Ca2+ Activation Kinetics of the Two Aspartate-Glutamate Mitochondrial Carriers, Aralar and Citrin

Ca2+ regulation of the Ca2+ binding mitochondrial carriers for aspartate/glutamate (AGCs) is provided by their N-terminal extensions, which face the intermembrane space. The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle. We report that their N-terminal extensions contain up to four pairs of EF-hand motifs plus a single vestigial EF-hand, and have no known homolog. Aralar and citrin contain one fully canonical EF-hand pair and aralar two additional half-pairs, in which a single EF-hand is predicted to bind Ca2+. Shuttle activity in brain or skeletal muscle mitochondria, which contain aralar as the major AGC, is activated by Ca2+ with S0.5 values of 280–350 nm; higher than those obtained in liver mitochondria (100–150 nm) that contain citrin as the major AGC. We have used aralar- and citrin-deficient mice to study the role of the two isoforms in heart, which expresses both AGCs. The S0.5 for Ca2+ activation of the shuttle in heart mitochondria is about 300 nm, and it remains essentially unchanged in citrin-deficient mice, although it undergoes a drastic reduction to about 100 nm in aralar-deficient mice. Therefore, aralar and citrin, when expressed as single isoforms in heart, confer differences in Ca2+ activation of shuttle activity, probably associated with their structural differences. In addition, the results reveal that the two AGCs fully account for shuttle activity in mouse heart mitochondria and that no other glutamate transporter can replace the AGCs in this pathway.

to calmodulin (CaM) and CaM-related proteins (6). Unlike SCaMCs, the N-terminal extensions of AGCs (2,3) are not closely related to CaM or any of the known members of the CaM superfamily (23). In the human proteome, the closest related protein is calcium binding atopy-related autoantigen, CBARA1 (24), with 23% identity and 43% similarity with aralar, and 24 and 40% with citrin, respectively. The AGCs catalyze an electrogenic 1:1 exchange of aspartate for glutamate plus a proton (8,9,(25)(26)(27)(28). Brain mitochondria have aralar as the only AGC isoform, and we have recently shown that MAS activity in brain mitochondria is stimulated by extramitochondrial Ca 2ϩ with an S 0.5 for Ca 2ϩ activation of around 320 nM (29), i.e. below the Ca 2ϩ concentrations at which the calcium uniporter is known to be active. This opened up the possibility that neuronal MAS might be activated by cytosolic Ca 2ϩ signals below the threshold of the calcium uniporter. Indeed, with the use of two photon microscopy imaging of mitochondrial NAD(P)H and neuronal cultures derived from aralar-deficient mice (30), we have shown that small Ca 2ϩ signals that do not reach the mitochondrial matrix were able to activate NADH accumulation in mitochondria from control but not aralar-deficient neurons (29) under conditions of lactate utilization.
Aralar and citrin have differences in primary sequence, especially along their N-terminal halves but a very high identity along their MC homology sequence (3), which explains the very similar transport activity of both isoforms (5). In the present work, we have studied whether the differences in their N-terminal sequences could lead to differences in calcium regulation. By studying Ca 2ϩ activation of MAS activity in tissues expressing the individual isoforms, we conclude that citrin has a smaller capacity to be activated by calcium than aralar, with an S 0.5 of about 100 -150 nM.
We have next used this information to study the role of aralar and citrin in heart MAS activity. MAS is the dominant NADH shuttle in heart, (31)(32)(33)(34)(35)(36), including human heart (37). Unlike skeletal muscle, which expresses only aralar, the heart shows high levels of both isoforms, with a preferential enrichment of aralar in atria (38). The role of MAS activity in adult heart is somewhat controversial. It is lower in adults than in neonates, and this has been attributed to a postnatal decrease in the levels of the oxoglutarate-malate carrier (OGC) observed in pig and rabbit, which could limit shuttle function in adult heart (36,39). This contrasts with long standing evidence that AGC rate limits MAS function (8). Moreover, recent reports have introduced added complexity to heart MAS activity. Ralphe et al. (40,41) have proposed that an isoform of the excitatory amino acid transporter type 1 (EAAT1) is present in heart mitochondria where it acts as a glutamate carrier within MAS and is responsible for the up-regulation of MAS by thyroid hormone.
