Physiol Rev 87: 29 – 67, 2007;
doi:10.1152/physrev.00005.2006.
Mitochondrial Transporters as Novel Targets
for Intracellular Calcium Signaling
JORGINA SATRÚSTEGUI, BEATRIZ PARDO, AND ARACELI DEL ARCO
Departamento de Biologı́a Molecular Centro de Biologı́a Molecular “Severo Ochoa” UAM-CSIC, Facultad de
Ciencias, Universidad Autónoma, Madrid; and Área de Bioquı́mica, Centro Regional de Investigaciones
Biomédicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, Toledo, Spain
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I. Introduction
II. Calcium Binding Mitochondrial Carrier Gene and Proteins
III. Aspartate-Glutamate Carriers
A. Mammalian AGCs
B. Nonmammalian AGCs
C. Ca2⫹ regulation of the MAS
D. Other mechanisms of Ca2⫹ regulation of NAD(P)H transfer to mitochondria independent
of Ca2⫹ entry
E. Interplay between AGCs, GPS, and the Ca2⫹ uniporter in regulation of mitochondrial metabolism
in response to cytosolic Ca2⫹ signals
IV. ATP-Mg/Pi Carriers, SCaMC Genes, and Proteins
A. Mammalian SCaMCs
B. Hormonal and developmental changes in AdN Content in Liver Mitochondria
C. Nonmammalian ATP-Mg/Pi carriers
V. Evolution of Mechanisms for Calcium Signaling in Mitochondria
VI. Conclusions
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Satrústegui J, Pardo B, del Arco A. Mitochondrial Transporters as Novel Targets for Intracellular Calcium
Signaling. Physiol Rev 87: 29 – 67, 2007; doi:10.1152/physrev.00005.2006.—Ca2⫹ signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca2⫹ entry in
mitochondria via the Ca2⫹ uniporter followed by Ca2⫹ activation of three dehydrogenases in the mitochondrial
matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake
by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate
uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca2⫹ signals
via a mechanism that does not require the entry of Ca2⫹ in mitochondria, a mechanism depending on the activity of
Ca2⫹-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC)
and the ATP-Mg/Pi carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are
members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both
types of CaMCs are activated by Ca2⫹ in the intermembrane space and function together with the Ca2⫹ uniporter
in decoding the Ca2⫹ signal into a mitochondrial response.
I. INTRODUCTION
Ca2⫹ signaling in mitochondria is important to regulate mitochondrial function in response to intra- and extracellular cues. The main mechanism whereby Ca2⫹
modulates mitochondrial function involves Ca2⫹ entry in
mitochondria via the Ca2⫹ uniporter or rapid uptake
mode (RAM) mechanisms (143, 145, 190) followed by the
activation by Ca2⫹ of three dehydrogenases in the mitowww.prv.org
chondrial matrix (247). This causes an increase in mitochondrial NADH/NAD ratios which may result in increased energy available for mitochondrial functions. In
addition, matrix Ca2⫹ has been suggested to activate ATP
synthesis through a direct effect on F0F1-ATPase (31).
Here, we review work dating more than 30 years ago and
new evidence indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca2⫹ signals via a mech-
0031-9333/07 $18.00 Copyright © 2007 the American Physiological Society
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SATRÚSTEGUI, PARDO, AND DEL ARCO
neurotransmitters. These carriers exist in organisms such
as Saccharomyces cerevisiae that lack a Ca2⫹ uniporter.
In particular, S. cerevisiae Sal1p appears to be the most
basic CaMC, in fact the only mechanism conveying Ca2⫹
signals to mitochondria in this organism.
II. CALCIUM BINDING MITOCHONDRIAL
CARRIER GENE AND PROTEINS
Metabolite transport in and out of mitochondria is
essential for mitochondrial function. The MC family of
proteins comprises a group of structurally related proteins that exist only in eukaryotes (see Refs. 96, 211, 283,
418 for recent reviews). MC members are integral proteins in the inner mitochondrial membrane, with a molecular mass of ⬃35 kDa, six transmembrane spanning segments, and a tripartite structure (408) that catalyze the
transport of metabolites, nucleotides and derivatives, and
cofactors in mitochondria. The CaMC are a subset of MCs
that have long hydrophilic amino-terminal extensions harboring EF-hand Ca2⫹-binding motifs that face the intermembrane space (92, 95) (Fig. 1).
The CaMC family as such was discovered when the
genome sequences of S. cerevisiae (136) and Caenorhabditis elegans (78) became available. At that time and still
now, the identity of the mitochondrial Ca2⫹ transporters
responsible for Ca2⫹ signaling to mitochondria was unknown despite extensive efforts to characterize these
proteins (342, 405; see also Refs. 63, 340 for review). The
genome of S. cerevisiae was found to contain sequences
for ⬃35 members of the MC superfamily (117), and database analysis revealed that the genome of C. elegans also
contained a number of similar sequences. A S. cerevisiae
gene, YNL083w (364) (Table 1), and three sequences in C.
2⫹
FIG. 1. Schematic diagram of Ca -dependent mitochondrial carrier (CaMC) secondary structure. In both CaMCs, the carboxy-terminal half
corresponds to the MC homology region, and the amino-terminal extension harbors Ca2⫹-binding EF-hand motifs. The MC homology region, ⬃300
amino acids long, is represented according to its homology with that of ANT in its carboxyatractyloside-bound form (290, 291). TM1– 6 correspond
to the six tilted transmembrane helices characteristic of all MC, three of which are kinked (1, 3, and 5) due to the presence of conserved prolines.
Three shorter helices in the loops between TM 1–2, TM 3– 4, and TM 5– 6 face the matrix and are parallel to the membrane surface. The MC region
forms a basket opening to the intermembrane space with a funnel-shaped cavity inside that ends on a narrow pit close to the matrix surface. The
amino-terminal extensions of aspartate-glutamate carriers (AGCs) are longer than those of short CaMCs (SCaMCs), and the overall distribution and
sequence of EF-hand motifs are unrelated to that of SCaMCs, which is very similar to calmodulin.
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anism that does not require the entry of Ca2⫹ in mitochondria, a mechanism depending on the activity of the Ca2⫹binding mitochondrial carriers.
Mitochondrial carriers (MCs) are integral proteins of
the mitochondrial inner membrane that function in the
shuttling of metabolites, nucleotides, and cofactors between the cytosol and mitochondria (408), a subset of
which are the Ca2⫹-binding MCs (CaMCs) (95). CaMCs
fall into two groups: the aspartate/glutamate carriers
(AGC) (92, 95, 195, 286) and the ATP-Mg/Pi carriers, also
named SCaMC (for short CaMC) (94, 122). The two mammalian AGCs, aralar (AGC1) and citrin (AGC2), are members of the malate-aspartate NADH shuttle, and citrin, the
liver AGC2, is also a member of the urea cycle (217). The
transport reaction catalyzed by the AGCs is the only
irreversible step in the malate-aspartate shuttle (217, 218)
and a main point of regulation. The ATP-Mg/Pi carriers
exchange cytosolic ATP-Mg for mitochondrial Pi and are
involved in loading newly formed mitochondria with adenine nucleotides and modulating mitochondrial adenine
nucleotide content in response to hormones (10, 13). The
activity of both types of CaMCs is stimulated by Ca2⫹
acting on the external face of the inner mitochondrial
membrane (286, 163, 276).
To perform a role in transducing Ca2⫹ signals to
mitochondria, CaMCs must be poised for activation under
normal cell conditions. The existing data indicate that the
reaction catalyzed by these carriers is energetically favored under resting conditions but limited by the low
activity of the carrier. Ca2⫹ activation removes this limitation, allowing the transport reaction to occur. Available
data indicate that such processes occur at the expense of
relatively small Ca2⫹ signals, with limited capacity to
activate Ca2⫹ entry in mitochondria, and may be responsible for the energizing role of low levels of hormones and
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MITOCHONDRIAL TRANSPORTERS
TABLE
1.
The two groups of CaMC genes
SCaMCs (ATP-Mg/Pi Carriers)
AGCs (Aspartate/Glutamate Carriers)
Gene
Protein
Gene
Protein
Saccharomyces cerevisiae
YNL083w
SAL1
Yn1083p/Sallp
545*aa/EF
YPR020c
AGC1
Agc1p
902aa/no EF
Homo sapiens
SLC25a24
SCaMC-1
Chr 1p13.3
SCaMC-1
455-477aa†/EF
SLC25a12
AGC1
Chr 2q31
AGC1/aralar1
628aa/EF
SLC25a25
SCaMC-2
Chr 9q34
SCaMC-2
344-503aa†/EF
SLC25a13
AGC2
Chr 7q21
AGC2/citrin
675aa/EF
SLC25a23
SCaMC-3
Chr 19q13
SCaMC-3
422-477aa*/EF
* The sequence of YNL083w is different in two yeast backgrounds (364, 427). The length of Yn1083p/Sal1p [545 amino acids (aa)] corresponds
to the longest of the two. † Variants of different sizes (91, 94, 96). EF or no EF refers to the presence or absence of EF-hand domains in the proteins.
elegans code for putative proteins containing sequence
motifs corresponding to EF-hands (92), a property not
described so far in members of the MC family. EF-hands
(where EF stands for the E and F alpha helices of parvalbumin) are Ca2⫹-binding domains present in different
Ca2⫹-binding proteins (269), and their presence in a putative MC protein opened up the possibility that the protein might be involved in the handling of Ca2⫹, or in
Ca2⫹-regulated metabolite transport in mitochondria. The
realization of the potential implications of this finding was
the starting point for the cloning and characterization of
all members of the CaMC subfamily. Soon after, it became
clear that CaMCs are not involved in Ca2⫹ transport.
The CaMC family is made up of two types of carriers:
the AGCs and the ATP-Mg/Pi carriers. The AGCs have
long amino-terminal extensions, hence the name of aralar,
coined by Satrústegui to designate the first of those carriers cloned by Araceli del Arco (Araceli, hiperlargo) (95).
AGCs are represented by two isoforms in mammals,
aralar (or aralar1) (93) encoded by SLC25A12, and citrin,
named after type II citrullinemia, a disease caused by
mutations in citrin (92, 195), encoded by SLC25A13 (195,
358). These isoforms are mainly expressed in brain, skeletal muscle, and heart (aralar, AGC1) and liver (citrin,
AGC2).
The ATP-Mg/Pi carriers were first described as
CaMCs with short amino-terminal extensions or SCaMCs
and are represented by three main isoforms: SCaMC 1–3
(91, 94, 122) with a number of variants arising from alternative splicing. The identification of the transporter function of SCaMCs was carried out by Palmieri’s group (122)
after reconstitution of the recombinant proteins into proteoliposomes. Throughout this manuscript the acronyms
SCaMC 1–3 will be used to designate the particular isoforms of the ATP-Mg/Pi carrier, rather than the acronyms
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used previously (APC 1–3, from ATP-Mg/Pi carrier, Ref.
122), to avoid confusions with the well-known APC protein, adenomatous polyposis coli, a tumor suppressor
multifunctional protein that is mutated in a majority of
colon cancers (160).
The transporter function of the AGCs was reported
long ago (27). These carriers are important for the urea
cycle and malate-aspartate NADH shuttle and have been
the subject of a number of studies including purification
and functional reconstitution in proteoliposomes. However, Ca2⫹ regulation of the AGCs had escaped attention.
An ATP transport pathway different from the adenine
nucleotide translocases was first described in fetal liver
mitochondria where it was found to be important for the
net uptake of adenine nucleotides immediately after birth
(299, 300, 373). A few years later, a series of careful
studies carried out by Aprille and co-workers (276) and
Haynes and co-workers (163) resulted in the discovery
that the ATP-Mg/Pi carrier was regulated by Ca2⫹ from the
external side of the inner mitochondrial membrane (163,
276). In retrospect, this seminal finding set the stage for
the identification of MCs endowed with Ca2⫹ regulation
properties.
III. ASPARTATE-GLUTAMATE CARRIERS
A. Mammalian AGCs
Aralar/AGC1 and citrin/AGC2 are coded for
SLC25a12 and SLC25a13, in human chromosomes 2q31
(80, 339) and 7q21.3 (358), respectively. The two genes
have the same genomic structure, with 18 exons (358),
also conserved in mice (180, 359).
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Organism
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SATRÚSTEGUI, PARDO, AND DEL ARCO
1. Development and tissue distribution of aralar/AGC1
and citrin/AGC2
2. Import of AGC precursor proteins to mitochondria
Like other MCs, AGC precursor proteins are synthesized in the cytosol and then imported to mitochondria.
Targeting information in CaMCs is contained in their carboxy-terminal half, and all amino-truncated variants are
targeted into mitochondrial membranes (91, 92, 94,
96, 286).
The mechanisms of import of MC precursor proteins
and CaMCs have been discussed in detail in recent reTABLE
2.
3. Transport activity
The AGCs aralar and citrin catalyze the exchange of
aspartate with glutamate plus a proton, as demonstrated
in intact mitochondria (215, 216, 218) and in proteolipo-
Distribution of AGC isoforms in adult mouse tissues
Aralar AGC1
Citrin AGC2
Aralar AGC1
Citrin AGC2
Skeletal
Muscle
Liver
Hepatocyte
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
ND
ND
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹
⫹⫹
ND
⫹⫹
ND
Brown
Adipose
Tissue
White
Adipose
Tissue
Testis
Ovary
Intestine
Pancreas
Pancreatic
Islets
Stomach
ND
ND
ND
ND
⫹
⫹
⫹⫹
⫹
⫹
⫹
⫹⫹
⫹⫹
⫹⫹
ND
⫹⫹
⫹⫹
Brain
Heart
⫹⫹⫹
ND
Kidney
Spleen
Bone
Marrow
Lung
The relative levels of immunoreactive citrin and aralar proteins are taken from del Arco et al. (92) and Begum et al. (35). ND, nondetectable.
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Aralar, citrin, or both isoforms are present in early
preimplantation embryos, as lactate supports development from the two-cell stage onwards, through the operation of the malate-aspartate NADH shuttle (214). Thus
the AGCs are present very early in mouse development.
Aralar and citrin have an overlapping distribution in embryogenesis (93, 358). Both isoforms are expressed
strongly in branchial arches, dermomyotome, limb, and
tail buds at early embryonic stages. The characteristic
strong expression of citrin in liver is acquired postnatally,
while that of aralar in heart and skeletal muscle begins
during embryonic development (93). In the adult mouse,
aralar is highly expressed in almost every tissue including
brain, skeletal muscle, and heart (95) as well as in cells
from the immune and hematopoietic tissues both at embryonic stages (embryonic liver) and in adults (Table 2).
Many tissues express both isoforms, except hepatocytes
from adult mouse liver where citrin is the only detectable
AGC (93).
Aralar and mitochondrial aspartate aminotransferase
expression decrease, whereas that of citrin increases in
heart from desmin-deficient mice (129), and the parallel
decreases of the former have been taken as an indication
of decreases in malate-aspartate NADH shuttle (MAS)
activity in these animals (129). Aralar expression decreases both in mouse and human skeletal muscle after
prolonged feeding on high-fat diet, in parallel with the
decrease of many other transcripts for oxidative phosphorylation proteins (359).
views (96, 196, 309). The insertion into the inner membrane of members of MC family is mediated by the TIM22
(translocase of the inner membrane) complex or carrier
translocase (Fig. 2B). The TIM22 pathway contains components at the inner membrane as well as in the intermembrane space, referred to as the small Tim proteins,
Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p (198). These
small Tims act as chaperone-like molecules to guide hydrophobic precursors across the aqueous intermembrane
space, whereas the inner membrane complex mediates
their insertion into the inner membrane (88, 89, 412). The
small Tim proteins assemble into 70-kDa complexes,
Tim8p with Tim13p and Tim9p with Tim10p in a 3:3 ratio
(Fig. 2B) (88, 89, 197). These 70-kDa complexes have
different substrate specificities, with the classical MC precursor proteins binding predominantly to the Tim9pTim10p complex (89, 289). In contrast, the Tim8p-Tim13p
complex binds to the human and yeast AGC precursor
proteins through their long hydrophilic amino-terminal
domains (318). Pathway selection seems to be determined
by the presence of the hydrophilic amino-terminal extension of CaMCs; thus the import of full-length aralar decreases 80% in mitochondria that lack the Tim8p-Tim13p
complex, and shortening of the aralar amino-terminal domain results in preferential binding of aralar to the Tim9pTim10p complex (318). Mutations in the locus for human
Tim8p (DDP1/TIMM8a locus) cause Mohr-Tranebjaerg
syndrome (MTS/DFN-1, deafness/dystonia syndrome)
(198, 317), and a lymphoblast cell line derived from an
MTS patient shows decreased mitochondrial NADH levels
due to defects in the MAS where AGC is the rate-limiting
component (318). The Tim8p-Tim13p complex probably
also participates in SCaMCs import as suggested by the
differences in import efficiency detected for overexpressed full-length and amino-truncated versions of
SCaMC3 protein (91).
