Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP4) from InsP5. Additionally, mIPMK forms InsP4 from Ins(1,4,5)P3 and InsP5 from Ins(1,3,4,5)P4.

Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase

Rab GTPases of the Arabidopsis Rab-E subclass are related to mammalian Rab8 and are implicated in membrane trafficking from the Golgi to the plasma membrane. Using a yeast two-hybrid assay, Arabidopsis phosphatidylinositol-4-phosphate 5-kinase 2 (PtdIns(4)P 5-kinase 2; also known as PIP5K2), was shown to interact with all five members of the Rab-E subclass but not with other Rab subclasses residing at the Golgi or trans-Golgi network. Interactions in yeast and in vitro were strongest with RAB-E1d[Q74L] and weakest with the RAB-E1d[S29N] suggesting that PIP5K2 interacts with the GTP-bound form. PIP5K2 exhibited kinase activity towards phosphatidylinositol phosphates with a free 5-hydroxyl group, consistent with PtdIns(4)P 5-kinase activity and this activity was stimulated by Rab binding. Rab-E proteins interacted with PIP5K2 via its membrane occupancy and recognition nexus (MORN) domain which is missing from animal and fungal PtdIns(4)P 5-kinases. In plant cells, GFP:PIP5K2 accumulated at the plasma membrane and caused YFP:RAB-E1d to relocate there from its usual position at the Golgi. GFP:PIP5K2 was rapidly turned over by proteasomal activity in planta, and overexpression of YFP:PIP5K2 caused pleiotropic growth abnormalities in transgenic Arabidopsis. We propose that plant cells exhibit a novel interaction in which PIP5K2 binds GTP-bound Rab-E proteins, which may stimulate temporally or spatially localized PtdIns(4,5)P(2) production at the plasma membrane.

Heat stress effects on ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco binding protein and Rubisco activase in wheat leaves

Changes in chlorophyll content, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) binding protein (RBP), Rubisco activase (RA), Rubisco large (LS) and small (SS) subunits, and electrolyte leakage were investigated in wheat leaf segments during heat stress (HS) for 1 h and for 24 h at 40 °C in darkness or in light, as well as after recovery from heat stress (HSR) for 24 h at 25 °C in light. The 24-h HS treatment in darkness decreased irreversibly photosynthetic pigments, soluble proteins, RBP, RA, Rubisco LS and SS. An increase in RA and RBP protein contents was observed under 24-h HS and HSR in light. This increase was in accordance with their role as chaperones and the function of RBP as a heat shock protein.

Dithiothreitol triggers photooxidative stress and fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact pea chloroplasts

In intact chloroplasts isolated from mature pea leaves (Pisum sativum L.), the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was rapidly fragmented into several products upon illumination in the presence of 1 mM dithiothreitol (DTT). Very similar effects on LSU stability could be observed when illuminated chloroplasts were poisoned with cyanide which, like DTT, inhibits important plastid antioxidant enzymes, or when a light-dependent hydroxyl radical-producing system was added to the incubation medium. Moreover, DTT-stimulated light degradation of LSU was markedly delayed in the presence of scavengers of active oxygen species (AOS). It is therefore suggested that light degradation of LSU in the presence of DTT is mainly due to inhibition of the chloroplast antioxidant defense system and the subsequent accumulation of AOS in intact organelles. When chloroplasts were isolated from nonsenescent or senescent leaves, LSU remained very stable upon incubation without DTT, indicating that the antioxidant system was still functional in the isolated chloroplasts during leaf ageing. Our data support the notion that AOS might be important for the degradation of Rubisco in vivo under oxidative stress.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves1

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m−2. Mesophyll conductance (μ) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on Et saturated at Et = 30 μmol active sites m−2 and μ = 11 mm s−1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a μ of only 6 to 8 mm s−1. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm−1 s−1. Our data show that the saturation of the relationship between Et and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.

Nuclear-Gene Mutations Suppress a Defect in the Expression of the Chloroplast-Encoded Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase1

The green alga Chlamydomonas reinhardtii mutant 76–5EN lacks photosynthesis because of a nuclear-gene mutation that specifically inhibits expression of the chloroplast gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Photosynthesis-competent revertants were selected from mutant 76–5EN to explore the possibility of increasing Rubisco expression. Genetic analysis of 10 revertants revealed that most arose from suppressor mutations in nuclear genes distinct from the original 76–5EN mutant gene. The revertant strains have regained various levels of Rubisco holoenzyme, but none of the suppressor mutations increased Rubisco expression above the wild-type level in either the presence or absence of the 76–5EN mutation. One suppressor mutation, S107–4B, caused a temperature-conditional, photosynthesis-deficient phenotype in the absence of the original 76–5EN mutation. The S107–4B strain was unable to grow photosynthetically at 35°C, but it expressed a substantial level of Rubisco holoenzyme. Whereas the 76–5EN gene encodes a nuclear factor that appears to be required for the transcription of the Rubisco large-subunit gene, the S107–4B nuclear gene may be required for the expression of other chloroplast genes.

4-Chloroanilinium 2-carboxy-4,5-dichlorobenzoate

The structure of the 1:1 proton-transfer compound of 4-chloroaniline with 4,5-dichlorophthalic acid (DCPA), viz. C6H7ClN+ C8H3Cl2O4-, has been determined at 130 K. The non-planar hydrogen phthalate anions and the 4-chloroanilinium cations form two-dimensional O-H...O and N-H...O hydrogen-bonded substructures which have no peripheral extension. Between the sheets there are weak \p--\p associations between alternating cation--anion aromatic ring systems [shortest centroid separation, 3.735(4)A].