To get a better understanding of the role of aralar and citrin in MAS heart activity and regulation by calcium, we have studied the effects of the selective deficiency of the two isoforms, aralar and citrin by employing mice with targeted disruption of each of the two genes. The results underscore a predominant role of aralar as the isoform that confers Ca 2ϩ regulation to heart MAS activity and strongly suggest that no other carrier can substitute for the AGCs in MAS.

Prediction of EF-hands in Aralar/AGC1 and Citrin/AGC2
Sequences-A multiple sequence alignment (MSA) of known EF-hands was extracted from the Pfam data base (42) and used to build hidden Markov models (HMMs) libraries. The HMMs were then used to search the N-terminal sequences of the aralar-like subfamily of proteins previously obtained from the Uniprot data base (43), using a methodology similar to that employed by Truong and Ikura (44) to study the distribution of EF-hand protein superfamilies. The putative structure of discovered EF-hands was contrasted with the secondary structure prediction of MSAs of aralar and citrin sequences to ensure structural compatibility.
Materials-Fura2-pentapotassium salt and CalciumGreen-5N were from Molecular Probes. MDH was from Boehringer, digitonin from Fluka, bovine serum albumin (fraction V) from Intergen, and the rest of the reagents were from Sigma.
Mice with targeted disruption of the citrin gene were obtained by gene trapping at Lexicon Genetics (The Woodlands, TX) in SVJ129 ES cells using the insertion vector method that was based on the gene trap technology of Lexicon (45) as described previously for Aralar-deficient mice (30). Slc25a13 ϩ/Ϫ ES cells were injected into C57BL blastocysts, and chimeric mice were bred to C57BL (albino) wild-type mice. Slc25a13 ϩ/Ϫ mice with a SVJ129xC57BL/6 inbred genetic background were backcrossed to C57BL/6 mice for at least 8 generations. The resulting slc25a13 ϩ/Ϫ (Citrin ϩ/Ϫ ) offspring were interbred to produce slc25a13 Ϫ/Ϫ (Citrin Ϫ/Ϫ ) mice and Citrin ϩ/ϩ mice in a C57BL/6 genetic background.
All mouse strains and male Wistar rats were housed with a 12-h light cycle and fed ad libitum on a standard chow. Animals were sacrificed by cervical dislocation, the tissue of interest quickly dissected and kept on ice-cold media for mitochondrial isolation carried out at 4°C. Rats were 3-months old. Unless indicated otherwise, mice were used at 15 days to allow comparisons between strains, as mice from one of the strains used, Aralar Ϫ/Ϫ , do not survive beyond 20 days (30). All animal procedures were approved by the Committee for Animal Experimentation, Kagoshima University and European guidelines.
Skeletal Muscle Mitochondria-Skeletal muscle mitochondria were obtained as described by Rolfe et al. (47) with minor modifications. Skeletal muscle was obtained from the four limbs, washed, and minced in IMM (mM; 100 sucrose, 9 EDTA, 1 EGTA, 100 Tris-HCl, 46 KCl, pH7.4). After 10 min of incubation with Nagarse (0.4 mg/ml IMM; stirring on ice), the tissue was homogenized and processed as described for brain and liver.
Heart Mitochondria-Hearts were treated the same way as skeletal muscle, except that the medium for isolation was IMH, mM: 230 manitol, 70 sucrose, 1 EDTA, 5 Tris-HCl, pH 7.4 (48). Proteins were measured by the Bradford method.