MITOCHONDRIAL TRANSPORTERS
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somes reconstituted with the native purified protein (97,
98) or recombinant AGCs (286). Thus there is one positive
charge that moves across the membrane together with the
two amino acids, and the exchange is electrophoretic
(114, 215–218, 352). This property of the AGC is rather
uncommon among the MC family, only shared by the
adenine nucleotide translocases and the uncoupling proteins. Because the aspartate-glutamate exchange reaction
is strongly dependent on the membrane potential, the
release of aspartate in exchange with glutamate is
strongly favored (97, 98, 215, 216, 218, 286). Thus, in
energized mitochondria, this carrier is essentially unidirectional. The affinity for glutamate is lower than that for
aspartate (Km values of ⬃0.2 and 0.05 mM, respectively),
both for the native or recombinant AGCs (97, 99, 286), and
these values do not change with the membrane potential
(286). The effect of the membrane potential is a pronounced increase in transport activity that depends on an
electrogenic modulation of the transport rate constant of
the negatively charged carrier-aspartate complex, and not
to a change in the substrate affinity of the carrier (286).
The two AGCs also catalyze 1:1 electroneutral homoexchange reactions with aspartate, glutamate, and cysteine
sulfinate and are strongly inhibited by pyridoxal phosphate (45, 101, 284, 286).
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4. Functional roles of AGCs in urea cycle and the MAS
AGCs are required for the transfer of mitochondrial aspartate to the cytosol, an important step in urea
synthesis (Fig. 3) and in gluconeogenesis from lactate
and amino acids. Arginino-succinate synthetase (ASS),
the cytosolic enzyme condensing citrulline with aspartate, uses preferentially but not exclusively, mitochondrial versus cytosolic aspartate (250, 319, 334, 413). For
recent reviews on this subject, see References 333 and
334. The role of citrin in the urea cycle is discussed in
section IIIA5.
AGCs are also needed for the transfer of mitochondrial aspartate to the cytosol in the MAS, described by
Borst (48). The shuttle transfers reducing equivalents of
NADH from cytosol to mitochondria and is required during glycolysis and lactate metabolism. Besides the cytosolic and mitochondrial isoforms of malate dehydrogenase (cMDH, mMDH) and aspartate amino transferases
(cAAT and mAAT), the shuttle uses two MCs, the AGC
and the malate/␣-ketoglutarate carrier (OGC) (Fig. 4). The
presence of the AGCs in this cycle provides a unidirectional substrate flow along the shuttle that functions in
the direction of reducing equivalent transfer from cytosol
to mitochondria. MAS is important in reducing equivalent
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FIG. 2. Topology and mitochondrial import pathways of CaMCs. Classical mitochondrial carriers (MCs) and CaMCs differ in the
presence of a long amino-terminal extension
with Ca2⫹-binding domains facing the intermembrane space (IMS) (A) and appear to use
different small Tim proteins to escort inner
membrane proteins across IMS (B). The outer
membrane translocase (TOM) transports mitochondrial precursor proteins from the cytosol
to IMS. In the inner membrane (IM) the TIM22
translocase mediates the import of inner membrane proteins. The movement of mitochondrial precursor proteins across the IMS is facilitated by the small Tim proteins. Classical
MCs use escort complexes made up of Tim9pTim10p proteins (pathway 1), whereas CaMCs
use escort complexes made up of Tim8pTim13p proteins (pathway 2). Tim8p-Tim13p
bind to the hydrophilic amino-terminal extensions of CaMCs, and Tim9p-Tim10p interact
with the hydrophobic domains of MCs.
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SATRÚSTEGUI, PARDO, AND DEL ARCO
transfer in many tissues including liver, particularly human liver, where other NADH shuttle systems such as the
glycerol-3-phosphate shuttle (GPS) have low activity,
⬃1/20 of that in rodents (330).
MAS is also the dominant NADH shuttle in heart (219,
221, 328, 331, 346 –348, 424), and both isoforms aralar and
citrin are expressed in heart, including human heart (380)
with a preferential enrichment of aralar in atria (93). In
the fetal and immediate perinatal period, the heart relies
on glycolysis as a source of energy, with ⬃40% of the ATP
production derived from glycolysis, but within days oxidative phosphorylation increases to provide 93% of the
ATP as fatty acid utilization increases (see Ref. 369 for
review). In neonatal hearts, lactate oxidation accounts for
the majority of oxygen consumption (127). MAS activity
of neonatal porcine heart mitochondria is about three
times higher than that of adult heart (346, 347), in which
fatty acid metabolism bypasses the NADH shuttles. Strikingly, the activity of the heart mitochondrial Ca2⫹
uniporter undergoes a similar decrease along postnatal
life in the rat (32). In contrast to the developmental
changes in MAS activity, mRNA and protein levels of
AGCs in porcine and mouse heart are higher in adult than
in neonates (35, 347), although a decrease in steady-state
aralar mRNA levels was observed from postnatal day 9
onward (35). Studies of the levels of all other MAS mem-
FIG. 4. Malate-aspartate NADH shuttle
(MAS). Role of the AGC in Ca2⫹ activation of
MAS activity. MAS is made up of four enzymes: mitochondrial and cytosolic aspartate
aminotransferases (AAT) (1), malate dehydrogenases (2), and two mitochondrial carriers, the aspartate/glutamate carrier (AGC)
and the ␣-ketoglutarate/malate carrier (OGC).
The site for Ca2⫹ activation of AGC is shown.
Mitochondrial matrix Ca2⫹, entering through
the Ca2⫹ uniporter (CU), activates pyruvate
dehydrogenase (PDH) (3), isocitrate dehydrogenase (IDH) (4), and ␣-ketoglutarate dehydrogenase (␣-KGDH) (5). AcCoA, acetyl CoA;
Asp, aspartate; Glut, glutamate; Isoc, isocitrate; ␣-KG, ␣-ketoglutarate; Mal, malate; OAA,
oxalacetic acid; Pyr, pyruvate; SuccCoA, succinil CoA.
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FIG. 3. Urea cycle. Role of citrin as
AGC in urea formation from ammonia.
mMDH, mitochondrial malate dehydrogenase; mAAT, mitochondrial aspartate
amino transferase; GDH, glutamate dehydrogenase; CAPS, carbamyl-phosphate synthetase; OTC, ornithine transcarbamylase;
ASS, argininosuccinate synthetase; ASAL,
argininosuccinate lyase; ARGase, arginase;
FUM, fumarase; DiC, dicarboxylate carrier;
PiC, phosphate carrier; GC, glutamate carrier; AGC, aspartate-glutamate carrier;
ORC, ornithine-citrulline carrier. [Redrawn
from Saheki et al. (333).]
MITOCHONDRIAL TRANSPORTERS
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particular developmental stage. Thus addition of exogenous aspartate enabled mouse zygotes to utilize lactate
and develop normally (214).
5. Mutations and polymorphisms
Much of the actual roles of aralar and citrin in humans and rodents have been unveiled through the discovery of human diseases involving the two carriers and with
the development of mouse models carrying disruptions in
the two genes.
A) CITRIN, AGC2, AND SLC25A13. A deficiency in citrin, the
AGC2 isoform specifically expressed in liver, kidney, and
heart encoded by gene SLC25A13, causes type 2 citrullinemia (CTLN2), an adult-onset liver disease predominantly found in the Japanese population (195). Unlike
classical citrullinemia in children, which is associated
with a deficiency in ASS, in CTLN2 the ASS gene is normal
and ASS deficiency is only found in liver, but not in other
organs that express ASS. Moreover, the residual amount
of enzyme present in the patients displays normal kinetic
properties (333, 335). The disorder was first reported in a
group of patients with high serum citrulline and ammonia
levels and neurological symptoms that closely resemble
those of hepatic encephalopathy. However, a different
disease, neonatal intrahepatic cholestasis caused by citrin
deficiency (NICCD), was found in a number of neonates
with idiopathic neonatal hepatitis that carry the same
mutations in citrin found in CTLN2 patients (280, 392).
These neonates have multiple metabolic abnormalities
including aminoacidemia, galactosemia, hypoproteinemia, hypoglycemia, and cholestasis. NICCD is usually not
severe, and it can be managed by nutritional manipulation, but a few patients develop CTLN2 after 10 years or
more (332).
In CTLN2 patients, mutations in citrin cause a complete lack of citrin protein (195). The generation of citrinknockout mice has revealed many of the consequences of
citrin deficiency (359). Liver mitochondria from citrin(⫺/⫺) mice had a drastic reduction of malate-aspartate
NADH shuttle activity and aspartate efflux with no compensatory changes in the glycerophosphate NADH shuttle. Gluconeogenesis from lactate, but not from pyruvate,
was severely impaired, in agreement with MAS requirement for gluconeogenesis from reduced substrates. Urea
production from ammonia was severely reduced in liver
from citrin(⫺/⫺) mice, and this was strongly alleviated in
the presence of asparagine, indicating that aspartate was
the major limiting factor. All of these metabolic characteristics are consistent with a primary failure in aspartate
efflux from mitochondria and malate-aspartate NADH
shuttle activity (359). However, despite the deficits in
AGC-dependent pathways, citrin(⫺/⫺) mice failed to show
any overt pathological consequences. In particular, these
mice had normal serum ammonia and citrulline levels and
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bers indicated that those of OGC decrease postnatally in
pig and rabbit (141, 347) and probably limit shuttle function in adult heart (347). An upregulation of MAS could be
of potential interest in heart failure and myocardium hypertrophy, when substrate utilization shifts away from
fatty acids toward glucose and lactate (328, 369).
Glucose metabolism drives the stimulation of insulin
secretion in pancreatic ␤-cells through increased ATP
levels that lead to closure of KATP channels (237, 238,
251). Lactate dehydrogenase activity is low in pancreatic
␤-cells (177, 178, 354), and NADH shuttle systems are
important for glucose-induced activation of mitochondrial metabolism and insulin secretion (112, 135, 377).
Among NADH shuttle systems, MAS is particularly important, as insulin secretion is normal in mice with no functional mitochondrial FAD-GPDH, the mitochondrial member of the glycerol-phosphate shuttle, whereas it is
blocked when MAS activity is inhibited in these transgenic mice (119). Aralar is the only AGC present in islets
and ␤-cells (93, 326), and its levels or activity appear to
limit MAS function in these cells. Thus overexpression of
aralar in INS-1E or pancreatic ␤-cell leads to increases in
glucose-induced reduction of mitochondrial NAD(P)H,
mitochondrial membrane potential, and ATP levels along
with an increase in glucose-induced insulin secretion
(326). Glucose utilization was not changed by aralar overexpression, but lactate formation was decreased, suggesting that MAS activity regulates the fraction of pyruvate
being either oxidized in mitochondria or reduced to lactate (326). On the other hand, aralar overexpression did
not modify insulin secretion when pyruvate, rather than
glucose, was used as fuel, showing that it contributes
primarily upstream of mitochondria to efficiently couple
glycolysis to mitochondrial metabolism (326). MAS is also
important in neurons, especially during lactate utilization,
and this is discussed in section IIIA5B.
Interestingly, MAS is also required in early embryos.
Studies on the preimplantation mouse embryo carried out
more than 30 years ago have shown that the zygote has an
absolute requirement for pyruvate to complete the first
cleavage division, whereas lactate and glucose are only
capable to support development from the two- and eightcell stages, respectively (56, 57). Mouse zygotes can use
both lactate and pyruvate (213), but only pyruvate supports the first cleavage. In zygotes, but not in blastocysts,
lactate oxidation and NAD/NADH ratios decreased with
increasing lactate concentration, suggesting that lactate
utilization by lactate dehydrogenase was limited due to an
inability to provide NAD for the reaction (213). Although
zygotes express all four MAS enzymes, the inability to
transfer reducing equivalents to mitochondria was found
to be due to a lack of MAS activity, aspartate being the
rate-limiting factor probably due to the high Km of the
cytosolic aspartate aminotransferase (214). This suggests
that AGC levels or activity may limit MAS function at this
35
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SATRÚSTEGUI, PARDO, AND DEL ARCO
high levels of citrulline and low levels of ornithine (265,
359). These findings suggest that the aspartate arising
from asparagine hydrolysis could serve as a substrate for
the ASS reaction. In addition, pyruvate administration
corrected the defect in ureogenesis, by lowering the cytosolic NADH/NAD ratio (265).
B) ARALAR, AGC1, AND SLC25A12. Aralar is an ubiquitous
AGC isoform highly expressed in brain and skeletal muscle. The liver parenchyma seems to be the only adult
tissue devoid of aralar, although it is present in Kupffer
cells, and also in the embryonic liver (93), a major hematopoietic organ.
The existence of aralar-knockout mice (180) has revealed new functions of this carrier, particularly a role in
the supply of brain N-acetyl aspartate (NAA) for myelin
lipid synthesis (Fig. 5). Aralar⫺/⫺ mouse embryos are
produced in normal numbers, as expected from the overlap of aralar and citrin expression in fetal tissues (35, 93).
However, soon after birth these mice develop severe
growth defects, neuromuscular deficiencies, and a reduced myelination in the central nervous system (CNS),
but not in peripheral nerves. Brain and muscle mitochondria from aralar-knockout mice have a complete loss of
MAS activity, a drastic decrease in respiration on glutamate plus malate, with no compensatory increase in respiration on pyruvate plus malate, and a pronounced decrease in aspartate efflux from mitochondria (180). Aralar
deficiency results in a large drop of NAA and aspartate in
the brain and primary neuronal cultures derived from
Aralar⫺/⫺ mice, together with a drop in myelin lipids and
similar reductions in the levels of the major proteins in
FIG. 5. Role of aralar in N-acetylaspartate (NAA) and myelin lipid synthesis. Aspartate (Asp) is synthesized in neuronal mitochondria and is transported out of the
mitochondrial matrix on the neuronal AGC1
aralar. Aspartate-N-acetyltransferase (AspNAT), the enzyme catalyzing NAA synthesis
from acetyl-CoA (Ac-CoA) and Asp, is enriched in microsomes and in mitochondrial
fractions. Ac-CoA arises from citrate (Cit) in
the classical lipogenic pathway, through the
ATP citrate lyase (ACL) and is transported
out of mitochondria through the citrate carrier (CC), SLC24A1, or an unrelated protein,
the tricarboxylate carrier with high expression in neurons, TCC (255). NAA shuttles
Ac-CoA from neurons to oligodendrocytes
that use it as myelin lipid synthesis precursor. Aspartoacylase (ASPA) is enriched in
oligodendrocytes and cleaves NAA giving
rise to acetate and Asp. ACS, acetylCoA synthetase; PDH, pyruvate dehydrogenase; CS,
citrate synthase; AAT, mitochondrial aspartate aminotransferase; MDH, malate
dehydrogenase.
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responded to increases in protein load in their diet by
increasing urea formation. This suggests that mitochondrial-derived aspartate is probably not essential for the
ASS reaction in mice (265, 359).
The characteristic decline in hepatic ASS levels and
activity of CTLN2 patients has been attributed to the
defect in the transport of aspartate from mitochondria to
the cytosol that could inhibit the ASS reaction and destabilize the liver enzyme (286). ASS protein has full activity
in NICCD neonates (381) and declines gradually during
the life of CTLN2 patients. Whether the shortage of aspartate by itself destabilizes ASS or other factors participate
in this defect is still unknown, but this particular defect in
hepatic ASS levels is also absent in the citrin(⫺/⫺) mouse
(359). Species-specific differences in gene regulation and
in alternative metabolic pathways have been proposed to
explain the different phenotypes. In particular, the glycerol phosphate shuttle is ⬃20-fold more active in rodent
than in human liver (330), suggesting that this shuttle
could compensate for the lack of MAS activity in mice.
There is indirect evidence that, to compensate for the lack
of MAS, CTLN2 patients rely heavily on an alternative
NADH shuttle, the malate-citrate shuttle pathway, which
is used for the transfer of acetyl CoA from mitochondria
to the cytosol for fatty acid synthesis (332, 333). Fat
accumulation that could be supported by increased
malate-citrate shuttle activity has been observed in the
livers of patients with citrin deficiencies (both NICCD and
CTLN2) (175, 334, 381, 392). The defects in ureogenesis in
the citrin(⫺/⫺) liver could be partially corrected by the
administration of asparagine, which also normalized the
MITOCHONDRIAL TRANSPORTERS
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Impaired aralar activity could also contribute to the
pathology of Mohr-Tranebjaerg syndrome (MTS/DFN-1,
deafness/dystonia syndrome) caused by mutations in the
locus for human Tim8p (DDP1/TIMM8a locus) (198, 317;
as discussed in sect. IIIA2). Together with Tim13, Tim8p
forms a 70-kDa complex specifically utilized by AGCs
(318), and possibly also the SCaMCs (89) on their way to
the inner membrane, a complex with prominent expression in large neurons in the brain (318). The mutations in
Tim8p in MTS/DFN-1 patients appear to result in impaired
import of aralar, as a lymphoblast cell line derived from a
MTS patient shows decreased mitochondrial NADH levels
due to defects in MAS activity (318).
Thus genetic variations that change the expression of
aralar, or an impaired import of aralar in mitochondria,
could lead to changes in NADH shuttle function, respiration on glutamate and malate, aspartate synthesis by neuronal mitochondria, and NAA production, which could be
related to these CNS diseases. A decrease in NAA in
different brain regions of autistic patients has been reported (130, 281).
B. Nonmammalian AGCs
A glutamate-aspartate MC is present in all known
eukaryotic genomes (96). In most higher eukaryotes
AGCs are highly similar to the mammalian genes and have
EF-hand Ca2⫹ binding motifs in equivalent positions. A
prominent exception to this rule is S. cerevisiae, in which
the long amino-terminal extension of the agc1p protein
lacks Ca2⫹-binding motifs (69) (Table 1).
