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

Abstract

Ca2؉ regulation of the Ca 2؉ 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 halfpairs, in which a single EF-hand is predicted to bind Ca 2؉. Shuttle activity in brain or skeletal muscle mitochondria, which contain aralar as the major AGC, is activated by Ca 2؉ with S 0.5 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 S 0.5 for Ca 2؉ 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 Ca 2؉ 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.

Calcium Signaling in Brain Mitochondria

“…In contrast, MAS activity in liver mitochondria is not affected by intramitochondrial Ca 2ϩ , as its activity is the same in the absence or presence of RR, although it is activated by extramitochondrial calcium to a smaller extent than in brain or heart (Fig. 1C) as reported earlier (36).…”

“…In addition, Ca 2ϩ binding to aralar on the external side of the inner mitochondrial membrane activates MAS (4,36) and explains the activation of respiration of brain mitochondria by extramitochondrial Ca 2ϩ when malate plus glutamate are used as substrates (69). In this study we report that the effect of matrix Ca 2ϩ on ␣KGDH results in an inhibition of MAS activity, at least in two tissues, brain and heart, but not in liver.…”

“…2ϩ -MAS activity was shown to increase in response to extramitochondrial calcium in brain and heart mitochondria (3,4,36). Paradoxically, it became inhibited in a calcium-dependent manner when calcium was allowed to enter the mitochondria through the Ca 2ϩ uniporter (4).…”

The control of brain mitochondrial energization by cytosolic calcium: The mitochondrial gas pedal

“…As in the case of a-KGDH, the sensitivity of ICDH increases with decreasing ATP/ADP ratios and Ca 2þ activation is realized through decreasing K m for the substrates without changes in V max values (27). (38)(39)(40)(41). Similarly, the oxidation of a-glycerophosphate (a-GP) by brain mitochondria is activated by Ca 2þ cyt , but in this case the V max is unchanged, and rather the affinity of mitochondrial a-GPDH to a-GP is increased (38).…”

“…The formation of pyruvate from its precursors is coupled to NAD þ regeneration via the MAS. The activity of aralar, the glutamate-aspartate carrier as the central enzyme of the MAS, is controlled by Ca 2þ cyt (38)(39)(40)(41). Aralar transports glutamate into the mitochondria, but under steady-state conditions this glutamate is not metabolised there since it will be regenerated within the complete MAS (36).…”