Reconstitution of the Malate-Aspartate NADH Shuttle Activity in Mitochondria-The reconstitution of the malate-aspartate NADH shuttle was based on published procedures (49 -51), modified as described in Pardo et al. (29) and Jalil et al. (30). Mitochondrial fractions (0.1-0.15 mg of protein, liver, and brain or 0.020 -0.030 mg of protein, heart and muscle) were suspended in 3 ml of MSK (and 100 M digitonin, in the case of brain preparations), and the shuttle was reconstituted as described (29,30). MAS activity was started by the addition of 5 mM glutamate, and was determined from the decay in NADH fluorescence at 37°C under constant stirring. To correct for any possible changes in free calcium concentration along the assay, the experiments have been also carried out in EGTA-calciumbuffered MSK medium, in which EDTA was replaced with 0.5 mM EGTA.

EF-hands in Citrin and
Aralar-Using an HMM-based methodology, similar to those used by Truong and Ikura (44) in the superfamily of EF-hands-containing proteins, we found a surprising amount of EF-hand compatible motifs in the sequence of both aralar (AGC1) and citrin (AGC2). Up to nine different EF-hands were predicted using this procedure, all located in the first 330 -340 amino acids of members of this family of proteins ( Fig. 1), the ninth EF-hand (positions 302-330 in human aralar), being not equally consistent. The sequence of these long N-terminal extensions in aralar and citrin, and the particular spacing of the set of EF-hands have no known homolog within the large family of calcium-binding proteins.
The analysis of sequence signatures of known Ca 2ϩ -binding proteins (22) compared with those of the proposed eight EFhands of aralar and citrin, yields a number of predictions about their functional characteristics ( Fig. 1). The first EF-hand, EF1 (residues 13-46 in human aralar), is predicted to be active both in aralar and citrin. In fact, the presence of Glu or Asp residues in all signature positions 1, 3 (Asn in citrin), 5, and 12 suggests an enhanced Ca 2ϩ coordination capability of EF1. A similar situation, but showing a more classical signature with Thr in coordination position 3, corresponds to EF2 (residues 54 -84 in human aralar). EF4, EF6, and EF7 (residues 127-154, 194 -223, and 228 -259 in human aralar) appear to be nonfunctional, both in aralar and citrin.
Interestingly, EF3 (corresponding to residues 88 -117 and 89 -118 in human aralar and citrin, respectively) and EF5 (positions 159 -189 (aralar) and 159 -190 (citrin)) are predicted to differ in terms of Ca 2ϩ binding, the canonical signature being present in aralar, but not in citrin (Ref. 22 and Fig. 1). Prediction of functionality of EF8 in aralar (residues 267-295) and citrin (residues 268 -296) based on the presence of the Ca 2ϩ binding signature gave an unclear result. This structure lacks aspartic or glutamic residues located at position 1, being substituted by Asp 276 (aralar) or Asp 277 (citrin) located in motif position 0. Whether or not this variant EF-hand structure could maintain functional ion binding properties cannot be deduced from sequence analysis.
The contribution to Ca 2ϩ binding of the canonical EF-hand motifs in aralar and citrin, respectively, is unknown. However, the sequence differences between the two isoforms may be associated with differences in Ca 2ϩ activation among the two AGCs.