1. Transport activity
The carboxy-terminal domain of yeast agc1p, that
encompasses the whole MC homologous region, has been
expressed in bacteria and reconstituted in phospholipid
vesicles. Its transport properties were found to be similar
to those of the mammalian AGCs in terms of substrate
specificity, affinity, inhibitor sensitivity, and voltage dependence but differed from mammalian AGCs in that
yeast agcp is able to catalyze the uniport of aspartate, and
to a greater extent of glutamate, in addition to the aspartate/glutamate exchange (69).
The carboxy-terminal domains of agc1p and mammalian AGCs are highly similar, suggesting that both arise
from a common ancestor. This ancestor probably duplicated in mammals to give rise to the AGCs that catalyze a
strict asp/glut exchange, and glutamate carriers (GCs)
that catalyze the uniport of glutamate driven by the pH
gradient (123), and evolved into agc1p in S. cerevisiae,
that catalyzes both transport activities (69). The second of
these activities, the uniport of glutamate, is thought to be
required to provide mitochondrial glutamate for transam-
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myelin, myelin basic protein (MBP), and myelin-associated oligodendrocyte basic protein (MOBP).
NAA plays a role in myelin lipid formation through
the supply of acetyl groups (71, 90, 249) (Fig. 5). NAA
produced in neurons (398) undergoes transaxonal transfer to oligodendrocytes where it supplies acetyl groups
for the synthesis of myelin lipids. The NAA cleavage
enzyme aspartoacylase is restricted primarily to oligodendrocytes (43, 191). Mutations in aspartoacylase cause
Canavan’s disease, characterized by a spongy degeneration of the CNS, increased levels of NAA in the brain and
body fluids, and an extensive loss of myelin. Aspartoacylase-deficient mice, generated as model for Canavan’s
disease (244), show decreased myelin lipid synthesis and
a very drastic (⬃80%) reduction in the brain acetate concentrations (235), showing that the lack of NAA results in
a lack of acetyl groups for myelin lipid synthesis.
The fall in brain aspartate levels and the lack of
aspartate production in brain mitochondria from
Aralar⫺/⫺ mice indicate that the major route of aspartate
production in the CNS is mitochondrial and that it depends on aralar for aspartate efflux to the cytosol. Indeed,
the CNS pathology in aralar-null mice appears at about
the time where the blood-brain barrier develops, preventing the entry of aspartate and other amino acids into most
regions of the adult brain except for selected circumventricular regions (302). Therefore, from the first week of
life onward, brain cells have to rely on their own endogenous production of aspartate, which takes place mostly
in neurons, the brain cells with highest aralar expression
(303). NAA is derived from neuronal aspartate, and its
synthesis is catalyzed by aspartate-N-acetyltransferase in
brain mitochondria (236, 334) and microsomes, which
have four to five times higher activity than mitochondria
(231). Thus the fall in NAA in brain and in cultured
neurons from Aralar⫺/⫺ mice is clearly associated with
the defect in aspartate production.
The important brain functions underscored from the
study of aralar-knockout mice may be relevant for two
human diseases of the nervous system. A strong linkage
and association of the gene for aralar, SLC25A12, with
autism has been reported (304, 353; but see Ref. 46). Two
polymorphisms that corresponded to single nucleotide
polymorphisms (SNPs) located in flanking intronic sequences of exons 4 and 16 of SLC25A12 were associated
with the disease, although the functional relevance of
these SNPs is yet unknown (304, 353). The expression of
a number of genes is regulated by cis-acting elements, and
inherited variation in gene expression may contribute to
disease (52). An altered regulation of gene expression
without variations in its coding sequence could affect
cellular functions when the levels of the gene product are
limiting, as appears to be the case for aralar, as an overexpression of this protein in ␤-cells increases MAS function (326).
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SATRÚSTEGUI, PARDO, AND DEL ARCO
ination reactions and as a carbon and nitrogen source for
ornithine synthesis, as discussed below (69).
2. Role of yeast AGC in amino acid biosynthesis and
in shuttling reducing equivalents to mitochondria
C. Ca2ⴙ Regulation of the MAS
In this section we review evidence gathered many
years ago suggesting that the MAS is regulated by Ca2⫹.
The mechanism was long considered to rely on Ca2⫹ entry
in mitochondria through the uniporter and subsequent
activation of mitochondrial dehydrogenases, or on direct
activation by calmodulin. After the molecular identification of the AGCs, it has been shown that the shuttle is
regulated by Ca2⫹ from the external side of the inner
mitochondrial membrane. However, the interactions between the Ca2⫹ uniporter-mediated Ca2⫹ signaling in mitochondria and the mechanism mediated by the AGCs are
complex and may vary from tissue to tissue.
Physiol Rev • VOL
It has been known for a long time that ␣-adrenergic
agents stimulate gluconeogenesis in hepatocytes. The
mechanism is independent of cAMP and related to
changes in cytosolic Ca2⫹ concentration ([Ca2⫹]i) (205,
372, 379). Increased gluconeogenesis is observed with
reduced substrates, such as glycerol, xylitol, and sorbitol,
that require the transfer of reducing equivalents to mitochondria, but is minimal or absent with oxidized substrates such as pyruvate or dihydroxyacetone phosphate
(193, 226). The proposed sites of regulation are at the
MAS or the GPS (194, 225, 379).
According to some groups, stimulation of rat hepatocytes or perfused liver with ␣-adrenergic agonists such as
phenylephrine leads to sustained activations of MAS and
GPS, or to a transient stimulation of MAS followed by a
stable stimulation of GPS (194, 225, 372, 379). Increased
MAS activity was typically inhibited by the transaminase
inhibitor aminooxyacetate. The mechanisms responsible
for increases in MAS activity were thought to involve
either changes in intermediates affecting AGC activity or
an increase in the mitochondrial membrane potential
caused by a direct stimulation of respiration that would
increase the driving force of the AGC and shuttle activity
(225). In fact, ␣-adrenergic agonists transiently increased
the cytosolic/mitochondrial aspartate concentration gradient and strongly reduced glutamate and especially ␣ketoglutarate concentrations (226, 370, 372), a finding
thought to reflect a rapid activation of ␣-KG dehydrogenase (␣-KGDH) activity (379). Careful studies by LaNoue’s group (371) showed that the mitochondrial membrane potential does not change significantly in glucagonstimulated hepatocytes, arguing in favor of changes in
AGC activity at the expense of changes in intermediates.
The fall in ␣-KG concentrations was regarded as the
mechanism responsible for ␣-adrenergic agonist-dependent increase in liver AGC activity (370, 371). The effect of
the decrease in ␣-KG levels was explained by the finding
that ␣-KG was a strong competitive inhibitor of aspartate formation (competitive with respect to oxaloacetate) via glutamate transamination in isolated liver mitochondria (371).
Hepatocyte activation by Ca2⫹-mobilizing agents was
thought to induce Ca2⫹ entry in mitochondria coupled to
Ca2⫹ activation of ␣-KGDH activity. Glucagon, which increases intracellular cAMP levels, synergistically stimulates net inflow of Ca2⫹ to hepatocytes through receptoractivated Ca2⫹ channels (4, 121, 167) and net uptake of
Ca2⫹ by hepatocyte mitochondria (4, 9, 121, 167, 264). As
Ca2⫹ increases the affinity for ␣-KG of ␣-KGDH (246, 273),
Ca2⫹ entry in mitochondria would result in a reduction of
␣-ketoglutarate levels, with subsequent activation of glutamate transamination and aspartate efflux from mitochondria, in spite of a concurrent decrease in oxalo-
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The activity of agc1p as a glutamate uniporter is
probably responsible for changes in amino acid levels and
labeling from [13C]acetate in yeast agc1p deletion mutants. These mutants had reduced valine, ornithine, and
citrulline levels (69) that were explained as a consequence of reduced uptake of glutamate in mitochondria,
with subsequent reduction in mitochondrial glutamate
levels, since glutamate dehydrogenase is not mitochondrial but cytosolic in S. cerevisiae (169). Mitochondrial
glutamate is required for transamination reactions within
mitochondria (valine synthesis), and as carbon and nitrogen source for ornithine synthesis, all of which are reduced in agc1p mutants (69).
The existence of a malate-aspartate NADH shuttle in
S. cerevisiae has been questioned for a long time, because
yeast mitochondria harbor external NADH dehydrogenases that oxidize NADH on the external face of the inner
mitochondrial membrane (233) and lack complex I in the
respiratory chain. However, all the components of the
shuttle exist in yeast, particularly the MCs for ␣-ketoglutarate (␣-KG) and malate exchange, Odc1 and Odc2,
oxodicarboxylate carriers that can transport ␣-KG,
␣-ketoadipate, and malate (285, 386, 396), and the aspartate-glutamate exchanger agc1p. Growth on oleic acid
produces large amounts of NADH in peroxisomes that
require shuttle systems for reducing equivalent transfer to
mitochondria. MAS is the shuttle system involved in the
transfer of reducing equivalents from the cytosol to the
mitochondria in this particular condition, as neither the
odc1⌬odc2⌬ double mutants (386) or agc1p deletion mutants (69) grow on oleic acid. Because growth on ethanol,
but not on acetate, is unimpaired (69), this defect is not
due to defects in gluconeogenesis, but rather to defect in
NADH shuttle function.
1. Ca2⫹ activation of MAS in liver
MITOCHONDRIAL TRANSPORTERS
2. Ca2⫹ regulation of MAS in heart
Liver is not the only organ where Ca2⫹ activates MAS
activity. Evidence for this was obtained in kidney (350)
and heart (342). However, the activation by Ca2⫹ of MAS
activity in heart has proven to be elusive. This is due to
the fact that in the heart, Ca2⫹ has opposing actions on
the two carriers of MAS, an activating role for the AGC,
which is observed only under specific conditions, and an
inhibitor role for the OGC. As a result, the overall activating role on the AGC or on MAS is not always apparent.
In studies with heart mitochondria, physiological increases in matrix Ca2⫹ resulted in a marked activation of
␣-KGDH and in a decreased accumulation of ␣-KG (409).
However, Scaduto (342) found that under conditions of
MAS operation (incubation with glutamate plus malate)
the decrease in ␣-KG levels led to an inhibition rather than
an increase of aspartate formation. Stimulation by Ca2⫹ of
aspartate efflux in rat heart mitochondria was only observed after imposing a rise in mitochondrial NADH/NAD
ratio through addition of pyruvate (together with malate
and glutamate), to decrease OAA levels and basal aspartate efflux rate [reduced to ⬃30% of control levels (342)].
Because ␣-KG is a competitive inhibitor of OAA transamination to aspartate, Ca2⫹-induced changes in ␣-KG levels
controlled aspartate formation only under conditions
where OAA levels were very low, as those provided by
pyruvate. In the absence of pyruvate, the influence of
Ca2⫹ on AGC activity was attributed to a balance between
an inhibitory effect, caused by the increase in the NADH/
Physiol Rev • VOL
NAD ratio and consequent reduction in OAA levels, and a
stimulatory effect caused by a decrease in ␣-KG levels (342).
Regarding the OGC, in heart mitochondria there is a
strict competition for ␣-KG between ␣-KGDH and this
carrier (Fig. 4), by virtue of the similarities of their apparent Km values. An apparent Km of ⬃1.5 mM for ␣-KG on
the matrix side of the OGC has been reported (360), while
the Km for ␣-KG in the case of ␣-KGDH is ⬃0.67 mM (219)
so that both steps will be very sensitive to the concentration of ␣-KG in the mitochondrial matrix. Consequently,
activation or inhibition of OGC transport will lead to a
reduction or stimulation in ␣-KGDH activity, respectively
(219). Conversely, in perfused rat heart and isolated heart
mitochondria, increased ␣-KGDH activity results in a decrease in ␣-KG efflux from mitochondria (278).
The relevance of the effects of Ca2⫹ on competition
for ␣-KG has been underscored by a recent study in which
heart metabolism was studied at both basal and high work
loads (279). Lewandowski’s group (279) showed that high
work loads, which lead to increases in mitochondrial
Ca2⫹ (see sect. III, C3B and E), resulted in a drastic reduction in OGC and MAS activity (to ⬃20% of basal levels).
Consistently, the decreased transfer of reducing equivalents to mitochondria was accompanied by increased tissue lactate levels (279).
On the basis of the above findings, it is interesting to
note that under physiological conditions of high cytosolic
and mitochondrial Ca2⫹ signals, MAS activity is activated
in the liver but not in the heart. There are several reasons
that could account for this difference. The competition
for ␣-KG between ␣-KGDH and the OGC has been detected in heart but not in the liver. There is only one OGC
gene (283), with no known variants, so that the difference
between heart and liver is unlikely due to kinetic differences among isoforms. Tissue specificity could arise from
differences between OGC levels in liver and heart; however, the activity and expression levels of OGC in heart
are severalfold higher than in liver (171).
Another reason is related to microcompartmentation
and substrate channeling, which may differ in heart and
liver. In liver but not in heart mitochondria, flux along
␣-KGDH is higher when ␣-KG arises from glutamate by
transamination rather than being provided directly as a
substrate (219, 361). As indicated above, interaction between mitochondrial transaminase and ␣-KGDH with
preferential ␣-KG channeling between the two enzymes
explains why Ca2⫹ activates MAS through stimulation of
␣-KGDH in the liver. Experiments in intact mitochondria
and in reconstituted systems suggest that ␣-KGDH can be
bound to mitochondrial aspartate aminotransferase and
that mitochondrial malate dehydrogenase can have a
higher affinity for this binary complex than for any of the
two constituents alone (120). It has been argued that this
ternary complex could facilitate direct transfer of oxaloacetate to the transaminase, activation of malate dehydro-
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acetate levels brought about by the increase in the NADH/
NAD ratio (370). Studies of LaNoue’s group have shown
that there is a preferential channeling of external glutamate to mitochondrial transaminase and to the active site
of ␣-KGDH, suggesting microcompartmentation or channeling between ␣-KGDH and the mitochondrial transaminase (351, 361 but see Ref. 267), especially in liver. This
could explain the high sensitivity of liver AGC activity to
a decrease of mitochondrial ␣-KG levels caused by Ca2⫹induced activation of ␣-KGDH. However, to the best of
our knowledge, a demonstration of this mechanism with
the use of Ca2⫹ uniporter blockers was never reported.
It should be noted that experiments carried out by
Sugano’s group (155) in perfused rat liver indicated that
MAS activation by ␣-adrenergic agonists and vasopressin
is completely suppressed by calmodulin antagonists
(155), whereas agonist-evoked Ca2⫹ signaling was not
modified. This was interpreted as a direct effect of calmodulin on MAS activity. While the molecular basis for
this effect is still unknown, it would be reasonable to
propose a cytosolic site for calmodulin-dependent effects
of Ca2⫹, in contrast to the interpretations based on a
mitochondrial matrix Ca2⫹ effect on ␣-KGDH.
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SATRÚSTEGUI, PARDO, AND DEL ARCO
genase due to a decrease in the Km of the enzyme within
the complex, and direct transfer of ␣-KG to ␣-KGDH
(120). The formation or stability of this complex may be
less important in heart than in liver mitochondria, and this
may explain the lack of a clear activation of the AGC upon
Ca2⫹ stimulation of ␣-KGDH in heart.
3. Ca2⫹ regulation of citrin and aralar
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The mechanism described above for the activation of
liver MAS by Ca2⫹, i.e., activation of ␣-KGDH by Ca2⫹
uptake on the Ca2⫹ uniporter (CU-␣-KGDH), has long
been the accepted explanation of MAS activation by Ca2⫹
mobilizing agents (370, 371). The discovery of the AGCs
and the existence of a mechanism to activate AGC by
Ca2⫹ acting on the external face of mitochondria (95, 286)
therefore came as a surprise.
It is self-evident that if this Ca2⫹ activation mechanism of the AGC is physiologically relevant it would need
to operate under conditions where the mitochondrial
Ca2⫹ uniporter is not active. This question has been addressed recently in liver mitochondria that contain a single AGC isoform, citrin, and in brain and skeletal muscle
mitochondria, that also have a single AGC isoform, aralar.
Ca2⫹ activation of reconstituted MAS activity has been
studied in isolated rat and mouse mitochondria incubated
with ruthenium red (RR), under conditions preventing
Ca2⫹ uptake in mitochondria (287).
Liver ACG coupled to MAS activity was found to be
modestly (⬃1.5-fold) activated by extramitochondrial
Ca2⫹, with an S0.5 (free Ca2⫹ concentration for half-maximal activation) of ⬃120 nM, close to the resting [Ca2⫹]i
(79). On the other hand, activation by Ca2⫹ of brain and
skeletal muscle MAS activity was more pronounced (⬃3fold) with an S0.5 of 324 ⫾ 114 nM for brain mitochondria
(287), with no changes in the Km for glutamate (79). These
S0.5 values are substantially lower than the Km of the Ca2⫹
uniporter of mitochondria [⬃10 –20 ␮M, (143, 145, 190)],
suggesting that Ca2⫹ activation of citrin- and aralar-coupled MAS activity may contribute to NADH reduction in
mitochondria at Ca2⫹ concentrations where the Ca2⫹
uniporter is still inactive.
Unlike the CU-␣-KGDH mechanism, in which the
control on AGC activity is secondary to an increase in
matrix aspartate level, i.e., through mass action ratio,
Ca2⫹ activation of the AGC from the external face of the
inner membrane leads to an increase in AGC flux and
MAS activity at constant substrate concentrations, as expected for a classical regulatory reaction.
