We showed that swelling of RLM was stimulated by the TSPO ligand PPIX and that Cbx significantly potentiated this effect (Fig. in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO. and apoptosis inducing factor [3C5]. Recently, we compared the mechanism of action of Cbx on Ca2+-induced mPTP opening in rat brain mitochondria (RBM, synaptic and non-synaptic) and rat liver mitochondria (RLM), in an attempt to identify the mitochondrial target of Cbx . Our data showed that Cbx altered the parameters of mPTP function by shortening the lag time of MPT onset (lowering the capacity to retain Ca2+ in the matrix) and initiating Ca2+-induced Ca2+ efflux from the mitochondrial matrix . Cbx increased Ca2+-induced high amplitude swelling of both RBM and RLM. LDN-27219 Cbx-stimulated Ca2+ efflux and Ca2+-induced high amplitude swelling of mitochondria were CsA sensitive . These effects of Cbx were not linked to ROS production, however, connexin43 (Cx43) was identified to be the target of Cbx . Connexins (Cx) are a family of proteins that form gap junction megachannels that mediate intercellular communication and allow inorganic ions and small organic signaling molecules to diffuse rapidly and directly from the cytoplasm of one cell to LDN-27219 another . The presence of connexin43 (Cx43) in mitochondria has been reported [7C11] and it was proposed that Cx43 may function in protective preconditioning mechanism [8,11]. Cbx is a universal effective water-soluble blocker of gap junctions . The presence of Cx43 in mitochondria suggested that connexins might be the target for Cbx in mitochondria. Indeed, we detected Cx43 in rat brain and heart mitochondria, but not in liver mitochondria. Col4a6 However, Cx26 and Cx32 were found in rat liver mitochondria and may also be targets for Cbx . Cbx being a gap junction inhibitor has a structural similarity to the LDN-27219 steroids . The initial steps of steroidogenesis take place in the mitochondria of steroid producing tissues, including adrenals, gonads, placenta, brain, and liver [13,14]. In these tissues, steroid formation is initiated with the transfer of the substrate cholesterol from intracellular stores to the inner mitochondrial membrane. Cholesterol transport into mitochondria is mediated by the translocator protein (18 kDa) TSPO, previously known as the peripheral-type benzodiazepine receptor, a high affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane [15,16]. Cholesterol binding to TSPO occurs at the cholesterol binding amino acid consensus sequence -L/V-(X1C5-Y-(X)1C5-R/K- [15,16]. Interestingly, a comparable cholesterol binding amino acid consensus sequence (CRAC motif) was detected in both Cx43 and Cx32 [17,18]. TSPO has been implicated in mPTP functions [14,19C21]. TSPO-associated mitochondrial proteins have been described, including the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) [22C24], which both are considered to be major modulators of mPTP. Modulation of mPTP by chemicals opening or closing the channel alters the ability of steroidogenic cells to form steroids . Moreover, TSPO ligands have been shown to modulate mPTP function [19,25]. We also reported that TSPO ligands modulate in a Ca2+- and CsA-dependent manner the phosphorylation of 43C46 kDa, 21 kDa and 17 kDa proteins, as well as of a 3.5 kDa peptide. The phosphorylation status of these proteins and peptide was shown to change depending on the opened/closed state of the pore . These phosphoproteins were identified: 46 kDa phosphoprotein is 2,3-cyclic nucleotide-3-phosphodiestearase , 21 kDa and 17 kDa phosphoproteins are isoforms of myelin basic protein (MBP) , and the 3.5 kDa phosphopeptide is subunit of ATP synthase . Incubation of the rat brain mitochondria (RBM) with anti-TSPO antibodies specifically prevented these phosphorylations, suggesting that TSPO participates in the modulation of mPTP opening. It was previously reported that in the presence of the anti-TSPO antibody there was strong suppression of the Ca2+.