Johan Haumann

Mitochondrial Free [Ca2+] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+](m)) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+](m) is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+](m), membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). Energized mitochondria showed a dose-dependent increase in [Ca2+](m) after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] ([Ca2+](e)); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased [Ca2+](m) up to twofold. The ADP effect on increasing [Ca2+](m) could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or P(i). Oligomycin largely reduced the increase in [Ca2+](m) by ADP compared to control, and [Ca2+](m) did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca2+](m) increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+](e). These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+](m) largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.

Buffer Magnesium Limits Mitochondrial Calcium Uptake but not Matrix Calcium Buffering in Response to ADP

Mg2+ is known to limit Ca2+ uptake by mitochondria through the Ca2+ uniporter. Changes in matrix Ca2+ concentration are an important signaling pathway in mitochondrial function as well as in apoptosis. In a previous study we showed an increase in matrix free Ca2+ in response to added ADP in MgCl2 free buffer. Because of the presumed role of Mg2+ in mitochondrial regulation of Ca2+ we explored the effects of buffer Mg2+ on matrix Ca2+ uptake and buffering in isolated mitochondria. Guinea pig heart mitochondria were isolated by differential centrifugation, loaded with the fluorescent dye Indo 1 AM and then suspended in respiration media, containing 1 mM of EGTA, with or without added 1 mM MgCl2. To the mitochondrial suspension was added 0.5 mM pyruvic acid, either 0.25, 0.5 or 0.75 mM CaCl2, and 250 μM ADP. Adding 0.25, 0.5 and 0.75 mM Ca2+ caused a dose-dependent increase in matrix Ca2+ of 14, 35 and 45%, respectively, in the group without Mg2+ in the buffer, and 6, 18 and 42%, respectively, in the group with Mg2+ in the buffer. The differences in uptake between Mg2+ and no Mg2+ groups were significant in the 0.25 and 0.5 mM groups, but not in the 0.75 mM group. The additional increase in matrix free Ca2+ in response to ADP without Mg2+ was 9, 11 and 9% for the 0.25, 0.5 and 0.75 mM Ca2+ groups, respectively. These additional increases in matrix free Ca2+ with ADP were not significantly altered by Mg2+. We conclude that external Mg2+ alters the uptake of Ca2+ into the mitochondrial matrix, but does not alter the increase in matrix ionized Ca2+ after addition of ADP.

Modeling Regulation of Mitochondrial Free Ca2+ by ATP/ADP-Dependent Ca2+ Buffering

Introduction: Mitochondrial free [Ca2+] ([Ca2+]m) is regulated by cation fluxes through the Ca2+ uniporter (CU), Na+/Ca2+ exchanger (NCE), Na+/H+ exchanger (NHE), and Ca2+/H+ exchanger (CHE) as well as via Ca2+ buffering by the mitochondrial proteins. However, the regulation of [Ca2+]m via ATP/ADP-dependent dynamic Ca2+ buffering mechanism inside the mitochondrial matrix during transient state-3 respiration is not well known. Methods: To gain a quantitative understanding of this Ca2+ buffering phenomenon, we developed a computational model of mitochondrial bioenergetics and Ca2+ handling by integrating our recent biophysical models of the CU, NCE, NHE, and CHE into our well-validated model of mitochondrial oxidative phosphorylation, TCA cycle, and electrophysiology. The model also accounts for binding and buffering of cations with metabolites, including ATP, ADP and Pi. Experiments were performed to spectrofluorometrically measure [Ca2+]m, pHm, membrane potential (ΔΨm), and NADH redox state in guinea pig heart mitochondria suspended in Na+ and Ca2+ free buffer medium (ensured with ∼50 μM of EGTA) with 0.5 mM pyruvic acid (HPyr). Dynamics were inferred with various addition of CaCl2 (0, 10, 25 μM of CaCl2; 16, 88, 130 nM of free [Ca2+] followed by 250 μM of ADP in the presence or absence of carboxyatractyloside (ANT blocker) and oligomycin (F1F0-ATPase blocker). Results and Discussion: Model analysis of the data on (i) initial decrease of [Ca2+]m with addition of Na+-independent substrate HPyr, and (ii) transient increases of [Ca2+]m with addition of ADP suggests ATP/ADP-dependent dynamic Ca2+ buffering inside the cardiac mitochondrial matrix. This model will be helpful to understand mechanisms by which [Ca2+]m both regulates, and is modulated by, mitochondrial energy metabolism.

ADP/ATP Antiport and ADP Phosphorylation Increase Mitochondrial Free Ca2+

Introduction: Matrix free [Ca2+] (m[Ca2+]) is believed to be a key regulator of mitochondrial function. The effect of differential buffering of calcium by ADP, ATP and Pi on m[Ca2+] levels has not been examined. We tested how m[Ca2+] is increased by ADP/ATP transport and phosphorylation, and if increased m[Ca2+] alters the bioenergetic state. Materials and Methods: Guinea pig heart mitochondria were isolated by differential centrifugation. Respiration and m[Ca2+], using indo-1 fluorescence, and corrected for NADH autofluorescence, were measured. After energizing mitochondria with 0.5 mM pyruvic acid, 0, 10, 25 μM CaCl2 (16, 88, 130 nM [Ca2+]) was added to the suspension before adding 250 μM ADP, in the presence or absence of ADP/ATP carrier blocker carboxyatractyloside (CATR) or F1F0ATPase blocker oligomycin (OMN). Results: m[Ca2+] increased proportionately with addition of CaCl2. ADP caused an additional increase to 100±6% in m[Ca2+] after 25 μM CaCl2. This was due to lesser binding of ADP vs. ATP to Ca2+. The rise in m[Ca2+] after ADP was reversed after all ADP was converted to ATP. With OMN the increase after ADP was lower (18±6%), but remained elevated as ADP was not phosphorylated to ATP. CATR completely blocked the ADP -induced increases in m[Ca2+] because matrix ADP transport was blocked. State 2 and 4 respiration, but not state 3, increased 14% and 18% with 25 μM CaCl2. NADH decreased with ADP alone, but NADH was not altered by adding CaCl2. Discussion: These results show that ADP transport into mitochondria and ADP conversion to ATP have significant effects on m[Ca2+]. Acutely changing buffer [CaCl2] has limited effects on redox state, although m[Ca2+] is believed to stimulate several dehydrogenases. However the k0.5 (1 μM) for this effect is only reached by adding ADP after 25 μM CaCl2.