A) ROLE OF ARALAR AND THE MALATE-ASPARTATE NADH SHUTTLE
2⫹
IN TRANSDUCTION OF SMALL Ca
SIGNALS TO MITOCHONDRIA. The
role of aralar-MAS pathway in transduction of small Ca2⫹
signals to mitochondria has been studied in neurons.
Pardo et al. (287) have employed primary neuronal cultures from control and aralar-deficient mice and
NAD(P)H imaging with two-photon excitation microscopy to study MAS activity. Lactate utilization involves
NADH shuttle activity and is important in neurons, as part
of the astrocyte-neuron lactate shuttle (292). Lactate is
produced by astrocytes and taken up by neurons that use
it as an oxidizable substrate, especially during periods of
high activity (187, 293, 294). Addition of 20 mM lactate
caused an increase in cytosolic and mitochondrial
NAD(P)H and a substantial transfer of NAD(P)H to mitochondria in control, but not in aralar-deficient neurons
(287), indicating that MAS is the main NADH shuttle in
neurons. This was also confirmed by the marked increase
of the lactate-to-pyruvate ratio observed in the culture
medium of aralar-deficient neurons (Pardo and Satrústegui, unpublished observations).
To study the effect of small [Ca2⫹]i signals on MAS
activity, neurons were incubated in Ca2⫹-free medium
plus 100 ␮M EGTA and exposed to lactate together with
100 ␮M ATP or different Ca2⫹-mobilizing agonists (Fig. 6).
ATP-induced [Ca2⫹]i transients were small, with departures of ⱕ100 nM from resting values that lasted 1 min at
most and were not accompanied by any detectable increase in mitochondrial Ca2⫹ concentration ([Ca2⫹]mit)
measured with rhod 2, in control or aralar-deficient neurons. However, this small Ca2⫹ signal resulted in a remarkable potentiation of lactate-dependent increase in
mitochondrial NAD(P)H fluorescence in control neurons,
which was notably absent in aralar-deficient neurons.
Other Ca2⫹-mobilizing agonists, such as carbachol (50
␮M) or thapsigargin (1 ␮M), also potentiated in a significant way the response to lactate in control neurons (287).
Since lactate-derived pyruvate contributes to
NAD(P)H generation, Pardo et al. (287) also tested
whether the effects of ATP involve Ca2⫹-stimulated pyruvate metabolism rather than Ca2⫹-stimulated MAS activity. Remarkably, the small [Ca2⫹]i signals triggered by
ATP had no effect on the increase in mitochondrial
NAD(P)H induced by pyruvate (Fig. 6) (287). Therefore,
the results clearly showed that the neuronal aralar-MAS
pathway is selectively activated by small Ca2⫹ signals that
are below the threshold for Ca2⫹ uniporter activation.
Synaptosomal mitochondria start taking up Ca2⫹ at a
global [Ca2⫹]i of ⬃350 – 400 nM (241). This indicates that
Ca2⫹ signaling through the aralar-MAS pathway in neuronal mitochondria, which displays an S0.5 of 324 nM, would
be significant only below these Ca2⫹ concentrations,
which is exactly what Pardo et al. found. On the other
hand, the fact that mitochondria respond to [Ca2⫹]i signals with a substantial increase in NAD(P)H through a
Ca2⫹ uniporter-independent pathway was new and surprising, especially because the magnitude of the response
via the aralar-MAS pathway is very large in neurons, not
far from that evoked by the activity of the Ca2⫹ uniportermitochondrial dehydrogenases pathway (see sect. IIIC3B).
The availability of aralar-knockout animals will make it
41
MITOCHONDRIAL TRANSPORTERS
possible to assess the contribution of this pathway to
Ca2⫹ signaling in different tissues. These studies may
provide novel insights in Ca2⫹ signaling in mitochondria.
The role of the citrin-MAS pathway in transduction of
Ca2⫹ signals to liver mitochondria has not been investigated so far, but based on the very low S0.5 for Ca2⫹ of
citrin (79), it may be relevant at small departures from the
basal Ca2⫹ concentration, which are below the range of
the Ca2⫹ uniporter. Larger Ca2⫹ signal increases would be
relayed to the mitochondrial matrix, resulting in Ca2⫹
activation of ␣-KGDH, decrease in ␣-KG, and subsequent
stimulation of aspartate efflux from mitochondria (370,
371) and MAS activity. Indirect evidence that this may be
so is examined in section IIIE.
Physiol Rev • VOL
2⫹
B) EFFECTS OF LARGE Ca
SIGNALS ON THE ARALAR-MAS PATHPardo et al. (287) studied the increase in mitochondrial NAD(P)H in neurons in response to large [Ca2⫹]i
signals brought about by depolarization-dependent activation of voltage-operated Ca2⫹ channels (VOCC) in the
presence of millimolar external Ca2⫹ concentrations. In
addition to Ca2⫹-mediated MAS activation, which was
abolished in aralar-deficient neurons, it was expected that
VOCC-dependent Ca2⫹ entry would result in Ca2⫹ uptake
by mitochondria and subsequent dehydrogenase activation (110) in both control and aralar-deficient neurons.
There was no difference between the increases in
[Ca2⫹]i and [Ca2⫹]mit obtained after lactate plus KCl addition in control and aralar-deficient neurons, in agreeWAY.
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2⫹
FIG. 6. Effect of small Ca
signals on aralar-malate-aspartate NADH shuttle activity. A: [Ca2⫹]i imaging of fura 2-loaded cortical neuronal
cultures from control (Ctr) and aralar-deficient (KO) mice, exposed to lactate and malate (LM) ⫹ ATP in Ca2⫹-free HCSS in the presence of 100 ␮M
EGTA. LM ⫹ ATP were added at the arrows. Each trace represents the [Ca2⫹]i from a single neuron. B: mitochondrial Ca2⫹ dynamics of the
responses to LM ⫹ ATP in rhod 2-loaded cortical neuronal cultures from control (Ctr) and aralar-deficient (KO) mice. C: NAD(P)H fluorescence
images corresponding to control (Ctr) and aralar-deficient (KO) neurons, before (Ctr-ATP, KO-ATP) or 80 s after LM ⫹ ATP addition (Ctr ⫹ ATP,
KO ⫹ ATP). Bar represents 20 ␮m. D and E: time course of changes in mitochondrial/cytosolic NAD(P)H fluorescence ratios in neuronal cultures
from control (D) and aralar-deficient neurons (E). F: changes in mitochondrial/cytosolic NAD(P)H fluorescence ratio in control neurons after the
addition of 2 mM pyruvate ⫹ 5 mM malate (Pyr 2) or Pyr 2 ⫹ 100 ␮M ATP (Pyr 2 ⫹ ATP), 10 mM pyruvate ⫹ 5 mM malate (Pyr 10), or Pyr 10 ⫹
100 ␮M ATP (Pyr 10 ⫹ ATP), in Ca2⫹-free HCSS in the presence of 100 ␮M EGTA. The arrow indicates the time of addition. [From Pardo et al. (287).]
42
SATRÚSTEGUI, PARDO, AND DEL ARCO
2⫹
FIG. 7. The activity of the Ca
uniporter prevents full Ca2⫹ activation of malate-aspartate NADH shuttle (MAS) activity in brain mitochondria at high Ca2⫹ concentrations. MAS activity (A) and Ca2⫹ uptake
(B) in mouse brain mitochondrial fractions measured at different free
Ca2⫹ concentrations in the presence (open bars) or absence (closed
bars) of 200 nM ruthenium red (RR). B: serial additions of 20 ␮M Ca2⫹
at 5-min intervals. In trace a, 200 nM RR was added at the arrow. In trace
b, no RR was added. In trace c, RR was present from the beginning.
[From Pardo et al. (287).]
Physiol Rev • VOL
incubated with different fuel secretagogues that bypass
glucose metabolism, each of them setting a maximal hyperpolarization of the mitochondrial membrane that correlated with insulin secretion (7).
The results obtained in neurons suggest that, as in
heart, large Ca2⫹ signals generate an increase in NAD(P)H
arising mainly from the activation of tricarboxylic acid
cycle dehydrogenases rather than from MAS activity. The
inhibition of MAS probably disappears when the activation by Ca2⫹ of mitochondrial dehydrogenases comes to
an end, and ␣-KG levels and mitochondrial proton electrochemical gradient return to control values. Thus it is
possible that after the decay of [Ca2⫹]mit transients, MAS
activity could prolong the increase in mitochondrial
NAD(P)H induced by high [Ca2⫹]i signals, and thus contribute to ATP synthesis and recovery of the resting state,
conditions that rely on neuronal lactate utilization (293).
D. Other Mechanisms of Ca2ⴙ Regulation
of NAD(P)H Transfer to Mitochondria
Independent of Ca2ⴙ Entry
1. Mitochondrial glycerol-3-phosphate dehydrogenases
The mitochondrial glycerol-3-phosphate dehydrogenases (FAD-GPDH), together with cytoplasmic NADlinked GPDH, catalyze the reactions of the GPS. FADGPDH is present at high levels in tissues capable of rapid
oxidative metabolism of glucose and is thought to be
preferentially used over other shuttle systems when glucose is used as the carbon source, since glycerol-3-phosphate arises directly from the glycolytic substrate dihydroxyacetone phosphate. FAD-GPDH enzymes from insect flight muscle (158), lung (125, 126), liver (414), brown
adipose tissue and brain (36), and beta-cell mitochondria
(329) are stimulated by Ca2⫹ (Fig. 8). Ca2⫹ activation in all
cases results in a decrease in the Km for glycerol-3-phosphate with no changes in the apparent Vmax. Half-maximal
activation occurs at ⬃100 nM free Ca2⫹, i.e., at around
resting Ca2⫹ concentration (174, 329), and similar values
were obtained for glycerol-3-phosphate-induced ATP production (174) or respiration (75) in mitochondria from
pancreatic ␤-islets. Inhibition of mitochondrial Ca2⫹ uptake with RR did not interfere with the stimulation in
blowfly flight muscle mitochondria (66), indicating that
Ca2⫹ exerts its effects on the extramitochondrial side of
the inner mitochondrial membrane. Since the apparent
Km of liver FAD-GPDH, 4 –5 mM (414), is far higher than
the concentration of glycerol-3-phosphate in liver of
fasted rats, ⬃0.1 mM (308), the Ca2⫹-dependent increase
in affinity will result in an increased glycerol-3-phosphate
oxidation in the liver.
Activation by Ca2⫹ of FAD-GPDH is hardly modified
in the presence of calmodulin and calmodulin inhibitors
(234, 414), suggesting that the enzyme reacts directly with
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ment with the lack of effect of aralar overexpression on
Ca2⫹ homeostatic mechanisms reported earlier (222). The
increase in the mit/cyt NAD(P)H fluorescence ratio obtained in the presence of KCl was significantly larger than
that obtained by lactate addition itself, but in this case,
there were no differences between control and aralardeficient neurons (287). As the increase in mitochondrial
NAD(P)H obtained with high K⫹ was expected to result
from the additive effects of Ca2⫹ activation on MAS activity and Ca2⫹ activation of mitochondrial dehydrogenases, the lack of difference between wild-type and
aralar-deficient neurons indicated that MAS activity contributes very little to the increase in mitochondrial
NAD(P)H under high-K⫹ depolarization, suggesting that
MAS activity may be inhibited under conditions allowing
mitochondrial Ca2⫹ uptake.
Studies of MAS activity in isolated mitochondria in
the presence or absence of 200 nM RR showed that Ca2⫹
activation of MAS was diminished if no RR was present
(Fig. 7). This concentration of RR inhibits mitochondrial
Ca2⫹ uptake completely (Fig. 7). Thus, in neurons, Ca2⫹
activation of MAS is blunted when Ca2⫹ is allowed to
enter the mitochondria (287). This is possibly due to the
effect of mitochondrial Ca2⫹ on the affinity for ␣-KG of
␣-KGDH and the competition for ␣-KG between ␣-KGDH
and the OGC (Fig. 4), as described earlier for the heart
MAS activity (see sect. IIIC2). Another factor that may
blunt MAS activation when all mitochondrial dehydrogenases are activated by Ca2⫹ is the thermodynamic constraint of proton gradient formation. If the proton electrochemical gradient obtained under those conditions is
close to maximal and cannot be increased, this would
limit the entry of reducing equivalents into mitochondria
through MAS. This situation has been described in ␤-cells
MITOCHONDRIAL TRANSPORTERS
8. Other Ca2⫹-dependent systems to transfer reducing equivalents to mitochondria that do not require the activity of the Ca2⫹
uniporter. FAD-GPDH, mitochondrial glycerol phosphate dehydrogenase; NDE1, Ca2⫹-dependent external NAD(P)H dehydrogenase from
Neurospora; ndb1, plant Ca2⫹-dependent external NAD(P)H dehydrogenase.
FIG.
Physiol Rev • VOL
the glucose-evoked rhythmical plasma membrane potential activity (338), and with fluctuations in the ATP/ADP
ratio, islet respiration, and insulin secretion (189).
Ca2⫹ entry mitochondria via the Ca2⫹ uniporter are
critical to maintain cytosolic Ca2⫹ oscillations in ␤-cells
(189), and although GPS could also participate in the
maintenance of the oscillatory behavior, studies with
mice deficient in FAD-GPDH have shown that this is not
the case. In these studies, the oscillatory [Ca2⫹]i response
and the fluctuations in plasma membrane potential with
bursts of action potentials in response to glucose were
unchanged by FAD-GPDH deficiency. Moreover, an imposed and controlled rise in [Ca2⫹]i in ␤-cells similarly
increased NAD(P)H fluorescence in control and GPDHdeficient islets (307). These results have led to the conclusion that a possible cyclic activation of the enzyme by
Ca2⫹ is not required for the oscillatory behavior (307).
However, because MAS and GPS shuttles play partly redundant roles, it is still possible that a cyclic, Ca2⫹-dependent activation of GPDH could be unmasked under conditions where MAS is inactive. Alternatively, it has been
argued that the high affinity for Ca2⫹ of GPDH causes a
constitutive activation of GPS (307).
2. External NAD(P)H dehydrogenases in fungi
and plants
Mitochondria from fungi and plants contain non-proton-pumping NAD(P)H dehydrogenases as alternative
systems to feed electrons from NAD(P)H to the respiratory chain in a rotenone-insensitive manner. These enzymes are the only system to feed electrons from
NAD(P)H to ubiquinone in those organisms, such as S.
cerevisiae, that lack complex I. Unlike complex I, nonproton-pumping NAD(P)H dehydrogenases are single
polypeptide chains that oxidize NAD(P)H in the cytosol
(external enzymes) or mitochondrial matrix (internal enzymes), and pass the electrons to FMN or FAD in the
mitochondrial membrane (67).
Plants contain four alternative NADH dehydrogenases, and S. cerevisiae has one internal and two external
enzymes, which oxidize matrix or extramitochondrial
NADH, respectively. The filamentous fungus Neurospora
crassa has complex I and three or four alternative
NAD(P)H dehydrogenases, a situation similar to that of
plants (67, 257). Two of these dehydrogenases are external, NDE1 and NDE2, and NDE1 was shown to be Ca2⫹
dependent (251). NDE1 is anchored to the inner mitochondrial membrane by an amino-terminal domain, and
its catalytic site faces the intermembrane space (Fig. 8). It
contains an insertion that harbors an EF-hand motif (252).
NDE1 is thought to be mainly involved in the oxidation of
cytosolic NADPH and is absolutely dependent on Ca2⫹ at
physiological pH (251). Ca2⫹-dependent NAD(P)H dehydrogenases exist in the genome of filamentous fungi As-
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Ca2⫹. In fact, the protein sequence of FAD-GPDH from
mammalian sources (58, 234) shows two EF-hand motifs
close to the carboxy terminus of the enzyme, a region
facing the intermembrane space. These putative Ca2⫹
binding domains are absent in the enzyme from S. cerevisiae (273, 321), which is not known to be Ca2⫹ sensitive.
EF-hand motifs are also absent in the FAD-GPDH proteins
from Candida albicans, Aspergillus nidulans, and Neurospora crassa databases. The mammalian enzyme shows
Ca2⫹ binding in 45Ca2⫹ overlay experiments, suggesting
that the EF-hand motifs bind Ca2⫹ and are responsible for
Ca2⫹ activation (234). A mutation in the Ca2⫹-binding
domain associated with a reduced Ca2⫹ activation was
found in a family of diabetic subjects (277). These results
suggest but do not prove that the carboxy-terminal Ca2⫹binding domain of FAD-GPDH is responsible for Ca2⫹
activation. The sequence of the Ca2⫹-regulated blowfly
FAD-GPDH (158) is still unknown, but it is interesting to
note that a Drosophila FAD-GPDH sequence with 62%
identity with respect to that of the human enzyme
(NP_611063, CG8256-PC, isoform C) also harbors two
EF-hand motifs in conserved positions.
Pancreatic ␤-cells have a highly active glycerol-3phosphate shuttle. NADH shuttles are important in glucose-stimulated insulin secretion, as glucose metabolism
in ␤-cells occurs essentially through aerobic metabolism,
and lactate dehydrogenase levels are extremely low (354).
