ANT located in the IMM mediates the exchange of ATP
ANT, located in the IMM, mediates the exchange of ATP/ADP between the mitochondrial matrix and the intermembrane space (IMS) (Brand et al., 2005; Palmieri and Pierri, 2010). Of the 4 known isoforms, ANT1 is the dominant isoform in the heart (Palmieri and Pierri, 2010). Under physiological conditions, VDAC and ANT are functionally coupled, which allows for the efficient transfer of metabolites across mitochondrial membranes (Allouche et al., 2012; Camara et al., 2017; Vyssokikh and Brdiczka, 2003). Using surface plasmon resonance (Allouche et al., 2012) ANT and VDAC1 were found to have direct structural interactions that were dependent on the ionic conditions and ionic strength of the experimental buffer. The binding of the ANT inhibitor atractyloside (ATR) or bongkrekic Vincristine (BKA) reduced the interaction of ANT and VDAC1 in vitro (Allouche et al., 2012). It is not known if oxidant stress conditions, such as excess peroxynitrite (ONOO−) production from the reaction of superoxide (O2•−) and nitric oxide (NO•) during I/R injury, induces modification of ANT by tyrosine nitration and alters its interaction with VDAC1. VDAC, the most abundant protein in the OMM, plays a crucial role in both mitochondrial metabolism and cell death (Colombini, 2012; Shoshan-Barmatz and Ben-Hail, 2012). In the open state, VDAC favors the transport of anions, such as metabolites, ATP, ADP, and Pi, but it also permits the free diffusion of cations, including Ca2+, K+, and Na+ (Mazure, 2016; O'Rourke, 2007), whereas in the closed state, VDAC favors cationic permeability, notably Ca2+ ions, which in excess impairs ADP/ATP transport (O'Rourke, 2007). VDAC exists as three isoforms in mammals, VDACs 1, 2 and 3; the protein spans the OMM with 19 β-strands (Messina et al., 2012; Raghavan et al., 2012). Among the three isoforms, VDAC1 is the most abundant in heart mitochondria and it plays a role in modulating cardiac IR injury (Das et al., 2012; McCommis and Baines, 2012; Shoshan-Barmatz and Ben-Hail, 2012; Shoshan-Barmatz et al., 2008). The mechanisms of how VDAC1 regulates cell death in cardiac IR injury remain incompletely understood. There are reports that reducing cytosolic ATP entry into mitochondria via VDAC during ischemia and its subsequent consumption by the F1F0-ATPase might better preserve cellular ATP, thereby reducing glycolysis, ischemic acidosis, and intracellular Ca2+ overload, to culminate in protection against IR injury (Das et al., 2012; McCommis and Baines, 2012; Murphy and Steenbergen, 2008). In addition, the regulation of VDAC1 function by protein-protein interactions with, for example ANT and hexokinase II (HK II), and the effects of deleterious post-translational modifications (dPTMs) on ANT and VDAC, may also play a major role in altering cell death pathways. Studies in cardiac and skeletal myocytes show that VDAC reversibly binds with several cytosolic proteins, including HK II (Das et al., 2012; Perevoshchikova et al., 2010; Zorov et al., 2009). The predominant isoform of hexokinase in the myocardium is HK II, which binds to mitochondria where it acts as an important regulator of mitochondria-mediated cell demise (Azoulay-Zohar et al., 2004; Sun et al., 2008). In this case, the association of HK II with mitochondria inhibits the mitochondrial translocation of Bax, a proapoptotic protein, and the release of cyt c (Majewski et al., 2004; Pastorino et al., 2002), thereby impeding cell apoptosis (Das et al., 2012; Zorov et al., 2009). It has been reported that ischemia (Pasdois et al., 2012) or glucose deprivation in adult hearts or isolated cardiac myocytes (Calmettes et al., 2013) induces HK II dissociation from mitochondria, possibly from VDAC, which destabilizes the mitochondrial contact sites between VDAC and ANT, causing OMM permeabilization and inducing mitochondrial cyt c loss to promote apoptosis (Pasdois et al., 2012). The mechanism that regulates association/dissociation of HK II with/from VDAC is not clear. Suffice it to say, activation of glycogen synthase kinase 3β (GSK-3β) phosphorylates VDAC and this may disrupt the binding of HK II to potentiate cell death (Pastorino et al., 2005). We have reported (Yang et al., 2012) that ONOO− produced during cardiac IR injury induced tyrosine nitration of VDAC1 on at least one specific tyrosine residue, but it is unknown if this modification also alters the association of HK II with VDAC1.