Myocardial ischemia and subsequent reperfusion trigger a deleterious cascade involving mitochondrial dysfunction, excessive reactive oxygen species (ROS) production, calcium overload, and mitochondrial permeability transition pore (mPTP) activation. In this context, the mitochondrial ATP-sensitive potassium channel (mitoKATP) emerges as a central regulator of cellular homeostasis, modulated by both endogenous pathways (ischemic preconditioning) and pharmacological interventions. This thesis meticulously investigated the molecular mechanisms of mitoKATP, focusing on: (i) its allosteric regulation by nucleotides (ATP/GTP); (ii) the pharmacology of specific ligands (diazoxide and glibenclamide); and (iii) its functional implications in protection against ischemia/reperfusion (I/R) injury. Through a multimethodological approach combining physiological, biochemical, and computational techniques, we obtained the following key findings: Glibenclamide inhibited mitoKATP with an IC50 of 42 ± 3 nM, acting as a competitive antagonist of diazoxide (Ki = 28 nM), as demonstrated in mitochondrial swelling assays and isolated heart perfusion. The IMP-A analog (lacking the cyclohexylurea moiety) showed complete loss of inhibitory activity (IC50 > 500 μM), confirming the essentiality of this domain for binding to the ABCB8 sulfonylurea site. ATP inhibited channel activity with an EC50 of 180 ± 15 μM, while GTP reversed this effect with an EC50 of 85 ± 7 μM, indicating biphasic modulation. Molecular docking simulations (ΔG = -8.2 kcal/mol for ATP vs -9.5 kcal/mol for GTP) revealed distinct binding sites on the ABCB8 NBD domain, explaining the observed competition. MitoKATP activation by diazoxide (100 μM) reduced infarct size by 58 ± 6% (p < 0.01 vs I/R control), an effect completely abolished by glibenclamide (10 μM). Diazoxide-treated hearts showed lower LDH release (124 ± 18 U/L vs 287 ± 24 U/L in I/R group) and better recovery of developed ventricular pressure (72 ± 5% vs 41 ± 4% of baseline). Channel opening decreased ROS production by 40 ± 5%, as measured by DCFH-DA probe in isolated mitochondria. These results not only elucidate the molecular determinants of mitoKATP regulation but also validate its relevance as a therapeutic target. The precise characterization of nucleotide and drug binding sites opens new perspectives for developing more selective modulators, with potential applications in acute ischemic syndromes and organ transplant protection.