By using aminooxyacetate to inhibit malate-aspartate
NADH shuttle function in mice that also have targeted
disruption in FAD-GPDH, glucose-induced insulin secretion was abrogated (119). Whereas the metabolic role of
the enzyme is clearly established, the actual role of Ca2⫹
regulation of FAD-GPDH is not yet known.
In ␤-cells, stimulation of insulin secretion by glucose
involves a rise in [Ca2⫹]i. This rise requires mitochondrial
metabolism to increase the ATP/ADP ratio and results in
the closure of ATP-sensitive K⫹ channels, membrane depolarization, and influx of Ca2⫹ through voltage-sensitive
channels (for review, see Ref. 189). The resulting increase
in cytosolic Ca2⫹ displays oscillations that correlate with
43
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SATRÚSTEGUI, PARDO, AND DEL ARCO
E. Interplay Between AGCs, GPS, and the Ca2ⴙ
Uniporter in Regulation of Mitochondrial
Metabolism in Response to Cytosolic
Ca2ⴙ Signals
Ca2⫹ signals in excitable and nonexcitable tissues
are made up of single or periodic fluctuations in cytosolic
Ca2⫹ concentrations. These are driven by electrical activation or by stimulation by hormones or paracrine agents
that evoke Ca2⫹ release from intracellular stores mainly
through the operation of inositol trisphosphate (IP3) and
ryanodine (Ry) receptors (see for reviews, see Refs. 40 –
42, 314).
Local Ca2⫹ signals are produced spontaneously or by
low levels of a locally applied stimulus and involve either
IP3Rs or RyRs together with different types of plasma
membrane channels. The elementary Ca2⫹ signal events
generated this way are thought to reflect the coordinate
opening of clusters of channels and have been termed
Physiol Rev • VOL
puffs, sparks, or QEDs depending on whether the channels are IP3Rs, RyRs, or voltage-operated channels, respectively (40, 42). These local signals are very brief Ca2⫹
pulses that do not extend spatially to the rest of the cell (42).
At higher level of stimulation, the Ca2⫹ transients last
longer and the resulting signal usually spreads out as a
Ca2⫹ wave to reach targets that are distributed throughout the cell. For the waves to occur, most of the IP3Rs or
RyRs must respond to each other through the process of
Ca2⫹-induced Ca2⫹ release (40, 42).
Hormones and agonists acting through receptors
coupled to phospholipase C (PLC) generate periodic Ca2⫹
spikes or oscillations whose amplitude is not affected by
the agonist dose, which rather modulates the spiking
frequency (314, 322, 421). This phenomenon described as
frequency-modulated Ca2⫹ signaling (384) was first found
in nonexcitable cells, particularly in hepatocytes (322,
421), exocrine pancreas (385), smooth muscle, and egg
cells from animals and plants, where fertilization elicits
single or repetitive Ca2⫹ transients in response to fertilizing sperm (40, 113). Once initiated at a particular subcellular locus, Ca2⫹ waves propagate in the cell at a fixed
velocity, regardless of the agonist dose. Because Ca2⫹ has
a limited diffusion coefficient in the cytosol, particularly
in cells with high levels of Ca2⫹-binding proteins, it is
thought that Ca2⫹ waves may provide a mechanism to
deliver localized Ca2⫹ release to distal parts of the cell
(42, 384).
Ca2⫹ waves arising from any of these mechanisms
can reach the whole cell and can even spread from cell to
cell through intercellular waves. However, in common
with the local Ca2⫹ signals, every single wave or oscillation is a brief pulse of Ca2⫹, arising from the balance of
reactions that introduce Ca2⫹ in the cytosol and those
that remove it. It has been suggested that many Ca2⫹sensitive processes can be regulated more accurately by
low-frequency Ca2⫹ oscillations than by an equivalent
time-averaged Ca2⫹ signal, which would yield a small
elevation of Ca2⫹ above basal levels.
Spatial contiguity between the site where the Ca2⫹
signal is generated and downstream effectors is critical
for rapid responses to global Ca2⫹ signals and especially
to respond to localized Ca2⫹ signals. This is especially
important for mitochondria, where the main mechanisms
whereby Ca2⫹ signals are transmitted are the Ca2⫹
uniporter and the RAM (for rapid uptake mode) (61, 144,
365). The Ca2⫹ uniporter has a low affinity for Ca2⫹, with
a K0.5 of 5–10 ␮M (145, 190), and is allosterically activated
by Ca2⫹ (206 –208), while the RAM has a slightly higher
affinity (145). The fact that there is a substantial mitochondrial Ca2⫹ uptake despite modest changes in peak
[Ca2⫹]i is explained by the strategic location of mitochondria close to endoplasmic reticulum (ER) Ca2⫹ release
channels, or plasma membrane Ca2⫹ channels, and therefore to microdomains of very high [Ca2⫹]i which escape
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pergillus nidulans (EAL87637 and EAA62080), and Magnaporte grisea (EAA50381), and even in Yarrowia lipolytica (XP_503592), an alkane-utilizing yeast that shares
several properties with filamentous fungi, but not in the
budding yeast S. cerevisiae or Schizosaccharomyces
pombe.
Non-proton-pumping, Ca2⫹-dependent NAD(P)H dehydrogenases are also found in plant mitochondria (257,
258). The plant enzymes are similar to fungal external and
internal NAD(P)H dehydrogenases and fall in the nda,
ndb, and ndc families (253, 254, 256, 258). The ndb family
is characterized by the presence of an insertion carrying
an EF-hand motif roughly in the same position of N.
crassa NDE1, and potato ndb1 has been shown to correspond to an external Ca2⫹-sensitive NADPH dehydrogenase (253). The external NADPH dehydrogenases of plant
mitochondria are activated by cytosolic Ca2⫹ with an S0.5
of ⬃0.3 ␮M or higher (257, 271, 306).
The kinetics of Ca2⫹ activation of NADPH oxidation
in N. crassa mitochondria have not been directly assessed, but it is possible that these external, Ca2⫹-dependent NAD(P)H dehydrogenases provide a pathway to oxidize cytosolic NADPH under conditions where cytosolic
Ca2⫹ levels increase. In the case of the plant enzymes, it
is believed that external NADPH dehydrogenases will be
inactive under normal resting conditions when the cytosolic Ca2⫹ concentration is ⬃100 nM. However, when the
plant is stressed, an increase in [Ca2⫹]i to 1–2 ␮M contributes to its activation (257). Since a high-reduction
level of the respiratory chain leads to reactive oxygen
species (ROS) production, it has been proposed that activation of external NADPH dehydrogenase may help minimize ROS production under stress conditions by keeping
the cytosolic NADP pool relatively oxidized (257).
MITOCHONDRIAL TRANSPORTERS
Physiol Rev • VOL
a mitochondrial decoding toolkit whereby each Ca2⫹ signal translates into a physiological response of mitochondria: an increase in substrate supply (NADH or FADH2) to
the electron transport chain. The decoding devices are all
different in their Ca2⫹ sensitivity. The AGCs and FADGPDH are activated by Ca2⫹ at concentrations below
those where the Ca2⫹ uniporter operates. For unitary
sparks, puffs, QEDs, or other brief and highly localized
Ca2⫹ signals, AGCs and FAD-GPDH could respond both
earlier than the Ca2⫹ uniporter and after the Ca2⫹ mark
has ended, and even under conditions where the Ca2⫹
uniporter does not respond. In this way, AGCs may prime
mitochondria to respond to Ca2⫹ entry by prolonging
mitochondrial energization beyond the duration of the
individual Ca2⫹ mark through an increase of the reduction state of mitochondrial NADH and a decrease of that
of cytosolic NADH. This allows for a localized increase in
glycolysis and pyruvate supply to mitochondria needed to
respond to the Ca2⫹ activation of mitochondrial dehydrogenases.
In the case of global Ca2⫹ signals of low intensity, the
present data suggest that AGCs and FAD-GPDH will operate as only Ca2⫹ signaling devices in mitochondria at
the very start and at the end of a single transient, or at the
front and tail of every Ca2⫹ wave, and may cooperate with
the Ca2⫹ uniporter-mitochondrial DH (CU-mitDH) mechanism to elicit a full mitochondrial response to Ca2⫹ along
the duration of the Ca2⫹ transient or wave. This is the
situation in liver in response to ␣-adrenergic agonists and
glucagon in which citrin, the Ca2⫹ uniporter, and the liver
ATP-Mg/Pi carrier (see sect. IVB), are all activated, resulting in increases in MAS, gluconeogenesis, and urea synthesis. Whether the Ca2⫹ activation of citrin or aralar
actually persists throughout the duration of the Ca2⫹
signal is unknown, but at least in the case of the aralarMAS pathway in heart and neurons there is evidence that
this may not be so. In these two cases, Ca2⫹ activation of
the aralar-MAS pathway is blunted when Ca2⫹ activation
of the mitochondrial dehydrogenases occurs so that the
emerging scenario is one in which Ca2⫹ signaling
switches from aralar-MAS to uniporter-mitochondrial DH
and back to aralar-MAS as the global Ca2⫹ signal is initiated, reaches a maximum, and ends. The recruitment of
the GPS which is not known to be inhibited at high Ca2⫹
concentrations could compensate for the inhibition of
MAS at high Ca2⫹ concentrations. The combined or sequential activation of the AGC-MAS and CU-MitDH pathways may explain observations in hippocampal neurons
(185, 186), hepatocytes (149, 301, 313), ␤-cells and adrenal
glomerulosa cells (301), and pancreatic acinar cells (407)
in which the responses to single or low-frequency cytosolic Ca2⫹ spikes evoke transient increases in mitochondrial NAD(P)H that outlast the duration of the mitochondrial and cytosolic Ca2⫹ spikes. The longer duration of the
NADH transients compared with the duration of the Ca2⫹
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detection by present imaging methods (44, 124, 133, 260,
311). These microdomains of high [Ca2⫹]i may be detected by an aequorin chimera targeted to the mitochondrial intermembrane space, where it is exposed to local
high [Ca2⫹]i (295). The access of IP3-linked Ca2⫹ spikes,
but not global Ca2⫹ signals, to the intermembrane space is
limited by the permeability of outer mitochondrial membrane (305, 83). This barrier may be overcome by increasing voltage-dependent anion channels or tBid levels (83,
305), resulting in increased delivery of IP3-linked Ca2⫹
spikes and oscillations to mitochondria. However, it has
been conclusively demonstrated that mitochondria from
rat luteal cells, insulin-producing cell lines, rat adrenal
glomerulosa cells, and osteosarcoma cells take up Ca2⫹ at
low submicromolar [Ca2⫹]i, suggesting that under appropriate conditions the Ca2⫹ uniporter may operate at modest departures from resting [Ca2⫹]i (296, 367, 375). Recent
findings indicate that the capacity for Ca2⫹ uptake in
mitochondria is also influenced by mitochondrial morphology (with lower capacity when the mitochondrial
network is fragmented) and kinase activity (261; reviewed
in Ref. 44). Once the cytosolic Ca2⫹ has returned to
resting levels, Ca2⫹ is also pumped out of mitochondria to
the cytosol on the Na⫹/Ca2⫹ exchanger or the more sluggish H⫹/Ca2⫹ transporter (143, 145). Efflux through a
possible low-conductance mode of the permeability transition pore (PTP) has also been suggested by some studies (172, 173) but not by others (376). Thus mitochondria
participate in Ca2⫹ buffering through multiple mechanisms (see Refs. 310, 312 for review).
The mitochondrial matrix Ca2⫹ spikes that result
from the coordinated function of uptake and efflux pathways may be the same all through the cell, but heterogeneous Ca2⫹ signals in the mitochondrial population may
occur as a result of heterogeneity of the driving force for
Ca2⫹ uptake, of the spacing between mitochondria and
Ca2⫹ release sites, and of the cytosolic Ca2⫹ signal delivered in each site (148). This heterogeneity has been directly visualized in fluorescence imaging studies at the
level of individual mitochondria, in response to global
Ca2⫹ signals in pancreatic acinar cells (288) and primary
cultured aortic myocytes (259) or to local sparks in H9c2
cardiac cells (282). The term Ca2⫹ marks has been coined
to design miniature increases in mitochondrial Ca2⫹ in
response to these elementary release events (148, 282).
The mean duration of Ca2⫹ marks in H9c2 cardiac cells
was longer than that of the Ca2⫹ sparks that evoked them,
in agreement with the slow Ca2⫹ extrusion through the
Na⫹/Ca2⫹ exchanger (282). These studies have led to the
conclusion that not all Ca2⫹ sparks in cardiac cells (282)
nor Ca2⫹ puffs in HeLa cells (77) elicit a response in
mitochondria.
This is the scenario in which the role of AGCs as
Ca2⫹ signaling mechanisms becomes apparent. The AGCs
together with FAD-GPDH and the Ca2⫹ uniporter make up
45
46
SATRÚSTEGUI, PARDO, AND DEL ARCO
Physiol Rev • VOL
[Ca2⫹]i that drives the increase in the work load (31, 315)
and may activate Ca2⫹-regulated mitochondrial dehydrogenases resulting in relatively slower tonic increases in
NADH (51). In intact heart muscle, an increase in work
load causes an initial decrease in mitochondrial NADH
levels, followed by a slow recovery towards control values (51). This recovery of NADH levels is Ca2⫹ dependent
(50). As mitochondrial Ca2⫹ beat-to-beat spikes might not
return to basal levels at high work loads (315), Ca2⫹
activation of mitochondrial dehydrogenases persists between spikes and has been considered responsible for the
recovery of NADH levels (50, 51). If glucose or lactate is
used as fuel, any increase in mitochondrial activity in
cardiac cells involves the AGC (aralar or citrin)-MAS pathway. However, the persistent presence of Ca2⫹ in mitochondria and of Ca2⫹ activation of mitochondrial dehydrogenases may impose a constraint on the operation of
the aralar-MAS pathway eventually leading to its inhibition at high work loads (279).
IV. ATP-Mg/Pi CARRIERS, SCaMC GENES,
AND PROTEINS
A. Mammalian SCaMCs
The ATP-Mg/Pi carriers were first identified as
CaMCs with short amino-terminal extensions, or short
CaMCs (SCaMCs), and are represented by three main
isoforms, SCaMC 1–3 encoded, respectively, by
SLC25A24, SLC25A25, and SLC25A23 (33, 91, 94, 122),
with a number of variants arising from alternative splicing. Like human AGC genes, SCaMCs share exon-intron
structure. SCaMC-1 maps at chromosome 1p13.3 and
SCaMC-2 and SCaMC-3 genes at positions 9q34.13 and
19p13.3, respectively (33, 94).
1. Extended diversity of mammalian SCaMCs
Mammalian SCaMCs are one of most complex subsets of MCs. All code for highly conserved proteins (70 –
80% identity), of ⬃500 amino acids, with an amino-terminal extension harboring EF-hand Ca2⫹-binding motifs homologous to calmodulin (94). Mammalian orthologs for
each SCaMC isoform are also highly conserved (96).
Human SCaMCs present amino- and carboxy-terminal variants generated by alternative splicing (33, 91, 94,
122) (Fig. 9). Two SCaMC-1 amino-terminal variants, of
477 and 458 amino acids, are generated from an alternative first exon (94, 122). The 477-amino acid-long
SCaMC-1 variant is ortholog to Efinal, a protein from
rabbit small intestine reported to be present both in peroxisomes and mitochondria (413). A more complex pattern of amino-end variants are found for human SCaMC-2
(94). Three SCaMC-2 products have been characterized
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transients is thought to have the advantage of prolonging
enhanced energy production for some time after the termination of the Ca2⫹ signal so that all of the energy
demands for the restoration of the steady state disturbed
by the Ca2⫹ signal may be met (407). A differential recruitment of the AGC-MAS pathway by specific agonists
(for example, by stimulating glycolytic flow) may explain
findings in rat luteal cells where agonists such as PGF2␣
and ATP (acting at metabotropic P2 receptors) induce
higher and longer lasting increases in mitochondrial
NADPH than others, in spite of similar transmission of the
Ca2⫹ signal to mitochondria (291). In pancreatic ␤-cells
(1) and RBL-2H3 mast cells (84) undergoing sequential
cytosolic Ca2⫹ transients, mitochondria respond to a second cytosolic Ca2⫹ transient with a larger increase in
mitochondrial Ca2⫹ than the first. Whether mitochondrial
priming through the AGC-MAS pathway could be involved
in the facilitation effect of the first Ca2⫹ transient is an
open question.
The role of the AGG-MAS pathway in response to
high-intensity global signals is still unknown. The initial
response to high-frequency stimulation in the CA1 area of
the hippocampus is a global NAD(P)H dip caused by rapid
stimulation of NADH oxidation by the respiratory chain in
the postsynaptic neuron (187, 356) as also found in other
neuronal types (110, 162). This initial response is attributed to the feedback regulation of oxidative phosphorylation in response to the fall of the ATP/ADP ratio and the
increase in cytosolic and mitochondrial Ca2⫹, and modeling evidence indicates that it is due to increased uptake
of extracellular lactate by neurons (25). Subsequently,
this is followed by an increase in lactate production in
astrocytes, which is accompanied by an overshoot of glial
NADH levels which fuels a gradual recovery of neuronal
NADH levels as a result of the activation of tricarboxylic
acid cycle dehydrogenases (187). Both phases of the neuronal NAD(P)H response require the MAS-aralar pathway
to utilize lactate, and the global sustained increase in
NAD(P)H that follows the initial dip is inhibited in the
presence of inhibitors of the neuronal monocarboxylate
transporter MCT2 (131). However, most of the sustained
increase in NADPH upon high-frequency stimulation
probably occurs at high Ca2⫹ levels, when the CU-mitDH
pathway is highly activated and where Ca2⫹ activation of
the aralar-MAS pathway is blunted (287). Thus the actual
role of the Ca2⫹ activation of the aralar-MAS pathway in
this situation is unknown and awaits investigation.