1,10-phenanthrolin-1-ium 2-carboxy-4,5-dichlorobenzoate

In the structure of the 1:1 proton-transfer compound of 1,10-phenanthroline with 4,5-dichlorophthalic acid, C12H9N2+ C8H3Cl2O4-, determined at 130 K, the 1,10-phenanthroline cation and the hydrogen 4,5-dichlorophthalate anion associate through a single N-H...O(carboxyl) hydrogen bond giving discrete units which have no extension except through a number of weak cation C-H...O(anion) associations and weak cation--anion aromatic ring pi-pi interactions [minimum centroid separation, 3.6815(12)A]. The anions are essentially planar [maximum deviation 0.214(1)A (a carboxyl O)] with the syn-related H atom of the carboxyl group forming a short intramolecular O-H...O(carboxyl) hydrogen bond.

Zero- one- and two-dimensional hydrogen-bonded structures in the 1:1 proton-transfer compounds of 4,5-dichlorophthalic acid with the monocyclic heteroaromatic Lewis bases 2-aminopyrimidine, nicotinamide and isonicotinamide

The structures of the anhydrous 1:1 proton-transfer compounds of 4,5-dichlorophthalic acid (DCPA) with the monocyclic heteroaromatic Lewis bases 2-aminopyrimidine, 3-(aminocarboxy) pyridine (nicotinamide) and 4-(aminocarbonyl) pyridine (isonicotinamide), namely 2-aminopyrimidinium 2-carboxy-4,5-dichlorobenzoate C4H6N3+ C8H3Cl2O4- (I), 3-(aminocarbonyl) pyridinium 2-carboxy-4,5-dichlorobenzoate C6H7N2O+ C8H3Cl2O4- (II) and the unusual salt adduct 4-(aminocarbonyl) pyridinium 2-carboxy-4,5-dichlorobenzoate 2-carboxymethyl-4,5-dichlorobenzoic acid (1/1/1) C6H7N2O+ C8H3Cl2O4-.C9H6Cl2O4 (III) have been determined at 130 K. Compound (I) forms discrete centrosymmetric hydrogen-bonded cyclic bis(cation--anion) units having both R2/2(8) and R2/1(4) N-H...O interactions. In compound (II) the primary N-H...O linked cation--anion units are extended into a two-dimensional sheet structure via amide-carboxyl and amide-carbonyl N-H...O interactions. The structure of (III) reveals the presence of an unusual and unexpected self-synthesized methyl monoester of the acid as an adduct molecule giving one-dimensional hydrogen-bonded chains. In all three structures the hydrogen phthalate anions are

Unusual hydrate stabilization in the two-dimensional layered structure of quinacrinium bis(2-carboxy-4,5-dichlorobenzoate) tetrahydrate, a proton-transfer compound of the drug quinacrine

The crystal structure of the hydrated proton-transfer compound of the drug quinacrine [rac-N'-(6-chloro-2-methoxyacridin-9-yl)-N,N-diethylpentane-1,4-diamine] with 4,5-dichlorophthalic acid, C23H32ClN3O2+ . 2(C8H3Cl2O4-).4H2O (I), has been determined at 200 K. The four labile water molecules of solvation form discrete ...O--H...O--H... hydrogen-bonded chains parallel to the quinacrine side chain, the two N--H groups of which act as hydrogen-bond donors for two of the water acceptor molecules. The other water molecules, as well as the acridinium H atom, also form hydrogen bonds with the two anion species and extend the structure into two-dimensional sheets. Between these sheets there are also weak cation--anion and anion--anion pi-pi aromatic ring interactions. This structure represents only the third example of a simple quinacrine derivative for which structural data are available but differs from the other two in that it is unstable in the X-ray beam due to efflorescence, probably associated with the destruction of the unusual four-membered water chain structures.

Isopropylaminium 2-carboxy-4, 5-dichlorobenzoate

In the structure of the 1:1 proton-transfer compound of isopropylamine with 4,5-dichlorophthalic acid, C3H10N+·C8H3Cl2O4-, the three cation H-atom donors associate with three separate carboxyl O-atom anion acceptors, giving conjoint cyclic R44(12), R44(16) hydrogen-bonding cation-anion interactions in a one-dimensional ribbon structure. In the anions, the carboxyl groups lie slightly out of the plane of the benzene ring [maximum deviations = 0.439 (1) for a carboxylic acid O atom and 0.433 (1) Å for a carboxylate O atom]. However, the syn-related proton of the carboxylic acid group forms the common short intramolecular O-HOcarboxyl hydrogen bond.

Ethane-1,2-diaminium 4,5-dichlorophthalate

In the structure of the title compound, C2H10N22+·C8H2Cl2O42-, the dications and dianions form hydrogen-bonded ribbon substructures which enclose conjoint cyclic R21(7), R12(7) and R42(8) associations and extend down the c-axis direction. These ribbons inter-associate down b, giving a two-dimensional sheet structure. In the dianions, one of the carboxylate groups is essentially coplanar with the benzene ring, while the other is normal to it [C-C-C-O torsion angles = 177.67 (12) and 81.94 (17)°, respectively].