Mitochondrial Ca2+ uptake has a key role in cellular Ca2+ homeostasis. Excessive matrix Ca2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH2-linked substrates. The functional consequences of Ca2+-induced mPTP opening were assessed by Ca2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca2+ overload.

A range of ligands displayed agonism at the long isoform of the human dopamine D 2 receptor, whether using receptor-G protein fusions or membranes of cells in which pertussis toxinresistant mutants of individual G␣ i -family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [35 S]GTP␥S onto each of G␣ i1 , G␣ i2 , G␣ i3 , and G␣ o1 . By contrast, (S)-(Ϫ)-3-(3-hydroxyphenyl)-N-propylpiperidine was a partial agonist when G␣ o1 was the target G protein but an antagonist/inverse agonist at G␣ i1 , G␣ i2 , and G␣ i3 . In ligand binding assays, dopamine identified both high-and lowaffinity states at each of the dopamine D 2 receptor-G protein fusion proteins, and the high-affinity state was eliminated by guanine nucleotide. (S)-(Ϫ)-3-(3-Hydroxyphenyl)-N-propylpiperidine bound to an apparent single state of the constructs in which the D 2 receptor was fused to G␣ i1 , G␣ i2 , or G␣ i3 . However, it bound to distinct high-and low-affinity states of the D 2 receptor-G␣ o1 fusion, with the high-affinity state being eliminated by guanine nucleotide. Likewise, although dopamine identified guanine nucleotide-sensitive high-affinity states of the D 2 receptor when expression of pertussis toxin-resistant forms of each of G␣ i1 , G␣ i2 , G␣ i3 , and G␣ o1 was induced, (S)-(Ϫ)-3-(3-hydroxyphenyl)-N-propylpiperidine identified a high-affinity site only in the presence of G␣ o1 . p-Tyramine displayed a protean ligand profile similar to that of (S)-(Ϫ)-3-(3-hydroxyphenyl)-N-propylpiperidine but with lower potency. These results demonstrate (S)-(Ϫ)-3-(3-hydroxyphenyl)-N-propylpiperidine to be a protean agonist at the D 2 receptor and may explain in vivo actions of this ligand.A large number of G protein-coupled receptors (GPCRs) are able to generate a variety of intracellular signals, and for those with a rich pharmacology of synthetic small-molecule ligands, it has often been possible to observe differential pharmacology for individual end points (Perez and Karnik, 2005). This has resulted in an appreciation that different ligands may stabilize distinct conformational states of GPCRs (Kenakin, 2001;Perez and Karnik, 2005) and in an expansion of the simple "active" or "inactive" "two-state" model (Leff, 1996) of GPCR function into "three-state" models (Leff et al., 1997) and subsequent chemical and physical considerations of GPCRs that allow the potential for an essentially unlimited number of states (Milligan and IJzerman, 2000;Vauquelin and Van Liefde, 2005). Although GPCRs are defined by their capacity to activate heterotrimeric G proteins, a number of ligand-induced signals seem not to require G protein interactions (Wei et al., 2003;Gesty-Palmer et al., 2006). In the case of the ␤ 2 -adrenoceptor, for example, such separation of signal transduction has resulted in the identification of ligands that can be defined as inverse agonists for their effects on adenylyl cyclase activity but as agonists for their capacity...

The human dopamine D 2L receptor couples promiscuously to multiple members of the G␣ i/o subfamily. Despite the high homology of the D 2L and D 3 receptors, the G protein coupling specificity of the human D 3 receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D 2L and D 3 receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of individual G␣ i -family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D 3 receptor to G␣ o1 . Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D 3 receptor when coupled to G␣ o1 compared with the D 2L receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D 2L receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D 3 /D 2 receptor in which the equivalent region of the D 3 receptor was exchanged for this sequence. This chimera displayed D 3 -like affinity for [ 3 H]spiperone and potency for agonists but gained D 2 -like ability to couple to each of G␣ i1-3 as well as G␣ o1 .In recent studies, we have shown that the long isoform of the human dopamine D 2 receptor (D 2L receptor) couples efficiently and promiscuously to all four G␣ i/o subtypes . This confirmed previous work by other groups, although the relative order of preference remains controversial (Watts et al., 1998;Jiang et al., 2001;Gazi et al., 2003;Senogles et al., 2004). Although the D 2 receptor is considered a key target for antipsychotic drugs, there has been considerable interest in the contribution that the dopamine D 3 receptor may provide (Gurevich et al., 1997;Joyce, 2001;Joyce and Millan, 2005;Sokoloff et al., 2006). Human dopamine D 2 and D 3 receptors have an overall amino acid similarity of 52%, which increases to 78% if only transmembrane regions are considered (Sokoloff et al., 1990). As a result, D 2 and D 3 receptors exhibit a similar pharmacological profile (Ahlgren-Beckendorf and Levant, 2004). However, signal transduction by the D 3 receptor is less clearly understood than that of the D 2 receptor. Studies to elucidate cellular responses to D 3 receptor activation are hampered by the coexpression of D 2 and D 3 receptors in brain tissue and the lack of definitively selective pharmacological tools. It follows, then, that the majority of data have been obtained by expression of the receptors in heterologous systems. Initial reports indicated that the D 3 receptor, when expressed in a variety of cell systems, did not exhibit a decrease in affinity for agonists in the presence of guanine nucleotides a...

Production of antisera able to recognize individual heterotrimeric G protein alpha subunits resulted in rapid expansion of information on their distribution and function. However, no antibodies that specifically recognize the active state have been available. Four-way primary screening of 763 hybridomas generated from mice immunized with guanosine 5'-O-(3-thio)triphosphate-loaded G alpha(i1) and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q(204)L, mutant but neither the constitutively inactive, G(203)A, mutant nor wild-type G alpha(i1). This profile extended to other closely related G(i) family G proteins but not to the less closely related G alpha(s) and G alpha(q)/G alpha(11) families. Each antibody was, however, also able to identify wild-type, GDP-bound G(i) family G proteins in the presence of fluoroaluminate, which mimics the presence of the terminal phosphate of GTP and hence generates an active/transition state conformation. Stimulation of cells coexpressing a wild-type G alpha(i) subunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformationally selective antibodies to bind the G protein. Such reagents allow the specific identification of activated G proteins in a native environment and may allow the development of label-free screening assays for G protein-coupled receptor-mediated activation of G(i) family G proteins.