The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells.

Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson's disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson's disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson's disease, providing novel opportunities for therapeutic intervention.

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-ß hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-ß hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Molecular modeling of the GABA/GABA(B) receptor complex.

A three-dimensional model of the extracellular domain of the GABA(B) receptor has been built by homology with the leucine/isoleucine/valine-binding protein. The complete putative GABA-binding site in the extracellular domain is described in both the open and closed states. The dynamics of the "Venus flytrap" mechanism has been studied, suggesting that the molecular dipole moments play a key role in GABA binding and receptor activation. Important residues putatively implicated either in ligand binding or in the dynamics of the receptor are pinpointed, thus highlighting target residues for mutagenesis experiments and model validation.

Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay.

The constitutive activation of G-protein-coupled receptors is a major new approach to investigating their physiopathology and pharmacology. A large number of spontaneous and site-directed mutations resulting in constitutive activity have been identified, but systematic mapping of the amino acids involved for a given receptor would be extremely useful for complete elucidation of the molecular mechanisms underlying its activation. We carried out such mapping for the angiotensin II type 1A (AT(1A)) receptor by screening a randomly mutated cDNA library after expressing the mutated clones in eukaryotic cells. To test the AT(1A) mutants generated, we developed an original, specific, and highly sensitive assay based on the properties of CGP42112A. This classical AT(2) agonist is a weak partial agonist of the wild-type AT(1A) receptor and becomes a full agonist for constitutively active AT(1A) mutants, as shown experimentally and in allostery-based theoretical models. Activation of the mutated receptors by CGP42112A was monitored by using the bioluminescent protein aequorin, a very sensitive and specific sensor of intracellular calcium mobilization. The screening of 4,800 clones, providing an exhaustive coverage of all of the mutations generated, led to the identification of 16 mutations in sequences encoding the transmembrane domains that were responsible for high sensitivity to CGP42112A. The constitutive activity was confirmed by agonist-independent production of inositol phosphates, which showed that at least half of the clones had significantly increased basal activity. These data demonstrate that this new type of approach is very efficient for the systematic identification of constitutively active mutants of G-protein-coupled receptors.

A live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity.

Endothelin-1 (ET-1) is formed from its precursor preproET-1 via the cleavage of the intermediate bigET-1 by endothelin-converting enzyme (ECE-1). However, the subcellular site at which this step occurs is not clear: It could occur intravesicularly along the secretory pathway or bigET-1 might be released and processed extracellularly. To address this point, we have developed an integrated autocrine system that uses a recombinant Chinese hamster ovary (CHO) luciferase reporter cell line that permanently expresses the human ET(A) receptor. Into these cells we transiently transfected human ECE-1a cDNA, either together with the human preproET-1 cDNA (as an endogenous source of bigET-1), or alone (in which case exogenous bigET-1 was added). Phosphoramidon inhibited the conversion of exogenous bigET-1 (IC50 = 5 to 30 micromol/L) much better than that of endogenous bigET-1 (IC50 > 1 mmol/L). Both conversions showed similar high yields (20% to 100%) that depended on the amount of ECE-1a expressed. Thus, ECE-1a has two equally relevant activities in this recombinant system for CHO cells: (1) an intracellular, probably intravesicular activity, corresponding to the ECE-1a-mediated step of ET-1 biosynthesis and (2) an extracellular activity at the plasma membrane. If this is also the case for endothelial cells, ECE-1a inhibitors would have to cross the plasma and vesicle membranes to be effective. The present system could be useful for screening such inhibitors.

High affinity binding of pyrethroids to the alpha subunit of brain sodium channels.

Na+ channels are the primary molecular targets of the pyrethroid insecticides. Na+ channels consisting of only a type IIA alpha subunit expressed in Chinese hamster ovary cells responded to pyrethroid treatment in a normal manner: a sustained Na+ current was induced progressively after each depolarizing pulse in a train of stimuli, and this Na+ current decayed slowly on repolarization. These modified Na+ channels could be reactivated at much more negative membrane potentials (V0.5 = -139 mV) than unmodified Na+ channels (V0.5 = -28 mV). These results indicate that pyrethroids can modify the functional properties of the Na+ channel alpha subunit expressed alone by blocking their inactivation, shifting their voltage dependence of activation, and slowing their deactivation. To demonstrate directly the specific interaction of pyrethroids with the alpha subunit of voltage-gated Na+ channels, a radioactive photosensitive derivative, [3H]RU58487, was used in binding and photolabeling studies. In the presence of a low concentration of the nonionic detergent Triton X-100, specific pyrethroid binding to Na+ channels in rat brain membrane preparations could be measured and reached 75% of total binding under optimal conditions. Binding approached equilibrium within 1 hr at 4 degrees, dissociated with a half-time of approximately 10 min, and had K(D) values of approximately 58-300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced by approximately 40% in the presence of 100 nM alpha-scorpion toxin, but no allosteric enhancement was observed in the presence of toxins acting at other Na+ channel receptor sites. Extensive membrane washing increased specific binding to 89%. Photolabeling with [3H]RU58487 under these optimal binding conditions revealed a radiolabeled band with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit. Anti-peptide antibodies recognizing sequences within the alpha subunit were able to specifically immunoprecipitate the covalently modified channel. Together, these results demonstrate that the pyrethroids can modify the properties of cells expressing only the alpha subunit of Na+ channels and can bind specifically to a receptor site on the alpha subunit.