Studies with mitochondrial and cytosolic expressed
aequorin in heart cells have shown that overall myocardial work correlates with increases or decreases in beatto-beat frequency and/or amplitude of cytosolic and mitochondrial Ca2⫹ transients (315), and with sustained
changes in mitochondrial Ca2⫹ levels between oscillations, i.e., “diastolic” increases or decreases in mitochondrial Ca2⫹ (51, 315). Mitochondrial matrix Ca2⫹ tracks the
MITOCHONDRIAL TRANSPORTERS
9. Domain structure and distribution of human SCaMCs and yeast Sal1p.
SCaMC-2a, -2b, and -2c of 469, 503, and 488 amino acids,
respectively, with amino-terminal ends encoded by three
different exons 1 (Fig. 9). As a result, SCaMC-2a and
SCaMC-2c lack the first EF-hand motif, but SCaMC-2b has
all four (Fig. 9). Other SCaMC-2 variants, SCaMC-2d, arise
from the use of an alternative 5⬘-splicing donor site in the
most proximal exon 1, which causes a frame shift. This
transcript is still able to generate two shortened proteins
of 366 and 344 amino acids using internal ATGs. Both
SCaMC-2d proteins lack EF-domains 1, 2, and 3 but maintain the whole carboxy-terminal end (Fig. 9) (94). SCaMC2a, -2b, and -2c specific sequences have also been detected in other mammalian species, and a rat SCaMC-2a
ortholog with predominant expression in liver and skeletal muscle has been identified (243).
The main product of SCaMC-3 is SCaMC-3a, of 468
amino acids in length and four EF-hand motifs (91, 94)
(Fig. 9). Three carboxy-end variants, SCaMC-3b, -3b⬘, and
-3c/3d, of 422, 456, and 435 amino acids, respectively, have
been described (33, 91). These shortened proteins are
encoded by alternatively spliced transcripts that bear 3⬘end sequences derived from repetitive transposable elements, Alu and a MaLR retrotransposon, instead of sequences derived from the last SCaMC-3 exon (33, 91). All
3⬘-spliced sequences introduce premature stop signals
into the SCaMC-3 transcripts. These carboxy-end variants
lack the last transmembrane domain (TM6), but that does
not change their location in mitochondria (33, 91).
Physiol Rev • VOL
The available data about SCaMCs expression and
tissue distribution are far from being conclusive (33, 91,
94, 122, 243, 413). Most SCaMC isoforms show overlapping expression patterns with no clear tissue specificity.
SCaMC-1, -2, and -3 transcripts are found in several
tissues such as skeletal muscle, brain, kidney, and pancreas and also in ESTs sequences derived from eye (417).
Some isoforms appear to have a prominent expression in
specific tissues. Thus a high level of SCaMC-1 expression
is found in small intestine from rabbit (413) and a high
SCaMC-2 expression is detected in skeletal muscle and
liver (94, 243). In pancreatic rat AR2J cells, SCaMC-2a has
been found to be upregulated by dexamethasone (243).
On the other hand, human SCaMC-1 and SCaMC-2
amino-variants, SCaMC-1a, SCaMC-2b, and SCaMC-2c
(Fig. 9), show a more restricted distribution than
SCaMC-1 and SCaMC-2a, the variants of each gene most
widely expressed. Thus SCaMC-1a variant is exclusively
expressed in high levels in testis (122). A brain-specific
pattern is observed for SCaMC-2c isoform (94). SCaMC2b, the only SCaMC-2 protein containing four EF-hand
domains, is detected at low levels in kidney, brain, and
lung.
2. Targeting of ATP-Mg/Pi precursor proteins
to mitochondria
The mechanisms of mitochondrial import of CaMC
precursor proteins have been discussed in section IIIA2
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FIG.
47
48
SATRÚSTEGUI, PARDO, AND DEL ARCO
3. Transport activity of mammalian
ATP-Mg/Pi carriers
A) NET INFLUX AND EFFLUX OF ADENINE NUCLEOTIDES IN MITOCHONDRIA.
The exchange of cytosolic ADP against mitochondrial ATP is essential to maintain oxidative phosphorylation and is carried out by two or more adenine
nucleotide translocases present in all eukaryotes (96, 105,
283). The translocase can also work in the “reverse”
mode, i.e., exchanging cytosolic ATP against mitochondrial ADP. In the reverse mode, the exchange of ATP4⫺ to
ADP3⫺ is electrogenic, and it is thought to generate the
mitochondrial membrane potential in yeast growing in
anaerobiosis and in mitochondrial DNA (mtDNA) depleted cells (8, 60, 76, 223, 230). An upregulation of a
chloride channel may also be involved in the generation of
mitochondrial membrane potential in rho° cells (20). The
reverse mode of the translocase can proceed as long as
ATP is hydrolyzed in mitochondria by the F1F0-ATPase.
Because proton pumping, which requires mtDNA encoded subunits of F1F0-ATPase, is not needed, this process operates in mtDNA depleted cells (8). The reverse
mode of the adenine nucleotide translocase is also required during heart ischemia, to maintain a partially polarized mitochondrial membrane potential generated by
proton pumping through the F1F0-ATPase at the expense
of ATP hydrolysis (2, 111, 325).
It should be emphasized that the one-to-one exchange reaction catalyzed by the ANT does not result in
net increase or decrease in adenine nucleotide (AdN)
content in mitochondria. Although the translocase can
also exchange AdNs against pyrophosphate, the affinity of
the translocase for pyrophosphate is low compared with
that of AdNs (200), and it is therefore doubtful that this
exchange operates in vivo, at least under resting condiPhysiol Rev • VOL
tions, i.e., with normal pyrophosphate and AdN levels (13,
247). Thus it is generally believed that the ATP-Mg/Pi
carrier found more than 20 years ago and only recently
found to be encoded by the newly discovered SCaMC 1–3
genes is the only transporter responsible for net changes
in AdN levels.
B) TRANSPORT ACTIVITY OF THE ATP-Mg/Pi CARRIER. In the late
1970s, Sutton and Pollak (374) found that during rat neonatal development the AdN content of hepatic mitochondria increased more than twofold within the first 2 h after
birth (374). The mechanism responsible for this rapid
accumulation was found to be a saturable, atractylosideresistant, and mersalyl-sensitive ATP transport (14, 26,
299, 300). ATP was the preferred substrate, and transport
was inhibited by dATP, ADP, and AMP, but not by cAMP,
adenosine 5⬘-[␤␥-imido]triphosphate (p[NH]ppA), GTP,
or CTP. ATP transport occurred in mitochondria incubated without added substrates but was inhibited by
CCCP and KCN, indicating that the proton-motive force
was required (300).
Studies carried out by Aprille (10, 13) established
that the carrier present in liver mitochondria catalyzes an
exchange of ATP-Mg for Pi across the inner mitochondrial
membrane in either direction. To study the activity of the
carrier, the AdN content of liver mitochondria is first
decreased by in vitro incubation with pyrophosphate that
enters mitochondria on the adenine nucleotide translocase in exchange with ATP or ADP, causing a net loss of
AdN. Alternatively, matrix AdN can be depleted in preparation for studies of transport by incubating with Pi
which catalyzes net flux over the ATP-Mg/Pi carrier itself.
The matrix AdN content can then be restored by incubation of liver mitochondria with ATP, Mg2⫹, and phosphate
(21). At equilibrium, the relationship between the concentration gradients of ATP-Mg and Pi across the inner mitochondrial membrane implies that, like Pi, ATP-Mg is taken
up and retained in mitochondria against a concentration
gradient relative to the cytosol. The pH gradient is the
energy source for Pi accumulation, via the phosphate
carrier, and this in turn drives ATP-Mg accumulation (via
the ATP-Mg/Pi carrier), just as it also drives the transport
of malate and other dicarboxylates through the dicarboxylate carrier. As a consequence, ATP-Mg uptake or
efflux depends on the pH gradient and is abolished with
nigericin and uncouplers while it is largely unaffected by
dissipation of the membrane potential (15).
Net uptake or loss of AdNs occurs when flux in one
direction exceeds that in the opposite direction. For example, if mitochondrial ATPase in inhibited by oligomycin, or mitochondrial ATP synthesis is low due to the lack
of oxidizable substrates, then ATP-Mg uptake will take
place. On the other hand, if cytosolic ATP levels are low,
ATP-Mg efflux will prevail (13). According to Aprille (13),
the activity of the ATP-Mg/Pi carrier increases or decreases the matrix AdN pool but does not substantially
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(Fig. 2). These same mechanisms, notably the use of small
Tim proteins Tim8p-Tim13p to escort the precursor proteins along the intermembrane space (Fig. 2), apply to
ATP-Mg/Pi carriers coded by the SCaMC genes.
Some of the SCaMCs lack one or several EF-hand
domains and even most of the amino-terminal extension,
while others lack one transmembrane domain (91, 94)
(Fig. 9). The lack of transmembrane domains does not
alter the import of these SCaMC variants (91), but it is still
unknown whether the actual transporter function of the
carrier may be affected, as the current model for a functional carrier based on the structure of the ADP/ATP
translocase predicts that all transmembrane helices and
the short surface helices on the internal face of the inner
membrane participate in the transporter function (290,
291). On the other hand, the total lack of the aminoterminal extension appears to redirect SCaMC precursor
proteins to the escort proteins used by most MCs, the
Tim9p-Tim10p proteins (91) (Fig. 2).
MITOCHONDRIAL TRANSPORTERS
Physiol Rev • VOL
A related transport activity, the Ca2⫹-activated dCTP
(and other dNTP) transporter, has been described in mitochondria from human acute lymphocytic leukemia cells
(56), following the demonstration that a mechanism for
mitochondrial dNTP uptake existed in isolated mitochondria that is essential for DNA synthesis (73, 118). The
protein coded by the gene SLC25A19 has been shown to
catalyze the transport of dNTP when reconstituted into
proteoliposomes (105) and termed deoxynucleotide carrier (DNC). Mutations in SLC25A19 cause congenital microcephaly (323), and DNC is involved in the transport of
ddNT and antiretroviral reverse transcriptase inhibitors
(227). However, the DNC protein has no amino-terminal
extension or Ca2⫹-binding domains, has a much lower
affinity for dNTPs than the Ca2⫹-activated dCTP carrier
(212), and overexpression or downregulation of DNC in
RKO cells did not affect Ca2⫹-dCTP transport in mitochondria (212). Thus the molecular identity of the Ca2⫹activated dCTP carrier is still unknown.
B. Hormonal and Developmental Changes in AdN
Content in Liver Mitochondria
1. Hormonal regulation of liver mitochondria
AdN content
Mitochondria isolated from hepatocytes stimulated
with glucagon and Ca2⫹ mobilizing agents such as vasopressin, thapsigargin, or Ca2⫹ ionophores have increased
AdN content (17, 59, 152, 156, 357, 362, 389), which is not
due to an increase in mitochondrial volume (108). Glucagon-induced Ca2⫹ signals are transient and much smaller
than those elicited by ␣-adrenergic agonists (phenylephrine) or vasopressin (82, 121, 368), but by increasing the
intracellular concentration of cAMP and protein kinase A
activity, glucagon and Ca2⫹ mobilizing agents synergistically stimulate the net inflow of Ca2⫹ to hepatocytes
through receptor-activated Ca2⫹ channels (9, 168). Studies on the effect of mitochondrial inhibitors and RR on the
effect of glucagon, forskolin, or dibutyryl cAMP on thapsigargin-stimulated Ca2⫹ inflow suggested that protein
kinase A regulates the fate of Ca2⫹ entering the cell
through store-activated Ca2⫹ channels by directing some
of this Ca2⫹ to mitochondria (121). In fact, the increase in
mitochondrial AdN content is not observed if hepatocytes
are pretreated with EGTA, and this effect has been attributed to the strict Ca2⫹ requirement for the activation of
the ATP-Mg/Pi carrier (163, 276). An alternative view held
by Halestrap and co-workers (247) was that the increase
in mitochondrial AdN content could also be caused by an
exchange of cytosolic AdN with mitochondrial pyrophosphate on the ANT, as pyrophosphate accumulates in liver
mitochondria after hormonal treatment as a consequence
of Ca2⫹ inhibition of mitochondrial pyrophosphatase.
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change the mitochondrial ATP to ADP ratio, because the
carrier is much slower than the ATPase and ANT reactions, which exert control over that ratio (13). However,
other authors have found that ATP-Mg accumulation increased the ATP/ADP ratio in the liver mitochondrial
matrix (343).
The carrier catalyzes an electroneutral exchange of
divalent phosphate (HPO2⫺
4 ) against divalent ATP-Mg
2⫺
(ATP-Mg ) or divalent protonated ADP (HADP2⫺) (182,
275). The apparent Km for ATP-Mg in the presence of 2
mM Pi is ⬃2.6 mM, and HADP2⫺ competes poorly with
ATP-Mg so that in vivo ATP-Mg and Pi probably are the
physiologically relevant transported species (10, 108,
275). Remarkably, ATP-Mg/Pi carriers reconstituted into
liposomes had the same substrate specificity (122). The
transport rate of the ATP-Mg/Pi carrier is relatively slow
(1–2 nmol · min⫺1 · mg protein⫺1) compared with that of
the phosphate carrier or adenine nucleotide translocase
(⬃150 –200 nmol · min⫺1 · mg⫺1; Ref. 217), a fact that was
initially attributed either to the low abundance of the
carrier or to a slow rate constant (10). In fact, the Vmax
values of two of these carriers expressed in E. coli and
reconstituted in liposomes (280 and 44 ␮mol · min⫺1 · mg
protein⫺1) for SCaMC-1/SLC25A24 and SCaMC-3/
SLC25A23, respectively (122), are much lower than that
of the phosphate carrier (0.61 mmol · min⫺1 · mg protein⫺1) (142, 420).
ATP-Mg/Pi transport activity is inhibited by mersalyl
and is insensitive to carboxyatractyloside (CAT) (10), a
result also obtained with the carrier proteins reconstituted in liposomes. In addition, these studies revealed that
pyridoxal-5-phosphate and bongkrekic acid considerably
inhibited one of the mammalian isoforms, SCaMC-3/
SLC25A23 (122).
2⫹
C) Ca
REGULATION OF ATP-Mg/Pi TRANSPORT IN MAMMALIAN
MITOCHONDRIA. Haynes et al. (163) were the first to report
that net AdN transport required micromolar Ca2⫹. In fact,
net nucleotide uptake or efflux through the ATP-Mg/Pi
carrier is completely blocked in the presence of excess
EGTA and increases when free Ca2⫹ rises from 1 to 4 ␮M.
This results in a lower apparent Km for ATP (4.4 and 2.4
mM at 1 and 2 ␮M Ca2⫹, respectively), with little changes
in Vmax (276). Importantly, increased activity did not require Ca2⫹ entry in mitochondria, as it was unaffected by
RR (276). Thus the ATP-Mg/Pi carrier was the first known
MC with an absolute requirement for extramitochondrial
Ca2⫹, a property which is not observed when the proteins
are reconstituted in liposomes (122). Calmodulin antagonists inhibit the activation by Ca2⫹, suggesting that Ca2⫹
may stimulate ATP-Mg/Pi carrier activity via a specific
calmodulin-related Ca2⫹ binding site (276). All SCaMCs
have a set of EF-hand motifs highly similar to calmodulin
(91, 94) that are thought to confer Ca2⫹ regulation to
ATP-Mg/Pi activity, raising the possibility that calmodulin
antagonists might directly interfere with the SCaMCs.
49
50
SATRÚSTEGUI, PARDO, AND DEL ARCO
2. Role of liver ATP-Mg/Pi transport in early
postnatal development
In the newborn rat liver, initial AdN content in mitochondria is very low, but soon after birth the cytosolic
ATP/ADP ratio and AdN content of mitochondria increase
rapidly (14, 373, 374), more than twofold within the first
2 h after birth (373). A similar increase was found in
Physiol Rev • VOL
neonatal rabbit liver (53, 327). Total AdN content of the
liver increases more modestly, by ⬃25% (83), so that the
increase in the mitochondrial matrix AdN results in a
decrease in AdN in the cytosolic compartment of ⬃10 –
13% (see Ref. 12 for review).
The postnatal increase in mitochondrial AdN occurs
shortly after catecholamine and glucagon are secreted at
the time of parturition, while insulin levels decrease (85,
104, 134, 209). This leads to activation of glucagon and
␤2-adrenergic receptors in fetal liver (324), to increases in
cAMP, and to increased rate of glycogenolysis and glycolysis-derived ATP production, resulting in an increased
cytosolic ATP/ADP ratio (374). Activation of ␣-adrenergic
receptors and increases in cAMP levels also lead to increases in free Ca2⫹ concentration in fetal hepatocyte
cultures and to a stimulation of mitochondrial functions
including the increase in mitochondrial AdN content
(132). The availability of oxygen appears to be another
factor controlling AdN uptake in postnatal mitochondria.