Calcium Activation of the Malate-Aspartate NADH Shuttle in Liver, Brain, and Skeletal Muscle Mitochondria-MAS activity in rat brain mitochondria is activated around 3-fold by Ca 2ϩ , with an S 0.5 for activation of around 320 nM (29). Calcium activation of mitochondrial dehydrogenases, such as isocitrate DH, ␣KGDH, and FAD-linked glycerol-3-phosphate DH, leads to an increase in substrate affinity, with no changes in V max (see Refs. 19 and 55 for reviews). In contrast with this mechanism, the results in Fig. 2, A and B show that Ca 2ϩ activation of MAS in mouse brain mitochondria does not result in changes in the affinity for glutamate. Indeed, the apparent K m for glutamate is the same: 2.76 Ϯ 0.4 mM in the absence of calcium and 2.6 Ϯ 0.2 mM at 10 M free calcium, whereas V max increases about 3-fold in the presence of calcium. To study calcium activation of the malate-aspartate NADH shuttle, MAS activity was reconstituted in mitochondria isolated from adult rat tissues containing a single major AGC isoform, brain with aralar and liver with citrin, respectively. The results obtained in brain have been already reported (29) and are shown in Fig. 2C for comparative purposes. Calcium activation of shuttle activity was assayed in the presence of 200 nM ruthenium red. At this concentration, ruthenium red completely blocked calcium uptake in rat brain mitochondria isolated from 3-month-old animals (29). MAS activity in rat liver mitochondria increased about 1.5-fold in response to extramitochondrial calcium, (from 38 Ϯ 5.7 to 53 Ϯ 6.5 nmol NADH min Ϫ1 mg prot Ϫ1 ), with an S 0,5 for Ca 2ϩ activation of 142 Ϯ 38 nM (Fig. 2C). In contrast, Ca 2ϩ stimulation of MAS activity from rat brain mitochondria was more pro- nounced, resulting in a 3-fold increase in activity (from 26.7 Ϯ 2.64 to 86.18 Ϯ 5.2 nmol NADH min Ϫ1 mg prot Ϫ1 ), with an S 0,5 of 324 Ϯ 57.4 nM (29). Ca 2ϩ activation of MAS activity and S 0,5 values did not vary when assays were performed in calciumbuffered media (results not shown). We have also studied Ca 2ϩ activation of MAS activity in mouse brain and skeletal muscle mitochondria (with aralar as single AGC) and mouse liver mitochondria (citrin as major single AGC) (2,3,4,38). Ca 2ϩ -stimulated MAS activity in all tissues (Fig. 3) and the S 0,5 for activation were higher in brain and skeletal muscle, which have aralar as AGC, compared with liver, which has citrin (316 Ϯ 68 nM brain mitochondria, 280 Ϯ 26 nM for muscle mitochondria, and 120 Ϯ 20 nM liver), in agreement with the results obtained in rat (Fig. 2C). On the other hand, the total activation was the same in all tissues, about 2.5-fold.
The higher S 0,5 value for Ca 2ϩ activation in tissues with aralar instead of citrin as the major AGC isoform suggests that the structural differences among isoforms may be related to their differences in Ca 2ϩ activation.
Ca 2ϩ Activation of Heart Malate-Aspartate NADH Shuttle-MAS is the dominant NADH shuttle in heart (31-36, 56, 57), and the two isoforms aralar and citrin are expressed in rat and mouse heart (2)(3)(4)38). However, the role of each isoform in MAS function remains unknown. Recent reports have suggested that the AGCs are only one of the possible players in the heart malate-aspartate shuttle at the step of glutamate uptake in mitochondria, and that the EAAT1, a plasma membrane glutamate carrier from brain, is localized to heart mitochondria and functions as a glutamate carrier within MAS (40,41). Therefore, it is necessary to clarify the contribution and role of each of the two AGC isoforms to understand MAS function in heart.
To address this point we have studied MAS activity in mouse strains with disrupted citrin or aralar genes. Citrin-deficient mice were generated by gene trapping, the gene trap vector inserted in intron 7 of slc25a13 gene (Fig. 4, A and B), and have a dose-dependent reduction in citrin mRNA and citrin protein levels in liver (Fig. 4, C and D). As observed for a different strain of citrin-deficient mice (58), disruption of the citrin gene also results in a dose-dependent reduction in citrin mRNA and protein levels in kidney and heart without any compensatory change in aralar levels (Fig. 4, C and D). Aralar-deficient mice were also generated by gene trapping as described previously (30). Aralar-deficient and citrin-deficient mice have different backgrounds (hybrid C57BL/6xSv129 and pure C57BL/6, respectively), that are named aralar wild-type and citrin wildtype for simplicity.
