response to added
CaCl2 (0, 0.25, 0.5, 0.6 mM),
in the absence (A) and
presence of 0.5 mM MgCl2
(B) and 1 mM MgCl2 (C)
added to the buffer medium.
Blue: 0 mM CaCl2
Green: 0.25 mM CaCl2
Red : 0.5 mM CaCl2
Pink: 0.6 mM CaCl2
(C)
Simulations of slow (timeindependent) and fast (timedependent) Ca2+ uniporter
fluxes for three levels of
extra-matrix MgCl2 and four
levels of extra-matrix CaCl2.
NCE was inactivated.
(D)
Comparison of model simulations to data on the dynamics of buffer (A) and matrix (B, C, D) free [Ca2+] in the
absence ([Mg2+]e = 0) and presence of 0.5 and 1 mM Mg2+ in Na+ free buffer mediums. Adding CaCl2 (0, 0.25, 0.5,
0.6 mM) caused a dose-dependent increase in [Ca2+]m, consistent with the increase in [Ca2+]e. Two different
phase of uptake (slow and fast) were observed during and after adding CaCl2. The slow phase of Ca2+ uptake
was through the low affinity Ca2+ uniporter and the fast phase was through the high affinity Ca2+ uniporter.
Model accurately describes the data for all Mg2+ levels and Mg2+ reduces [Ca2+]m. CaCl2 was added at t = 120 s.
(C)
(F)
In all the experiments and
simulations, MgCl2 was
added at t = 0 s and CaCl2
was added at t = 120 s. Ca2+
fluxes by both the slow and
fast uniporters were observed to be inhibited by extramatrix Mg2+. In addition, the
fast uniporter is slowly
inactivating depending on
the matrix free [Ca2+].