At birth, as lungs inflate, there is a change in fetal liver
circulation that results in sudden perfusion of the liver
with well-oxygenated blood (12). This appears to be a
permissive condition for mitochondrial uptake of AdN,
since animals subjected to prolonged postnatal hypoxia
fail to increase mitochondrial AdN content and related
functions (12, 16, 54, 397).
The rapid postnatal accumulation of AdN in mitochondria was found to be due to a saturable, atractyloside-resistant, and mersalyl-sensitive ATP transport (11,
26, 299, 300, 373), carried out by the ATP-Mg/Pi carrier
(reviewed in Ref. 13), which is thought to represent one of
the targets of glucagon and catecholamines in fetal liver
mitochondria. In preterm neonates that have lower ATP
levels than term fetuses, the increase in liver mitochondrial AdN content is delayed by 2 h and is preceded by an
equally delayed increase in cytosolic ATP (402), an increase required to drive the ATP-Mg/Pi carrier towards
mitochondrial ATP accumulation (13). Control strength
studies with appropriate inhibitors led to the finding that
the early postnatal AdN accumulation in mitochondria
results in an increase in the respiratory control index
(298) mainly as a consequence of an increase in state 3
respiration (373, 400, 401) and also from structural
changes in the inner mitochondrial membrane that result
in decreases in mitochondrial volume (400) and permeability to protons (28, 298, 399, 401). The increase in state
3 respiration resulted from an increase in ANT activity
(13, 14, 327) and/or F1-ATPase activity (13, 28, 401), which
have been attributed both to a simple mass action ratio
effect of the increase in matrix AdN content and to rapid
de novo synthesis of mitochondrial proteins, particularly
F0F1-ATP synthase (3, 248, 400), a process dependent on
an increased stability and translational efficiency of mitochondrial transcripts (179, 232), but also requiring ade-
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The fact that hormones can trigger a Ca2⫹-dependent
activation of the ATP-Mg/Pi carrier in normal resting cells
has been taken as an indication that the matrix/cytosol
ATP-Mg gradient is lower than the Pi gradient in resting
cells and ATP-Mg uptake is favored over release (108).
However, the ATP-Mg/Pi carrier stays inactive unless triggered by an appropriate Ca2⫹ signal (108). By changing
the matrix AdN content, the ATP-Mg/Pi carrier may regulate mitochondrial metabolic pathways that depend on
AdNs either as substrates or cofactors, such as gluconeogenesis from lactate, urea synthesis, synthesis of mitochondrial proteins, RNA and DNA, and import of mitochondrial precursor proteins, among other processes. The
first reactions in gluconeogenesis and urea synthesis are
ATP-dependent carboxylations. In urea synthesis, CO2 is
fixed into carbamoyl phosphate, which is formed by carbamoyl phosphate synthetase I, an ATP-dependent reaction. Citrulline arises from carbamoyl phosphate and ornithine, and citrulline synthesis in mitochondria isolated
from hepatocytes treated with glucagon and ␣-adrenergic
agonists is strongly activated (137, 156, 389). There is a
correlation between AdN pool size and citrulline synthesis in mitochondria that suggests that the AdN pool size
may control citrulline synthesis (137), although this has
been questioned (156). Because the direction of net AdN
movement occurs in both directions depending on the
relative ATP/Pi concentration on each side, it has been
suggested that in situations where energy status is low
(i.e., hypoxia), Ca2⫹ activation of the carrier results in
AdN loss to the cytoplasm. The result, at least in liver,
would be downregulation of metabolic pathways that are
energy consumers but are not necessary for survival of
the liver cell itself (i.e., gluconeogenesis and urea synthesis). When favorable energy conditions (hypoxia to normoxia) are restored, mitochondria would take up AdN
and intermediary metabolic pathways would be active
again (13).
Interestingly, the emerging picture is one in which
glucagon and ␣-adrenergic agents activate the two CaMCs
existing in hepatocytes, causing both an increase in AdN
content through Ca2⫹ activation of ATP-Mg/Pi carrier
(which results in increased gluconeogenesis through
an increase in pyruvate carboxylase and ureogenesis
through an increase in citrulline synthesis) and increase
in AGC activity, also required to activate MAS, gluconeogenesis, and ureogenesis (see sect. IIIA4).
MITOCHONDRIAL TRANSPORTERS
3. ATP-Mg/Pi transport in extrahepatic tissues
A mechanism for net AdN transport similar to the one
described in liver mitochondria has also been observed in
rat kidney (146, 165). In kidney mitochondria, net AdN
uptake and efflux takes place through two different mechanisms, a CAT-resistant and Ca2⫹-regulated pathway similar to the ATP-Mg/Pi carrier from liver mitochondria, and
a CAT-sensitive pathway (inhibited at 5 ␮M CAT) that is
resistant to EGTA (146). ATP efflux or uptake through
these pathways was inhibited (25–50%) by high (10 ␮M)
cyclosporin A concentrations (146, 165) but not by 1 ␮M
cyclosporin A (146), a concentration that is adequate to
fully desensitize the permeability transition pore in liver
Physiol Rev • VOL
mitochondria to cyclophilin D (34). From its CAT sensitivity, it has been suggested that the mechanism responsible for this second pathway could be an isoform of the
ANT different from that of liver (146).
Net changes in AdN content occur in heart mitochondria, but the mechanisms seem to be different from those
in liver mitochondria. Net decreases in AdN content have
been observed in isolated heart mitochondria exposed to
high Pi concentrations, but the transport mechanisms are
to a large extent CAT sensitive and do not match that of
the liver mitochondria ATP-Mg/Pi carrier. AdN efflux has
been shown to proceed through two pathways: a fast,
Pi-dependent pathway, which is blocked by CAT, and a
slow and CAT-resistant pathway (23, 415), to be discussed
in section IVB3A. Unlike liver mitochondria, rat heart mitochondria were unable to accumulate AdN when incubated with 2 mM ATP (24). On the other hand, the postnatal increase in mitochondrial AdN pool size that has
served as metabolic signature of the ATP-Mg/Pi carrier
activity is a rapid process only in liver, but it is much
slower in heart (349). Therefore, the identity of the possible carriers involved in AdN loading of mitochondria in
extrahepatic organs is still unknown.
The SCaMC proteins are obvious candidates for such
a role. Compared with other MCs, SCaMC-1 to -3 have an
unusually large number of variants. These variants probably differ in their Ca2⫹ binding properties, as Ca2⫹ binding motifs are lost in some of them (Fig. 9) (91, 94), and
perhaps also in catalytic activity, since some SCaMC-3
lack one transmembrane domain (91) (see Fig. 9).
Fiermonte et al. (122) studied the catalytic activity of the
proteoliposome-reconstituted carboxy-terminal regions
of SCaMC-3 and SCaMC-1 and found that SCaMC-3 activity is about eight times lower than that of SCaMC-1,
whereas the affinities for substrates were the same (122).
SCaMC-1 and -3 were equally resistant to CAT (10 ␮M),
but another translocase inhibitor, bongkrekic acid (10
␮M), considerably inhibited SCaMC-3 but not SCaMC-1.
This opens up the possibility that the different SCaMC
isoforms and variants may have on the one hand differences in Ca2⫹ sensitivity, and on the other, partial sensitivity towards the classical translocase inhibitors. The
particular set of SCaMC genes and variants in kidney and
heart may be responsible for the changes in AdN content
observed in isolated kidney or heart mitochondria.
As indicated in section IVB2, in intact cells the matrix/
cytosol ATP-Mg gradient is lower than the Pi gradient, and
the ATP-Mg/Pi carrier remains inactive in the resting
state, unless stimulated by an appropriate Ca2⫹ signal
(108). The activated carrier would direct ATP-Mg into
mitochondria, and the resulting increase in mitochondrial
AdN should increase ANT and F1-ATPase activity through
mass action ratio, resulting in an increase in mitochondrial ATP synthesis (10). This should actually result in a
Ca2⫹-dependent increase in mitochondrial ATP synthesis.
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quate ATP levels for chaperonin-assisted folding of the
mitochondrial proteins.
The increase in mitochondrial AdN content produced
in the postnatal liver could stimulate pyruvate carboxylase and citrulline formation in response to glucagon, as
discussed in section IVB1. Pyruvate carboxylase in mitochondria and phosphoenolpyruvate carboxykinase are
rate limiting for gluconeogenesis immediately after birth
(54, 86, 87), and pyruvate carboxylation rates increase in
concert with the increase in AdN content (18). Increasing
AdN content of newborn mitochondria in vitro mimicked
the developmental changes in pyruvate carboxylase activity, indicating that pyruvate carboxylation is one of the
consequences of postnatal activation of the mitochondrial
ATP-Mg/Pi carrier (12, 18, 53). Citrulline synthesis is another candidate for regulation by the postnatal increase in
mitochondrial AdN content, as it is low in newborn mitochondria and increases along with the AdN content (12).
Intramitochondrial protein synthesis, which increases
severalfold along the first postnatal hours, is also regulated by the mitochondrial AdN pool, and it greatly increases when 0-h newborn mitochondria are preincubated to accumulate AdN (183).
In sum, cAMP and Ca2⫹ converge to stimulate the
ATP-Mg/Pi carrier of newborn liver mitochondria, through
an increase in cytosolic ATP (mainly through cAMP-mediated stimulation of glucogenolysis and glycolytic ATP
production) and an increase in cytosolic free Ca2⫹, which
is required to trigger transport activity of the carrier. The
consequences of the postnatal increase in liver mitochondria AdN content may vary in different species. In the rat,
the mitochondrial AdN pool is very small at birth, and its
rapid postnatal increase correlates with an increase in
ANT activity and state 3 respiration (13). On the other
hand, in the rabbit the mitochondrial AdN content at birth
is larger and its rapid increase is not followed by changes
in state 3 respiration (327). Therefore, the consequences
of the small mitochondria AdN pool at birth depend on its
size with respect to a certain threshold that may vary
depending on the particular mitochondrial function.
51
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SATRÚSTEGUI, PARDO, AND DEL ARCO
Physiol Rev • VOL
AMP, ADP, or ATP across the inner mitochondrial membrane. Since 5⬘-nucleotidase, which is responsible for the
dephosphorylation of AMP and IMP to adenosine and
inosine, is not found in the mitochondrial matrix (192)
and in vitro studies of AdN efflux from isolated heart
mitochondria incubated with Pi showed a correlation between the loss of mitochondrial AdN and the appearance
of extramitochondrial AdN (23), it was concluded that the
most probable mechanism was AdN efflux (337). AdN
efflux has been shown to proceed through two pathways:
a fast, Pi-dependent pathway, which is blocked by CAT,
and a slow Pi-independent and CAT-resistant pathway
(337). The Pi-dependent pathway has many of the characteristics of the liver ATP-Mg/Pi carrier, including the requirement for Mg2⫹ (23, 355), but appeared to be a different mechanism, as it was inhibited by atractyloside.
The extent of AdN loss from mitochondria and its
implications for a decrease in state 3 respiration in ischemic heart are somewhat controversial. Dransfield and
Aprille (107) did not find any decrease in the mitochondrial AdN pool in hepatocytes incubated under chemical
hypoxia, while the mitochondrial AdN shifted to AMP
(107). Not all studies show a clear-cut decrease in the
activity of the respiratory chain during ischemia, and in
some studies state 3 respiration was found to increase
after 30 min of ischemia (49, 228; see Ref. 363 for review).
These variations are perhaps associated with the preparation of heart mitochondria, which may not reflect that
of the original tissue, particularly in substrate and ion
content, especially in ischemic heart, that have more fragile mitochondria than those of normal heart (181). In
addition, the procedures for isolation of heart mitochondria yield largely subsarcolemmal mitochondria rather
than interfibrillar mitochondria, which provide most of
the energy for the contractile apparatus (see Ref. 363 for
review).
Although a role for ATP-Mg/Pi carriers in efflux of
mitochondrial AdN to the cytosol during ischemia is uncertain, these carriers could be involved in the events
triggered during reperfusion, particularly the opening of
the permeability transition pore (see Refs. 37, 38, 81, 102,
153, 154, 274 for reviews). This pore, which allows the
passage of solutes smaller than 1.5 kDa across the inner
mitochondrial membrane, opens under the influence of
Ca2⫹, lack of AdNs, and oxidative stress and is inhibited
by cyclosporin A (CSA), an inhibitor of cyclophylin D
(CypD), a peptidyl-prolyl cis-trans isomerase (PPIase) in
the mitochondrial matrix (5, 378). The pore has been
shown to open during reperfusion, but not ischemia (102,
153), possibly as a consequence of the increased ROS
formation that accompanies reperfusion, and the critical
role of mitochondrial-generated reactive oxygen species
in PTP opening (201).
As the total level of mitochondrial AdN regulates PTP
opening, Hagen et al. (147) have proposed that the ATP-
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Increases in mitochondrial ATP synthesis that depend on Ca2⫹ signals have been reported in heart at
different work loads (382, 383; see Ref. 31 for review).
31
P-NMR studies have shown that these increases occur
without the variations in ADP, ATP, Pi, or phosphocreatine concentrations that normally control oxidative
phosphorylation in skeletal muscle and have been attributed to metabolic consequences of increase in mitochondrial Ca2⫹ (31). In intact myocardial cells, mitochondrial
Ca2⫹ tracks the rapid changes in cytosolic Ca2⫹ levels
occurring in each cycle of contraction (70, 176), and
increased frequency or amplitude of cytosolic Ca2⫹
spikes is mirrored by similar changes in mitochondrial
Ca2⫹ spikes (315, 395), and by elevations in interspike
Ca2⫹ levels (diastolic increase in mitochondrial Ca2⫹)
(103, 315). The mechanisms proposed for mitochondrial
Ca2⫹ signaling in heart are a Ca2⫹-induced increased activity of mitochondrial dehydrogenases (but see Ref. 410),
and activation of F0F1-ATPase (31, 161, 345, 382, 383, but
see Ref. 159 for review), and ANT (262) by matrix Ca2⫹. It
has been proposed that F1-ATPase is activated by Ca2⫹induced release of a Ca2⫹-binding inhibitor protein, CaBI
(422, 423), and Ca2⫹ binding to the ␤-subunit (170), or
e-subunit of F1-ATPase (19). Experiments in which RR
inhibited the activation of F1-ATPases at high work loads
implicated matrix Ca2⫹ as the primary effector (161, 383).
However, the effects of RR in intact cells are not specific
for the Ca2⫹ uniporter (110), and Robert et al. (315) found
RR or R360 to be totally inefficient in inhibiting the Ca2⫹
uniporter in intact neonatal cardiomyocytes. Thus a possible upregulation of heart ATP-Mg/Pi carriers by cytosolic Ca2⫹ emerges as an alternative possibility contributing
to increased ATP synthesis with increasing work loads.
A) ISCHEMIA. Early studies with mitochondria isolated
from ischemic organs showed a diminished content of
AdN in mitochondria from heart (22, 220), liver (270, 411),
and kidney (164, 165) associated with a decrease in oxidative phosphorylation typically found in mitochondria
isolated from ischemic tissues. After 20 min of ischemia,
the levels of heart mitochondrial AdN decreased to ⬃20%
of control values, with a rapid loss between 10 and 20 min
(22). State 3 respiration was also inhibited, and titration
with CAT of state 3 respiration in mitochondria from
control and ischemic heart suggested that the decrease in
state 3 and uncoupled respiration caused by ischemia
cannot be attributed to a loss of ANT activity but rather to
the decrease of intramitochondrial AdN levels (22).
As Pi can induce an efflux of AdN from heart mitochondria, and Pi doubles within 1 min after the onset of
heart ischemia (210, 272, 337), a number of studies by
Asimakis and co-workers (23, 337, 355) have addressed
the involvement of the ATP-Mg/Pi carrier in loss of mitochondrial AdN. The loss of AdN in rat heart mitochondria
has been suggested to occur either by enzymatic degradation in the mitochondrial matrix or by movement of
MITOCHONDRIAL TRANSPORTERS
Physiol Rev • VOL
but not atractyloside sensitivity, at least not for isoforms
SCaMC-1 and SCaMC-3 (122). However, one of the ATPMg/Pi isoforms, SCaMC-3, is sensitive to bongkrekic acid
(122) so that the similarity among the ANT and ATP-Mg/Pi
carriers could be even larger. As with all other MCs, all
three SCaMC isoforms have prolines at conserved positions in the boundary of the TM domains facing the matrix
(91, 94), that could interact with the mitochondrial PPIase
CypD. In fact, ATP-Mg loading and efflux from kidney
mitochondria through the ATP-Mg/Pi carrier have been
shown to be inhibited by CSA, albeit at relatively high
concentrations (146, 166). Being strictly Mg2⫹ dependent,
the ATP-Mg/Pi carriers might also confer Mg2⫹ sensitivity
to the PTP, a known property of the pore (37). PTP
opening is triggered by Ca2⫹, and there is mounting evidence that this involves matrix Ca2⫹ rather than Ca2⫹ in
the intermembrane space, the compartment sensed by the
ATP-Mg/Pi carrier. However, under certain conditions,
PTP opening has revealed an extramitochondrial site for
Ca2⫹ regulation. Induction of PTP by thiol cross-linkers
such as phenylarsine oxide does not require the presence
of Ca2⫹ in the mitochondrial matrix and is activated by
extramitochondrial Ca2⫹ (200, 201, 224a).