Extramitochondrial Ca 2ϩ -activated MAS in heart mitochondria from both wild-type strains as shown in Fig. 5 and Table 1. The S 0,5 for activation was 323 Ϯ 44 nM in wild-type aralar and 367 Ϯ 77 nM in wild-type citrin strains. MAS activity in heart mitochondria from Aralar ϩ/Ϫ mice decreased to about one-half, with no change in Ca 2ϩ activation (Fig. 5A), and it was drastically reduced in heart mitochondria from Aralar Ϫ/Ϫ mice, which have only citrin as the AGC isoform (from 207 Ϯ 31 to 54 Ϯ 13 nmol NADH min Ϫ1 mg prot Ϫ1 as V max values in wild-type and Aralar Ϫ/Ϫ , respectively). Strikingly, Ca 2ϩ activation of MAS was almost lost in Aralar Ϫ/Ϫ mice, the  maximal increase in activity being only 1.3-fold (Fig. 5A), with an S 0,5 of 94 Ϯ 28 nM. This suggests that in heart mitochondria, Ca 2ϩ activation is mainly conferred by the presence of aralar.
MAS activity in heart mitochondria from Citrin ϩ/Ϫ and specially Citrin Ϫ/Ϫ mice was reduced with respect to the wild-type (V o and V max 34 Ϯ 5 and 90 Ϯ 9 nmol NADH min Ϫ1 mg prot Ϫ1 , respectively, in the wild type, versus V o and V max 24 Ϯ 1.6 and 66 Ϯ 4.3 nmol NADH min Ϫ1 mg prot Ϫ1 , respectively, in Citrin Ϫ/Ϫ ) but still activated by Ca 2ϩ with an S 0,5 of 270 Ϯ 38 nM; essentially the same as the wild-type strain (Fig. 5B). Because aralar is the only AGC isoform in the heart of Citrin Ϫ/Ϫ mice, this result further confirms that calcium activation of MAS in heart mitochondria is conferred mainly by aralar.
Strain-dependent Variations in Aralar and Citrin Levels-Unexpectedly, MAS activity in aralar and citrin wild-type mice was quite different, about 2-fold higher in aralar wild-type animals (note the different scales on the y-axis in Fig. 5, A and B and Table 1). As indicated above, the genetic backgrounds of the two types of null animals were different. Aralar wild-type mice, a C57BL/6xSv129 mixed strain (30), whereas citrin-deficient mice, a pure C57BL/6 strain. To study the cause of increased MAS activity in the mixed background, aralar and citrin levels were determined by Western blotting using the ␤-subunit of F1-ATPase as the mitochondrial marker in pure Sv129 and C57BL/6 and mixed C57BL/ 6xSv129 backgrounds (Fig. 5C). Both citrin and aralar were significantly increased in heart mitochondria, from the mixed background with respect to either of the pure backgrounds (Fig. 5, D and E). A similar increase in citrin levels was noted in liver mitochondria (results not shown). However, brain aralar levels were the same in the three backgrounds (results not shown). From these results, we have calculated the contribution of single doses of aralar and citrin to MAS activity in heart mitochondria from mixed C57BL/6xSv129 and pure C57BL/6 strains. As shown in Table 2, one dose of aralar or citrin contributes an activity of about 76.5 or 27 nmol NADH min Ϫ1 mg protein Ϫ1 , respec-  . Ca 2؉ activation of MAS in heart mitochondria. MAS activity was determined in heart mitochondria isolated from 15-day-old mice. A, kinetics of Ca 2ϩ activation in mitochondria from Aralar ϩ/ϩ (circles), Aralar ϩ/Ϫ (triangles), and Aralar Ϫ/Ϫ (squares) mice in C57BL/6xSv129 background. B, kinetics of Ca 2ϩ activation in mitochondria from Citrin ϩ/ϩ (circles), Citrin ϩ/Ϫ (triangles), and Citrin Ϫ/Ϫ (squares) mice in C57BL/6 background. C, isolated heart mitochondria from wild-type mice of different backgrounds were probed by Western blotting against aralar (1:10000) or citrin (1:2000) and with antibodies against ␤F1- ATPase (1:20000). D and E, histograms representing the ratio of aralar/␤F1-ATPase and citrin/␤F1-ATPase, respectively as means Ϯ S.E. (n ϭ 4). Significant differences to the C57BL/6xSv129 background is denoted by *** (p Ͻ 0.005).
