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Gene targeting via homologous recombination can occasionally result in incomplete disruption of the targeted gene. Here, we show that a widely used Nur77-deficient transgenic mouse model expresses a truncated protein encoding for part of the N-terminal domain of nuclear receptor Nur77. This truncated Nur77 protein is absent in a newly developed Nur77-deficient mouse strain generated using Cre-Lox recombination. Comparison of these two mouse strains using immunohistochemistry, flow cytometry, and colony-forming assays shows that homologous recombination–derived Nur77-deficient mice, but not WT or Cre-Lox–derived Nur77-deficient mice, suffer from liver immune cell infiltrates, loss of splenic architecture, and increased numbers of bone marrow hematopoietic stem cells and splenic colony–forming cells with age. Mechanistically, we demonstrate that the truncated Nur77 N-terminal domain protein maintains the stability and activity of hypoxia-inducible factor (HIF)-1, a transcription factor known to regulate bone marrow homeostasis. Additionally, a previously discovered, but uncharacterized, human Nur77 transcript variant that encodes solely for its N-terminal domain, designated TR3β, can also stabilize and activate HIF-1α. Meta-analysis of publicly available microarray data sets shows that TR3β is highly expressed in human bone marrow cells and acute myeloid leukemia samples. In conclusion, our study provides evidence that a transgenic mouse model commonly used to study the biological function of Nur77 has several major drawbacks, while simultaneously identifying the importance of nongenomic Nur77 activity in the regulation of bone marrow homeostasis.

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40–47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.

International audience; T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.

International audience; We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca(v)2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca(v)2.1. As they are known to affect inactivation, we also analyzed the effects of beta subunits on these interactions. In experiments in which the beta(4) subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the beta subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the beta subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.

Maurotoxin (MTX) is a scorpion toxin acting on several K+ channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an “uncommon” arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general α/β scaffold of scorpion toxins.

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.

The voltage-gated calcium channel beta subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, alpha 1, in several ways. Complementary binding sites on alpha 1 and beta have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the beta subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of alpha 1A and showing specificity for the beta 4 (and to a lesser extent beta 2a) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of beta 4, poorly conserved between beta isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.

N-, P- and Q-type voltage-dependent Ca2+ channels control neurotransmitter release in the nervous system and are blocked by ω-conotoxin MVIIC. In this study, both a high affinity and a low affinity binding site for ω-conotoxin MVIIC were detected in rabbit brain. The low affinity binding site is shown to be present on the N-type Ca2+ channel. Using optimized conditions for specific labeling of the high affinity ω-conotoxin MVIIC receptor and a panel of subunit specific antibodies, the molecular structure of the high affinity receptor was investigated. We demonstrate for the first time that this receptor is composed of at least α1A, α2δ, and any one of the four brain β subunits. Such association of different β subunits with α1A and α2δ components may produce Ca2+ channels with distinct functional properties, such as P- and Q-type.

beta Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of alpha 1-beta subunit interaction have been localized recently to cytoplasmic domains of both subunits. The alpha 1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all alpha 1 subunits which is essential for the binding of beta subunits. In order to further study the interaction of beta subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the alpha 1A subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized 35S-labeled beta 2 and beta 3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to beta subunits. The affinity bead assay was also used to identify and characterize native beta subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind beta subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of beta 1a in skeletal muscle, beta 2 and beta 3 in cardiac muscle, and beta 1b, beta 3, and beta 4 in brain. The assays also demonstrate the AIDA beads bind to beta subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of beta subunits which is not always associated with alpha 1 subunits. Also, beta subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native beta subunits from detergent and non-detergent tissue extracts illustrating that this domain on the alpha 1 subunit is the major anchoring site for the beta subunit.

In voltage-dependent Ca2+ channels, the beta subunit interacts with the alpha 1 subunit via a cytoplasmic site. A biochemical assay has been developed to quantitatively describe the interaction between both subunits. In vitro synthesized 35S-labeled beta subunits specifically bind to a glutathione S-transferase (GST) fusion protein containing the alpha 1A interaction domain (AIDA, located between the amino-acids 383 and 400 of the cytoplasmic loop between the hydrophobic domains I and II). Kinetic analysis demonstrates that the association of 35S-labeled beta 1b subunit to the AIDA GST fusion protein occurs with a fast rate constant at 4 degrees C. The binding is almost irreversible as demonstrated by the absence of dissociation observed after an 8-h incubation with an 18-amino acid synthetic AIDA peptide. The alpha 1-beta binding site does not seem to be a target for cytoplasmic regulation. The interaction is mostly unaffected by changes in ionic strength, pH, and Ca2+ concentration or by protein kinase C phosphorylation. The specificity of subunit interaction in voltage-dependent Ca2+ channels was also followed by saturation analyses. The data obtained show that the AIDA GST fusion protein binds to a single site on the beta 1b with an apparent Kd of 5 nM. The affinities of AIDA GST fusion protein for various beta subunits was measured and demonstrate that beta subunits associate with different affinities to each alpha 1 interaction domain. The rank order of AIDA affinity for each beta subunit is as follows: beta 4beta 2abeta 1bbeta 3. The binding of the beta subunit to alpha 1 subunit can be inhibited in vitro by the AIDA synthetic peptide with an apparent Ki of 285 nM. This interaction can also be prevented in heterologous Ca2+ channels by the injection of the AIDA GST fusion protein into Xenopus oocytes. Our results demonstrate that the site of interaction between AID and beta subunit is responsible for anchoring the beta subunit to the alpha 1 subunit and thus allowing the beta subunit to modify Ca2+ channel activity.

