**6. Mitochondrial PTP gating by TH**

In testing the role played by PTP gating in TH action, a straightforward approach would be to examine whether TH-induced uncoupling is inhibited by the PTP specific inhibitor, cyclosporin A (CSA). CSA acts as a potent inhibitor of PTP gating due to its binding to CypD, resulting in interfering with CypD interaction with PTP-ANT (64, 65). Indeed, TH-induced lowering of mitochondrial membrane potential and proton gradient followed by mitochondrial swelling are all eliminated by added CSA, pointing to PTP involvement in TH mitochondrial activity (66, 67). In addition, liver mitochondria of hypothyroid rats show decrease in mitochondrial Ca+2 efflux, swelling and protein release, being restored by TH treatment (68-70). Furthermore, TH treatment of Jurkat cells induces induce LC-PTP gating (71), implying that mitochondrial PTP may serve as target for TH in inducing mitochondrial uncoupling. However, as described below, TH activity in gating PTP is not accounted for by modulating gene expression of structural components of mitochondrial PTP.

**Adenine Nucleotide Translocase (ANT):** ANT is a central player in oxidative phosphorylation due to its primary function in translocating adenine-nucleotides via the IMM. ANT function in the PTP context has been verified by its direct association with CypD and VDAC (72), as well as by PTP gating being activated and inhibited by the ANT ligands Atractylate and Bongrekic acid, respectively (73). Moreover, ANT/CypD/VDAC- reconstituted liposomes show PTP characteristics in terms of sensitivity to Ca+2, CSA and ANT ligands ((74, 75) but see also (76)). Also, over-expression of ANT isoforms (ANT1, ANT3) promotes apoptosis, being inhibited by CSA (77, 78). Moreover, ANT expression levels affect mitochondrial IMM potential, with high ANT levels resulting in IMM depolarization and mitochondrial proton leak (71, 77, 79). Hence, in light of ANT structural and regulatory functions in the PTP context, and since the expression level of ANT2, the only ANT isoform expressed in liver, is increased in hyperthyroidism and decreased by hypothyroidism (80), TH-induced ANT expression could apparently account for liver TH-induced LC-PTP gating. However, over-expression of ANT2 in HeLa cell line or in rat primary hepatocytes resulted in extensive mitochondrial depolarization that was not inhibited by CSA (71), implying the formation of PTP-nonrelated ANT channels (81), or of CSA-insensitive PTP (82). Lack of an obligatory linkage between PTP and ANT conforms to other findings pointing to PTP gating by proapoptotic ligands in liver cells or isolated mitochondria that lack ANT (76).

280 Thyroid Hormone

dependent anion channel (VDAC, in the OMM), cyclophylin D (CypD, in the mitochondrial matrix) and the Bcl2 family of proteins (in the OMM). PTP gating may present itself in definitive or transient modes, differing in reversibility and synchronization (51, 52). Definitive synchronized PTP gating is induced by intramitochondrial Ca+2 load (53), and is enhanced by oxidative stress, depletion of adenine nucleotides, increased inorganic phosphate, increased matrix pH, and depolarization of the IMM (54-56). This opening/gating mode results in high-conductance PTP (HC-PTP), extensive depolarization of the IMM (~70% decrease in IMM potential), rapid passage of ions and solutes of less than 1500 Da across the IMM, and mitochondrial swelling. These may lead to rupture of the OMM, release of mitochondrial proapoptotic proteins (such as cytochrome c, apoptotic intrinsic factor), followed by programmed cell death/apoptosis (57). Alternatively, spontaneous, non-synchronized, transient/flickering PTP gating due to cyclic opening and closure of individual PTP channels may result in reversible and limited depolarization of the IMM (~30% decrease in IMM potential), moderate decrease in proton motive force, and passage of solutes of less than 300 Da, accompanied by mitochondrial contraction rather than swelling (58-63). Most importantly, in contrast to the irreversible proapoptotic depolarization inflicted by definitive PTP gating, transient low conductance PTP (LC-PTP) gating is innocuous and reversible, leading to mild mitochondrial uncoupling. These findings may indicate that LC-PTP may serve as mitochondrial target of TH in inducing