tively, to heart MAS activity in C57BL/6xSv129 mice, but only about half those values in C57BL/6 strains. The difference in activities between strains (about 2-fold each) agrees with the increase in aralar and citrin protein levels in the mixed C57BL/ 6xSv129 background observed in Western blots (about 1.4-fold) ( Table 2). Moreover, the kinetics of Ca 2ϩ activation in Aralar ϩ/ϩ , Aralar ϩ/Ϫ , Citrin ϩ/ϩ , and Citrin ϩ/Ϫ mice closely matches that obtained, assuming that individual doses of each AGC contribute independently to the activity and Ca 2ϩ regulation of MAS (compare calculated S 0.5 values for Aralar ϩ/Ϫ and Citrin ϩ/Ϫ mice in Table 2 with experimental S 0.5 values in Table 1). In summary, the results suggest that MAS activity in mouse heart mitochondria can be fully accounted for by the two AGCs, aralar and citrin, as glutamate carriers, because the disruption of each results in a residual MAS activity, which agrees with the level of the undisrupted AGC isoform. Moreover, each isoform appears to contribute independently to MAS activity and Ca 2ϩ regulation. Thus, while other mitochondrial glutamate carriers may be present in heart mitochondria (the glutamate/hydroxyl carrier (59) or the plasma membrane EAAT1 reported to be present in rat heart mitochondria (40, 41)), our results do no support that glutamate carriers that are different from the AGCs function in mouse heart MAS.

DISCUSSION
We report that the AGC members of the CaMC subfamily belong to a novel family of EF-hand Ca 2ϩ -binding proteins. These proteins contain four pairs of EF-hands, and a single nonfunctional hypothetical EF9. Most EF-hand Ca 2ϩ binding motifs occur in pairs, and the two-EF-hand domain is considered to be the functional unit (60,61). In aralar and citrin, only one of these pairs, made up of EF1 and EF2, is predicted to be functional, and deletion studies suggest that in citrin, Ca 2ϩ binding is mostly conferred by the EF1-EF2 pair (3), suggesting that the remaining EF-hands do not play a major role in the absence of EF1-EF2. However, aralar contains two additional EF half-pairs, i.e. two pairs of EF-hands, EF3-EF4 and EF5-EF6, in which only one EF is predicted to bind Ca 2ϩ , occupying the odd position. It is possible that these extra half-pairs contribute to modulate the calcium affinity of the EF1-EF2 pair. Thus, the presence of these two additional EF half-pairs in aralar may result in Ca 2ϩ binding properties that are different than those of citrin.
Indeed, we have found a consistent difference between Ca 2ϩ activation kinetics of MAS in mitochondria containing either aralar or citrin as the only AGC isoform. The S 0.5 for activation in liver, which contains only citrin, is about 100 -150 nM (rat and mouse liver: 142 nM and 120 nM, respectively), but it is significantly higher, 280 -350 nM in brain and skeletal muscle mitochondria, which contain only aralar (rat and mouse brain 324 nM and 316 nM, respectively; skeletal muscle 280 nM) Although these results cannot exclude that interactions with other yet unknown proteins in the intermembrane space are involved in defining the Ca 2ϩ activation values, they suggest that the structural differences among isoforms are associated with their different sensitivity to Ca 2ϩ .