N-type Ca2+ channels control a variety of key neuronal functions including transmitter release at synaptic terminals. The purified omega-conotoxin receptor from rabbit brain is a multisubunit complex composed of alpha 1B, alpha 2 delta, beta 3, and 95-kDa subunits. Immunoadsorption experiments confirm that the purified preparation does not contain alpha 1 subunits other than the omega-conotoxin-sensitive class B isoform. The functional properties of the purified channel have been analyzed further in lipid bilayers, and similarities to or differences from the native N-type Ca2+ channel have been outlined. Conserved properties include ion selectivity, open-time duration, and pharmacology (insensitivity to drugs affecting skeletal muscle L-type Ca2+ channels). Observed properties of the reconstituted channel which differ from the native channel include (a) sustained channel activity without Ca(2+)- or voltage-induced inactivation; (b) examples of extremely high open-state probability; (c) the absence of "run-down"; and (d) voltage independence of the Ca2+ channel gating. In addition, the conductance of the purified receptor is comprised between 7 and 27 picosiemens. Our results suggest that cellular components may play critical roles in the regulation of several biophysical properties and neuronal function of the native N-type Ca2+ channel.

Poly(3-hydroxyalkanoates) (PHAs) were isolated from Pseudomonas putida KT2442 cultivated on petroselenic acid, oleic acid, and linoleic acid to study beta-oxidation of unsaturated fatty acids. Both saturated and unsaturated medium chain length 3-hydroxy fatty acids were found to be constituents of these polymers. With the aid of proton-detected multiple quantum coherence and proton-detected multiple bond coherence NMR spectra the structures of the unsaturated monomers were identified as 3-hydroxy-5-cis-tetradecanoate for PHA produced on oleic acid, and 3-hydroxy-6-cis-dodecanoate and 3-hydroxy-5-cis-8-cis-tetradecadienoate for PHA produced on linoleic acid. The identified structures, which are derived from fatty acid degradation intermediates, indicate a degradation of oleic acid via the enoyl-CoA isomerase-dependent route and a degradation of linoleic acid via the dienoyl-CoA reductase-dependent route.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination, and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols, which are nonsulfated, sulfated, and/or phosphorylated, were obtained from the carbohydrate-protein linkage region. Six compounds, containing 0 or 1 sulfate and/or phosphate residue, represent approximately 40% of the isolated linkage hexasaccharide alditols. They were analyzed by chondroitinase ACII or alkaline phosphatase digestion in conjunction with high performance liquid chromatography, and by 500 MHz one- and two-dimensional 1H NMR spectroscopy. All six compounds have the conventional structure in common. Delta 4,5-GlcA beta 1-3GalNAc beta 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol One compound has no sulfate nor phosphate. Two of the monosulfated compounds have a O- sulfate on C-6 or on C-4 of the GalNAc residue. The third monosulfated compound has a novel O-sulfate on C-6 of the Gal residue attached to xylitol. The two phosphorylated compounds have O-phosphate on C-2 of Xyl-ol, and one of them has in addition sulfate on C-6 of GalNAc.

The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F (PNGase-F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and analyzed by 500-MHz 1H NMR spectroscopy. The major acidic compounds are mono- and disialylated, fucosylated diantennary compounds terminated with alpha(2-6)-linked sialic acid on the Man alpha(1-3) branch. The Man alpha(1-6) branch shows a large heterogeneity. It can be terminated with Man-4', GlcNAc-5', or Gal-6', whereas the Gal-6' residue may be extended with Gal alpha(1-3), NeuAc alpha(2-3), or Sia alpha (2-6). In the major structures 8% of alpha(2-6)-linked sialic acid was found as NeuGc instead of NeuAc. The main compounds have sulfated homologues bearing a sulfate group (6-20%) at C-3 of Gal-6' or at C-6 of GlcNAc-5 as follows. [formula: see text]