In conclusion, although largely speculative, the notion that the ATP-Mg/Pi carriers may be involved in the
formation of the permeability transition pore deserves
further work and consideration.
C. Nonmammalian ATP-Mg/Pi Carriers
ATP-Mg/Pi carriers are widely conserved, with orthologs in most of eukaryotic organisms (94). Yeast contains a single isoform, whereas higher eukaryotes have
two (C. elegans or chicken) or three isoforms (Danio
rerio, Xenopus, or Arabidopsis) with high similarity
among each other (96). Except for the S. pombe ortholog,
all proteins display EF-hand motifs at equivalent positions
(94). It has been suggested that these isoforms arose by a
gene fusion process between a primitive ANT gene and
calmodulin-like sequences (96).
1. Transport activity of yeast Sal1p
Sal1p is the product of baker’s yeast gene YNL083w,
renamed SAL1 (for suppressor of aac2 lethality) by Chen
(74). YNL083w was identified during the sequencing of
the genome of S. cerevisiae, and it was found to be
nonessential (364, 427). The sequences of human SCaMCs
and Sal1p are very similar, with 34 –37% identity at the
amino acid level along the 300-amino acid-long carboxy
terminus (68, 94).
Like its human counterparts, Sal1p is localized to
mitochondria (68). The transport activity of Sal1p was
studied in mitochondria obtained from wild-type and
Sal1p-deficient yeast (68) and in yeast deficient in all three
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Mg/Pi carrier could modulate the probability of PTP opening by changing mitochondrial AdN levels. Pi and Ca2⫹
increase and ATP decreases during ischemia, and under
these conditions, the Ca2⫹-dependent efflux of mitochondrial ATP-Mg in exchange for cytosolic Pi on the ATPMg/Pi carrier was found to increase the susceptibility of
PTP opening by high Ca2⫹, atractyloside, and prooxidants
in liver mitochondria (147). However, whether this mechanism operates in vivo and its relevance to heart ischemia-reperfusion is still an open question.
The role of CypD in PTP opening has been proven
conclusively after the generation of mice deficient in
CypD (30, 34, 268, 344). Mitochondria from these mice
require much higher Ca2⫹ loads to induce PTP opening
(30, 34, 268, 344). Cell death in heart and brain as a
consequence of ischemia-reperfusion was greatly reduced
by CypD deficiency, indicating that PTP opening, which
recruits CypD to the pore, is involved specifically in this
type of necrotic death (30, 268, 344; reviewed in Refs. 39,
140, 150).
An involvement of ANT in PTP formation was initially suggested from the pore properties: sensitivity to
the ANT substrates ATP and ADP (as closers of the pore)
and inhibitors (CAT that locks ANT in a “c” conformation
and favors pore opening and bongkrekic acid that promotes the “m” conformation of ANT and favors pore
closure; see Ref. 426 for review). A favored model envisions that the ANT turns into a nonselective channel, an
idea supported by the finding that a number of MCs, such
as the ANT, PiC, and the AGC can behave as relatively
unspecific uniporters by treatment with SH reagents (99,
100, 204). However, recent work by Kokoszka et al. (199)
has shown that mitochondria from livers of ANT-knockout mice in which the two ANT genes expressed in liver,
ANT1 and 2, were genetically inactivated, still have PTP
activity. From this it was concluded that PTP can form in
the absence of ANT, although regulation of PTP activity
by atractyloside and ADP is lost and its sensitivity to Ca2⫹
is also decreased. Thus it appears that ANTs are nonessential components of the PTP but can regulate PTP
activity in response to Ca2⫹ and ANT agonists (199). This
opens the possibility that other MCs related to these ANT
isoforms could take over part of the role, both in normal
liver function and in PTP formation (151). Indeed, a most
surprising finding was that these ANT-knockout mice survived normally for more than a year (199). Since mice do
not have ANT3 (199), this could include the newly described ANT4 (106, 316), although this isoform is not
normally expressed in mouse liver (316).
The ATP-Mg/Pi carriers are the closest homologs of
ANT and are widely expressed in every tissue. In S. cerevisiae, Sal1p, the yeast ATP-Mg/Pi carrier, can partially
take over the role of ANT, and vice versa (68, 74), and this
opens up the possibility that this can also occur in mammalian tissues. It shares with ANT the sensitivity to AdNs,
53
54
SATRÚSTEGUI, PARDO, AND DEL ARCO
ANTs, aac1,2,3⌬ (109), in which the main mechanism for
ATP uptake in mitochondria is Sal1p. Freshly isolated
wild-type yeast mitochondria incubated in the presence of
CAT increased their total AdN content by about threefold,
after 5- to 10-min incubation in the presence of 4 mM ATP,
and this was reduced by 40 –50% in Sal1p-deficient mitochondria (68). ATP uptake on Sal1p was Mg2⫹ dependent,
and ATP efflux was stimulated by external Pi (68), as
reported for mammalian ATP-Mg/Pi carriers.
2. Ca2⫹ regulation of ATP-Mg/Pi transport
in yeast mitochondria
3. Physiological role of yeast Sal1p
As there is a large number of mitochondrial functions
that require ATP in the mitochondrial matrix, it was surprising that a triple deletant of all three ANTs (aac1,2,3⌬)
was still able to grow on glucose (109). This finding led to
the proposal of the existence of another transporter that
could supply mitochondria with AdNs in these conditions
FIG. 10. Role of Sal1p in glucose-induced
Ca2⫹ signaling in Saccharomyces cerevisiae.
Glucose readdition to starved yeast results in
the activation of a G protein-coupled receptor
signaling pathway in which the Gpr1 is the
receptor, Gpa2 is a yeast homolog of mammalian heterotrimeric G␣ proteins, and Plc1 is a
phosphatidylinositol (PI)-specific phospholipase C with high homology to the mammalian phospholipase C-␦ isoform (188, 320, 387).
Inositol trisphosphate (IP3) produced by Plc1
is required as a second messenger for glucoseinduced transient elevation of [Ca2⫹]i (388), a
process depending on the Cch1-Mid1 pathway
(391), in which Cch1 is a homolog of mammalian high-affinity voltage-gated Ca2⫹ channels.
Full activation of this pathway requires glucose uptake by plasma membrane hexose
transporters (Hxt) and phosphorylation by
hexokinase or glucokinase (Hxk1, Hxk2,
Glk1) (391). Further glucose metabolism results in glycolytic ATP production. Sal1p acts
as a Ca2⫹ sensor to promote ATP-Mg uptake
in mitocondria in exchange for Pi, a process
required to maintain mitochondrial functions
during growth on glucose in the absence of
ANT (68).
Physiol Rev • VOL
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ATP-Mg uptake on Sal1p was stimulated by extramitochondrial Ca2⫹, with an S0.5 of ⬃30 ␮M (68), a value
somewhat higher than that reported for the rat liver ATPMg/Pi carrier (163, 276). This is perhaps not too surprising
as Sal1p lacks two (out of four) canonical EF-hand motifs
present in most human SCaMCs (94). EF-hands 1 and 4
have two amino acids that do not satisfy the coordination
bond of canonical EF-hands. EF-hands 2 and 3 are canonical, and Chen (74) showed that they are required for Ca2⫹
binding and other Sal1p functions. Thus the lower affinity
for Ca2⫹ of Sal1p with respect to the liver ATP-Mg/Pi
carrier may be explained on this basis.
(109). Wild-type and aac1,2,3⌬ yeast express Sal1p during
growth on glucose, and Sal1p expression was found to be
required for growth in the absence of all three translocases (68, 74). Indeed, any combination of aac2⌬ and
sal1p⌬ is lethal. These findings support the idea that
Sal1p is the ATP carrier that compensates for the lack of
ANTs, particularly Aac2p, during growth on glucose.
Glucose sensing in yeast is required to tune growth
and metabolic activity to amount and type of sugars available. Glucose uses a signal transduction pathway similar
to that employed by hormones in higher eukaryotes, in
which both cAMP and Ca2⫹ are early second messengers
(139, 320). Glucose binds to the G protein-coupled receptor Gpr1p (224) inducing a transient elevation of cytosolic
Ca2⫹ through a pathway involving PLC stimulation (6,
387) and IP3 production (388). Although glucose-induced
Ca2⫹ transient involves mainly Ca2⫹ influx from the external medium through the Cch1p/Mid1p plasma membrane Ca2⫹ channel (391), Ca2⫹ entry was not required to
generate glucose-induced physiological changes as none
of them was blocked in the presence of EGTA (138). In
the face of these contrasting facts, the role of Ca2⫹ signaling in glucose sensing and the relevant targets, if any,
of the glucose-induced Ca2⫹ transient have remained
largely unknown.
Cavero et al. (68) tested the hypothesis that Sal1p
may be a target of the glucose-induced Ca2⫹ transient that
would be revealed in the absence of ANTs. By studying
the kinetics of glucose-induced bud formation, it was
found that in cells lacking all three translocases, the
presence of EGTA during preexposure to glucose resulted
in a total block of the effect of glucose. This effect was
not observed for wild-type or Sal1p-deficient yeast. In
MITOCHONDRIAL TRANSPORTERS
V. EVOLUTION OF MECHANISMS FOR
CALCIUM SIGNALING IN MITOCHONDRIA
Admittedly, the Ca2⫹ uniporter pathway is by and
large the most potent system for Ca2⫹ signaling in mitochondria. Mitochondria from vertebrate tissues have the
capacity for respiration-linked Ca2⫹ uptake (65), a capacity also present in insect flight muscle mitochondria (62,
66, 157, 419) and trypanosomatids (263, 266). However,
this capacity does not appear to be universal. Ca2⫹ transport varies between species in plant mitochondria and
between tissues within the same species (128). Mitochondria isolated from hypocotyls of plants such as coffee,
soybean, and corn, but not from potato tubers and white
cabbage leaves, have the ability to transport Ca2⫹ (128,
242). Neither energy-linked Ca2⫹ transport, high-affinity
Ca2⫹ binding, nor Ca2⫹-stimulated respiration occurs in
mitochondria isolated from S. cerevisiae or Torulopsis
utilis (Candida utilis) (64, 65). The absence of a Ca2⫹
uniporter in mitochondria from S. cerevisiae has also
been confirmed in later studies (184). Because mitochondria from N. crassa showed a slow respiration-dependent
Ca2⫹ accumulation, a limited capacity to accumulate
Ca2⫹, and lacked high-affinity Ca2⫹ binding sites, it is
uncertain whether they have a real Ca2⫹ uniporter (65).
Since the molecular identity of the Ca2⫹ uniporter or the
RAM (365) are still unknown, studies demonstrating that
such transport system is actually present or absent in
Neurospora mitochondria could provide helpful clues.
Physiol Rev • VOL
A separate set of mechanisms for Ca2⫹ signaling in
mitochondria relies on Ca2⫹ signals that reach the external face of the inner mitochondrial membrane. Three of
these are functionally related, in that all three supply
extra reducing equivalents to mitochondria, the Ca2⫹sensitive external NAD(P)H dehydrogenases from fungi
and plants (which transfer electrons to complex III), FADGPDH (which also transfers electrons to complex III),
and the AGC-MAS pathway which supplies electrons to
complex I.
The first two pathways are energetically equivalent,
and either one of them appears to be present in eukaryotes. Thus most fungi and plants have the external
Ca2⫹-dependent NAD(P)H DH system, but not the Ca2⫹regulated FAD-GPDH system, whereas animals have the
Ca2⫹-regulated FAD-GPDH system but lack Ca2⫹-regulated external NAD(P)H DHs. An interesting exception to
this rule is the yeast S. cerevisiae. Mitochondria from this
organism do not have a Ca2⫹-regulated external NAD(P)H
DH or a Ca2⫹-regulated FAD-GPDH and thus are completely devoid of a mechanism whereby cytosolic Ca2⫹
can regulate the transfer of reducing equivalents from
cytosolic NAD(P)H to complex III. The AGC-MAS pathway is energetically superior to the other two, in that the
reducing power is transferred to NADH that can be oxidized in complex I. The Ca2⫹-regulated AGC-MAS system
is present in all known plant and animal genomes, with
the perhaps not surprising exception of S. cerevisiae,
where agc1p is not regulated by Ca2⫹.
The singularity of these two exceptions is probably
related to the uniqueness and extreme simplicity of the
Ca2⫹ signaling mechanisms in Saccharomyces. Indeed,
the Ca2⫹-signaling apparatus of S. cerevisiae is much
simpler than that of animal and plant cells and is involved
in the regulation of the cell cycle, mating, sensing of
glucose, resistance to salt stress, and cell survival. In
filamentous fungi with more complex growth patterns,
Ca2⫹ is involved in other processes such as secretion,
cytoskeletal organization, hyphal tip growth, branching,
sporulation, and circadian clocks (47, 425). Thus filamentous fungi have a substantive “toolkit” of Ca2⫹-signaling
proteins, far more complex than that present in budding
yeast (47, 425).
An additional mechanism whereby cytosolic Ca2⫹
signals to mitochondria from the external face of the
inner mitochondrial membrane is the Ca2⫹-regulated
ATP-Mg/Pi carrier. The functional outcome of this regulation is quite different; it is not related to reducing equivalent transfer to mitochondria, but rather to loading or
unloading of ATP in mitochondria, the consequences of
which are still to be established. Interestingly, this mechanism for Ca2⫹ signaling is most probably the most basic
of them, including the Ca2⫹ uniporter, as it is the only one
present in the very small repertoire of Ca2⫹ signaling
devices of S. cerevisiae.
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fact, the kinetics of bud emergence in aac1,2,3⌬ yeast
that rely on Sal1p for ATP loading of mitochondria was
the same as that obtained in the absence of glucose (68).
These results show that Ca2⫹ binding to Sal1p is a component of the glucose sensing system that becomes essential in yeast lacking ANTs (Fig. 10). This is also supported by the results of Chen (74), who showed that
mutations in the EF-hand motifs of Sal1p that eliminated
Ca2⫹ binding also abrogated the ability of the protein to
suppress the lethality of aac2⌬ sal1⌬ double mutants,
suggesting that Ca2⫹ binding by the protein is critical for
its function during growth on glucose (74). This vital and
Ca2⫹-dependent function of Sal1p is ATP entry in yeast
mitochondria, a function that can be taken over by ANT,
possibly by exchanging cytosolic ATP for mitochondrial
pyrophosphate in a Ca2⫹-independent way.
Baker and Schatz (30) proposed that the only mitochondrial proteins essential for viability in S. cerevisiae
are those involved in protein import. Import and assembly
of mitochondrial proteins requires matrix ATP hydrolysis
at various steps including mHsp70 at TIM23 to translocate
preproteins to the mitochondrial matrix (196), and for
chaperonin-assisted folding of mitochondrial proteins
(240). This requirement probably explains the vital role of
Sal1p in the absence of ANTs.
55
56
SATRÚSTEGUI, PARDO, AND DEL ARCO
VI. CONCLUSIONS
There are many mechanisms for Ca2⫹ signaling in
mitochondria independent of Ca2⫹ entry. Some of them
have been known for a long time, particularly FAD-GPDH,
the external Ca2⫹-dependent NADPH dehydrogenases,
and the liver ATP-Mg/Pi carrier, while others, the AGCs,
are relatively new. However, their physiological role as
mechanisms for Ca2⫹ signaling in mitochondria has remained in the dark, probably because the Ca2⫹ uniporter
Physiol Rev • VOL
(CU) has been considered the one and only mechanism
responsible for that role. Indeed, the consequences of
their activation are either increases in mitochondrial redox potential (NADH or flavines), or ATP levels, such as
those expected to occur upon Ca2⫹ entry in mitochondria.
It is thus not surprising that the existence and importance
of some of them in relation to the CU has only become
evident when data from genetically modified organisms
has emerged. This is the case for two CaMCs: neuronal
AGC1, aralar, which through its participation in the
malate-aspartate NADH shuttle plays a prominent role in
transducing small Ca2⫹ signals to neuronal mitochondria,
and that of the yeast ATP-Mg/Pi carrier Sal1p and its role
in sensing glucose-induced Ca2⫹ signals. A more complete
understanding of the interplay between these different
mechanisms requires the molecular identification of the
CU, the development of more specific tools for the analysis of transporter function, and genetically engineered
organisms, and this should establish the actual role of
each of these CaMC in Ca2⫹ signaling in mitochondria.
ACKNOWLEDGMENTS
We are indebted to Dr. Kathryn F. LaNoue, Dr. José M.
Cuezva, and Dr. June Aprille for critical reading of the manuscript and for many helpful suggestions; to Laura Contreras,
Javier Traba, Patricia Mármol, Dr. Santiago Cavero, and Dr.
Milagros Ramos for their excellent work in quest for the role of
CaMCs; and to José Belio for assistance with the illustrations.
Address for reprint requests and other correspondence: J.
Satrústegui. Dept. Biologı́a Molecular, Centro de Biologı́a Molecular Severo Ochoa, UAM-CSIC, Campus Cantoblanco, 28049
Madrid, Spain (e-mail: [email protected]).
GRANTS
We are thankful for financial support received from the
Ministerio de Educación y Ciencia, Fondo de Investigaciones
Sanitarias del Ministerio de Sanidad (FIS), Comunidad de Madrid and Quı́mica Farmaceútica Bayer, and the institutional
support of the Ramón Areces Foundation to the Centro de
Biologı́a Molecular Severo Ochoa.
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