Conversion of big-endothelin-1 elicits an endothelin ETA receptor-mediated response in endothelial cells.

Functional conversion of big-endothelin-1 to endothelin-1 and characterization of endothelin receptor subtype were investigated in cultured rat aortic endothelial cells. Exogenous endothelin-1 and big-endothelin-1 both increased arachidonic acid release and inositol phosphate production dose dependently. Endothelin-1 was more potent than big-endothelin-1 as indicated by EC50 values: 0.5 +/- 0.1 nM and 10.0 +/- 2.0 nM for endothelin-1-induced arachidonic acid release and inositol phosphate formation, respectively, versus 1.0 +/- 0.4 nM and 35.0 +/- 6.0 nM for big-endothelin-1-induced responses. Big-endothelin-1, but not endothelin-1 actions were inhibited by phosphoramidon. Comparative studies of endothelin receptor agonists and antagonists showed that endothelin-3 but not sarafotoxin S6c stimulated arachidonic acid release and inositol phosphate formation. The responses to big-endothelin-1 and endothelin-1 were specifically inhibited by the selective endothelin ETA receptor antagonist, [cyclo-D-Trp-D-Asp-Pro-D-Val-Leu] (BQ-123) but not by the selective endothelin ETB receptor antagonist [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma- methyl-Leu-D-Trp-(COMe)-D-NLeu-ONa] (BQ-788). [125I]Endothelin-1 binding was inhibited by endothelin-1, endothelin-3 and BQ-123 but not by BQ-788. These results indicate that the pharmacological responses to big-endothelin-1 in aortic endothelial cells are due to the extracellular phosphoramidon-sensitive conversion to endothelin-1. Endothelin effects are mediated through endothelin ETA receptors in these cells.

Direct block of voltage-sensitive sodium channels by genistein, a tyrosine kinase inhibitor.

Genistein, an isoflavone inhibitor of tyrosine-specific protein kinases, was shown to specifically block the 22Na+ influx through voltage-sensitive Na+ channels in cultured rat brain neurons, whereas other tyrosine kinase antagonists such as lavendustin A, compound 5, tyrphostin A47 and an erbstatin analog were inactive at concentrations known to block kinase activity in other neuronal systems. Dose-response curves for genistein indicated a half-maximum effect at 60 microM. Daidzein, an inactive analog of genistein, had a similar inhibitory effect on the 22Na+ influx with a half-maximum effect at 195 microM. The time course of genistein action was rapid, because maximum effect on 22Na+ influx was obtained in less than 20 s at 100 microM. Analysis of Na+ currents by the whole-cell recording technique showed that 20 microM genistein reduced the sodium current and shifted the voltage dependence of both activation and inactivation curves. No competition with [3H]saxitoxin binding was observed, whereas the binding of [3H]batrachotoxinin A 20-alpha-benzoate to rat brain synaptosomal membranes was partially inhibited, which suggested a direct or allosteric interaction with neurotoxin binding site 2. These data taken together clearly indicate that the inhibition of voltage-sensitive sodium channels by genistein is not mediated by tyrosine kinase inhibition.

Interaction of endothelin-1 with cloned bovine ETA receptors: biochemical parameters and functional consequences.

This paper defines the properties of interaction of endothelin-1 (Et-1) with cloned bovine ETA receptors. The Kd value of Et-1/ETA receptor complexes was estimated in membrane preparations to 20 pM using kinetic experiments and saturation experiments performed under quasi equilibrium conditions. Competition experiments yield a wide range of apparent Kd(Et-1) values from 20 pM to 1 nM which were in fact measures of the receptor concentrations rather than of Kd values. This resulted from the fact that complex second-order rate kinetics rather than pseudo-first-order kinetics control the association of Et-1 to its receptor when the receptor concentration is larger than Kd(Et-1). Et-1 induced a production of inositol phosphates with an apparent affinity of 2.3 nM, 100 times higher than the Kd(Et-1) value determined previously. Numerical simulation suggested that under time-limited conditions, sub-nanomolar rather than picomolar concentrations of Et-1 are necessary to occupy an important fraction of picomolar sites. It is concluded that bovine ETA receptors have a single affinity state for Et-1 (Kd = 20 pM) and that this affinity state can account for nanomolar actions of Et-1 in intact cells. It is suggested that the sensitivity of a preparation to Et-1 is a cell property rather than a receptor property. It is also suggested that the main advantage of high-affinity Et-1 binding is to promote autocrine actions rather than a high potency of the peptide.

