Abstract
Ca(2+) 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 half-pairs, 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.
Highlights
Mitochondrial carriers, MCs,3 are integral proteins of the mitochondrial inner membrane that function in the shuttling of
Brain mitochondria have aralar as the only aspartate/glutamate carriers (AGC) isoform, and we have recently shown that malate-aspartate NADH shuttle (MAS) activity in brain mitochondria is stimulated by extramitochondrial Ca2ϩ with an S0.5 for Ca2ϩ activation of around 320 nM [29], i.e. below the Ca2ϩ concentrations at which the calcium uniporter is known to be active
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 Ca2ϩ 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
Summary
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. 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]. Nuclei and cell debris were first removed by 10 min of centrifugation at 700 ϫ g, and mitochondrial fractions were spun down (15 min, 10,000 ϫ g), and washed in MSK (mM: 75 mannitol, 25 sucrose, 5 potassium phosphate, 20 Tris-HCl, 0.5 EDTA, 100 KCl, and 0.1% bovine serum albumin, pH 7.4). 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). Membranes were incubated for 45 min with a goat anti-rabbit IgG conjugated with horseradish peroxidase (1:10000, Bio-Rad), and the signal was detected by chemiluminescence (ECL, PerkinElmer), as described earlier [38]
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