Human adenine nucleotide translocator isoforms mediate functionally redundant mitochondrial ADP/ATP exchange

The adenine nucleotide translocator (ANT) is essential for cellular energy metabolism, mediating the exchange of ADP and ATP across the inner mitochondrial membrane. While mammals express multiple ANT isoforms, it remains unclear whether they possess distinct properties or whether their diverse expression patterns reflect functional specialization as widely posited. In this study, we employed a controlled human cell system to directly compare the transport kinetics and bioenergetic contributions of individual human ANT isoforms. By evaluating endogenous knockout cells and systematically re-expressing each isoform, we demonstrate that ANT1, ANT2, and ANT3 are functionally redundant. The three ANT isoforms exhibited near-identical transport rates in both forward (ADPin/ATPout) and reverse (ATPin/ADPout) exchanges. While transport kinetics were equivalent, cells solely expressing ANT2 or ANT3 exhibited slightly different resilience to respiratory inhibition, suggesting isoform-specific roles that may be related to non-transport functions. Our findings provide a refined view of ANT-mediated mitochondrial nucleotide flux and suggest that ANT isoform-specific differences, if they exist, are likely subtle and not innately tied to ADP/ATP exchange.