Et-1 and Et-3 actions mediated by cloned ETA endothelin receptors exhibit different sensitivities to BQ-123.

Et-1 and Et-3 activate phospholipase C in fibroblasts expressing cloned ETA receptors of bovine, rat and human origins. BQ-123 competitively antagonizes both responses but Et-3 actions are 10 times more sensitive to BQ-123 than Et-1 actions. It is suggested that differential sensitivity to BQ-123 is an intrinsic property of Et-1 and Et-3 activated ETA receptors and that there is no need to postulate the existence of new ETA receptor isoforms to account for singular actions of BQ-123.

Properties of an endothelin-3-sensitive Eta-like endothelin receptor in brain capillary endothelial cells.

[125I]Et-1 binding experiments showed the presence in rat brain capillary endothelial cells of typical ETA receptors that recognized Et-1, BQ-123 and FR139317 but not IRL1620, [Ala1,3.11,15]ET-1 or Et-3. [125I]Et-3 binding experiments showed that the same cells expressed high affinity Et-3 binding sites (Kd=1 nM) that recognized Et-1] (Kd=1 nM), BQ-123 (Kd = 5 nM) and FR139317 (Kd=2 nM) but not low concentrations of IRL1620 or of [Ala 1,3,11,15]Et-1. Et-3 activated N+/H+ exchange activity in a manner that was sensitive to BQ-123 (Ki=6 nM) and FR 139317 (Ki = 0.6 nM). The results suggest the existence of a high affinity Et-3 receptor that is insensitive to ETB receptor agonists and sensitive to ETA receptor antagonists and that is functionally coupled to Na+/H+ exchange activity.

Why are circulating concentrations of endothelin-1 so low?

Physiological and pathophysiological roles of endothelins are still unclear. One reason is that circulating endothelin levels in normal and pathological states are much lower than the concentrations necessary to elicit contractions in vitro. It is usually assumed that endothelin accumulates in diseased tissues and that, because of its degradation, only a small fraction of it reaches the systemic circulation. Such a hypothesis does not fit with recent observations showing that low circulating endothelin levels may be active. We show here that most of the current inferences about the actions of endothelin assume that the peptide acts in the vessel wall under conditions known as non-stoichiometric binding conditions, that is, under conditions in which the receptor concentration in tissues ([Ro]) is smaller than the equilibrium dissociation constant of endothelin receptor complexes (Kd). Under stoichiometric binding conditions (defined by the condition [Ro] > Kd), most ligand molecules are bound to receptors and cannot be present in a free form. Estimates of [Ro] and Kd from the literature suggests that in vivo endothelin probably binds stoichiometrically to its receptors. Under this condition, most of tissue endothelin is probably bound to receptors. It is therefore suggested that plasma endothelin levels are low probably because tissue free endothelin levels are low, and this is not inconsistent with the presence of high tissue levels of active (that is, bound) endothelin. When the topology of the vessels with respect to the site of production (or of delivery) of endothelin is considered, stoichiometric binding may also account for the higher sensitivity to Et-1 of in vivo preparations. It also suggests that autocrine and paracrine actions of Et-1 are favoured at low and high secretory rates respectively, thus providing an explanation for the dual (vasodilator and vasoconstricting) actions of endothelin. Finally, the stoichiometric binding model predicts that functional receptors also act as clearance receptors and provides an explanation for the observation that antagonists of endothelin receptors are also clearance antagonists.

Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels.

Purified and reconstituted sodium channels have previously been shown to be functional in voltage-dependent ion conductance and in high affinity binding of tetrodotoxin and saxitoxin at neurotoxin receptor site 1 and alpha-scorpion toxins at receptor site 3, but high affinity binding of neurotoxins at receptor sites 2, 4, and 5 has not been demonstrated. The pyrethroid insecticide RU39568 enhances the specific binding of [3H]batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 on purified and reconstituted sodium channels up to 500-fold, reducing the Kd to 1.5 nM. Brevetoxins and alpha-scorpion toxins cause further allosteric enhancement of BTX-B binding. The pyrethroids deltamethrin and bifenthrin and the nonpyrethroid insecticide 2,2-bis(p-chlorophenyl)trichloroethane can partially substitute for RU39568 in enhancing BTX-B binding, but other pyrethroids are inactive. The brevetoxin PbTx-1 binds specifically to neurotoxin receptor site 5 on purified and reconstituted sodium channels with a Kd value of approximately 30 nM. Brevetoxin binding is enhanced up to 2-fold by the combination of batrachotoxin and RU39568. The allosteric enhancement of BTX-B binding by RU39568 is voltage dependent, decreasing progressively with depolarization to 0 mV. In contrast, PbTx-1 binding is not voltage dependent and PbTx-1 reduces the voltage dependence of the effect of RU39568. The results demonstrate restoration of high affinity binding and allosteric interactions of ligands at neurotoxin receptor sites 2 and 5 on purified and reconstituted sodium channels and provide an experimental approach to covalent labeling and identification of the peptide components of those receptor sites.