In testing the role played by PTP gating in TH action, a straightforward approach would be to examine whether TH-induced uncoupling is inhibited by the PTP specific inhibitor, cyclosporin A (CSA). CSA acts as a potent inhibitor of PTP gating due to its binding to CypD, resulting in interfering with CypD interaction with PTP-ANT (64, 65). Indeed, TH-induced lowering of mitochondrial membrane potential and proton gradient followed by mitochondrial swelling are all eliminated by added CSA, pointing to PTP involvement in TH mitochondrial activity (66, 67). In addition, liver mitochondria of hypothyroid rats show decrease in mitochondrial Ca+2 efflux, swelling and protein release, being restored by TH treatment (68-70). Furthermore, TH treatment of Jurkat cells induces induce LC-PTP gating (71), implying that mitochondrial PTP may serve as target for TH in inducing mitochondrial uncoupling. However, as described below, TH activity in gating PTP is not accounted for by

**Adenine Nucleotide Translocase (ANT):** ANT is a central player in oxidative phosphorylation due to its primary function in translocating adenine-nucleotides via the IMM. ANT function in the PTP context has been verified by its direct association with CypD and VDAC (72), as well as by PTP gating being activated and inhibited by the ANT ligands Atractylate and Bongrekic acid, respectively (73). Moreover, ANT/CypD/VDAC- reconstituted liposomes show PTP characteristics in terms of sensitivity to Ca+2, CSA and ANT ligands ((74, 75) but see also (76)). Also, over-expression of ANT isoforms (ANT1, ANT3) promotes apoptosis, being inhibited by CSA (77, 78). Moreover, ANT expression levels affect

modulating gene expression of structural components of mitochondrial PTP.

physiological mitochondrial uncoupling and calorigenesis.

**6. Mitochondrial PTP gating by TH** 

**Cyclophilin D (CypD):** CypD is a member of the family of peptidyl-prolyl cis-trans isomerases (PPIase) (83). The CypD protein contains a mitochondrial-targeting sequence that directs it specifically to the mitochondrial matrix. The link between CypD and PTP has been verified by CypD direct association with ANT (72) as well as by CSA inhibition of PTP gating due to its interaction and inhibition of CypD activity (64, 65, 84). Moreover, PTP opening by Ca+2 and oxidative stress was enhanced in isolated mitochondria of neurons over-expressing CypD (85), while CSA-sensitive PTP opening was abrogated in isolated mitochondria of CypD knock-out mice (86). These CypD characteristics may indicate that CypD could apparently serve as protein target of TH in inducing PTP opening and mitochondrial uncoupling. Indeed, liver mitochondria of hyperthyroid rats show increased expression of CypD as well as its PPIase enzymatic activity (71), while opposite effects prevailed in liver mitochondria isolated from hypothyroid rats. However, over-expression of CypD in HeLa cell line, or in rat primary hepatocytes, resulted in mitochondrial hyperpolarization rather than PTP opening (71, 87). Moreover, over-expressed CypD was found to desensitize cells to apoptotic stimuli or to protect cells from mitochondrial depolarization and apoptosis induced by over-expression of ANT1 (77, 78). Hence, THinduced CypD expression may not account for TH-induced PTP gating and calorigenesis. CypD induction by TH may reflect TH activity in inducing peptidyl-prolyl cis-trans isomerase activity and protein folding rather than PTP opening (88).

**Voltage Dependent Anion Channel (VDAC):** VDAC is a highly abundant protein of the OMM. Its primary function consists of exchanging anions between the cytosol and the intermembrane mitochondrial space (89). Previous findings have indicated its putative role in gating mitochondrial PTP (90-94). However, its expression level is not changed by *in vivo* TH treatment (71), excluding VDAC from being a molecular target of TH in inducing mitochondrial uncoupling.