A more straightforward proof of the role of structural differences between aralar and citrin in explaining the difference in Ca 2ϩ regulation arises from the study in the heart, an organ where both isoforms are expressed at high levels, using mice deficient in the two isoforms. While the loss of citrin results in MAS activity with similar Ca 2ϩ activation kinetics as the wildtype animals, and an S 0.5 close to the aralar values (280 -350

TABLE 2 Calculated contributions of individual AGC isoforms to MAS activity in heart mitochondria
The contribution of one dose of Aralar to MAS activity in C57BL/6xSv129 mice was calculated as the difference in MAS activity between Aralar ϩ/ϩ and Aralar Ϫ/Ϫ mice divided by two. The contribution of one dose of citrin in the C57BL/6 strain was calculated in a similar way from the activities in the Citrin ϩ/ϩ and Citrin Ϫ/Ϫ mice. In C57BL/6xSv129 mice, the contribution of one dose of citrin was calculated as half of the MAS activity in Aralar Ϫ/Ϫ mice, and a similar procedure was used to calculate one dose of aralar in C57BL/6 citrin Ϫ/Ϫ mice. The V max numbers are shown. Total MAS activity in each background was calculated as the sum of four independent equations , each one corresponding to one of the four possible AGC doses, in which V 0 and V max were the calculated values, S 0.5 for aralar was that obtained in the Citrin Ϫ/Ϫ mouse (Table I), and the S 0.5 for citrin was that obtained in the Aralar Ϫ/Ϫ mouse ( nM), the loss of aralar, which results in MAS activity exclusively dependent on citrin, is associated with a drastic loss of Ca 2ϩ activation of MAS in heart mitochondria and an S 0.5 for activation corresponding to the citrin values (100 -150 nM). This proves that the Ca 2ϩ activation properties of citrin and aralar when tested in one tissue, the heart, are indeed quite different; a result that strongly emphasizes the notion that the differences between the two AGCs are caused by their structural differences rather than to tissue-specific interactions.
On the other hand, the variations of Ca 2ϩ activation values for the same AGC in different tissues (citrin in rat and mouse liver and aralar-deficient mouse heart, aralar in brain, and skeletal muscle or citrin-deficient heart), may reflect tissue-specific interactions with yet unknown proteins in the intermembrane space, or the formation of homodimers and tetramers in some of these tissues as has been recently reported (62).
What is the functional role of these differences in Ca 2ϩ activation between isoforms? It is self-evident that an S 0.5 close to resting cytosolic Ca 2ϩ levels will maintain AGC fully active in the resting state. The AGC-dependent changes in MAS activity will merely reflect the changes in substrates and products, i.e. follow the changes in mass action ratio, but it is doubtful that calcium activation plays any significant role. This is the probable situation with citrin, at least in those tissues, such as rat liver, where the S 0.5 for calcium activation is lowest. On the other hand, higher S 0.5 values, as those found in mitochondria where aralar is the only isoform (like brain), are better suited to provide Ca 2ϩ activation of MAS in response to small Ca 2ϩ signals, under conditions where the Ca 2ϩ uniporter is not operating, or at the beginning and end of each miniature increase in mitochondrial Ca 2ϩ or Ca 2ϩ mark (63). In serving as the forefront of the mitochondrial response to a Ca 2ϩ signal, aralar may prime mitochondria to respond to Ca 2ϩ by prolonging mitochondrial energization beyond the duration of the Ca 2ϩ mark (19). This is the situation found in CNS neurons (29).
The role of the two isoforms, aralar and citrin, present together in heart is intriguing. We have previously shown that aralar is enriched in atria (38), while citrin appears to be enriched in ventricles, 4 suggesting a differential localization of the two isoforms within the heart as a whole. Moreover, it is possible that these isoforms are distributed within specialized subsets of mitochondria or even within different subcompartments in the inner mitochondrial membrane (64). It is known that atrial cell mitochondria are heterogeneous with regards to their responses to excitation contraction coupling (65). Unlike ventricular myocytes, that have a well developed t-tubule system with voltage-operated Ca 2ϩ channels (VOCs) juxtaposed to ryanodine receptors (RyR) in the neighboring sarcoplasmic reticulum, in atrial cells VOCs are only present on the outer membrane surrounding the cell, and only small numbers of RyR are located close to the VOCs (65-67). These "junctional RyR" located at the periphery of the cell respond to the opening of VOCs originating a sub-sarcolemmal Ca 2ϩ signal that does not propagate into the center of atrial myocytes. Mitochondria are located both at the periphery and center of atrial myocytes, but only those located at the periphery, close to the junctional RyR, are involved in calcium uptake and buffering under normal pacing conditions (65,68). Whether aralar and citrin have a differential distribution within these specialized mitochondria and participate in shaping the mitochondrial response to Ca 2ϩ signals are key questions requiring further investigation.