Katrin Watschinger

Pharmacological disruption of calcium channel trafficking by the α2δ ligand gabapentin

The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) binds to an exofacial epitope of the α2δ-1 and α2δ-2 auxiliary subunits of voltage-gated calcium channels, but acute inhibition of calcium currents by GBP is either very minor or absent. We formulated the hypothesis that GBP impairs the ability of α2δ subunits to enhance voltage-gated Ca2+channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of α2δ only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal root-ganglion neurons. GBP acts primarily at an intracellular location, requiring uptake, because the effect of chronically applied GBP is blocked by an inhibitor of the system-L neutral amino acid transporters and enhanced by coexpression of a transporter. However, it is mediated by α2δ subunits, being prevented by mutations in either α2δ-1 or α2δ-2 that abolish GBP binding, and is not observed for α2δ-3, which does not bind GBP. Furthermore, the trafficking of α2δ-2 and CaV2 channels is disrupted both by GBP and by the mutation in α2δ-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of α2δ-2 and CaV2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of α2δ subunit function, thereby impairing the trafficking function of the α2δ subunits to which it binds.

A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

Channelopathies are often linked to defective protein folding and trafficking. Among them, the calcium channelopathy episodic ataxia type-2 (EA2) is an autosomal dominant disorder related to mutations in the pore-forming Cav2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Cav2.1 channel activity, the molecular mechanism underlying dominant inheritance remains unclear. Here, we show that EA2 mutants as well as a truncated form (DI-II) of the Cav3.2 subunit of T-type calcium channel are misfolded, retained in the endoplasmic reticulum, and subject to proteasomal degradation. Pulse-chase experiments revealed that misfolded mutants bind to nascent wild-type Cav subunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Cav mutants is likely to occur in EA2 and in other inherited dominant channelopathies.

IDO and Regulatory T Cell Support Are Critical for Cytotoxic T Lymphocyte-Associated Ag-4 Ig-Mediated Long-Term Solid Organ Allograft Survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.

Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin.

The mechanism of action of gabapentin is still not well understood. It binds to the α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels but has little acute effect on calcium currents in several systems. However, our recent results conclusively demonstrated that gabapentin inhibited calcium currents when applied chronically but not acutely, both in heterologous expression systems and in dorsal root ganglion neurons 1. In that study we only examined a 40 hour time point of incubation with gabapentin, and here we have extended these results to include the effect of up to 6 and 20 hours incubation with gabapentin on calcium channel currents formed from CaV2.1/β4/α2δ-2 subunits. Gabapentin was significantly effective to inhibit the currents if included for 17-20 hours prior to recording, but it did not produce a significant inhibition if included for 3-6 hours. We previously concluded that gabapentin acts primarily at an intracellular location, requiring uptake into cells. However, this effect is mediated by α2δ subunits, being prevented by mutations in either α2δ-1 or α2δ-2 that abolish gabapentin binding 1. Furthermore, we also showed that the trafficking of α2δ-2 and CaV2 channels was disrupted by gabapentin. Here we have also extended that study, to show that the cell-surface expression of CaV2.1 is not reduced by chronic gabapentin if it is co-expressed with α2δ-2 containing a point mutation (R282A) that prevents gabapentin binding

Orphan enzymes in ether lipid metabolism

Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They have important roles as membrane components of e.g. lens, brain and testis, and as mediators such as platelet-activating factor. The metabolic enzymes for biosynthesis and degradation have been investigated to some extent. As most involved enzymes are integral membrane proteins they are tricky to handle in biochemical protocols. The sequence of some ether lipid metabolising enzymes has only recently been reported and other sequences still remain obscure. Defined enzymes without assigned sequence are known as orphan enzymes. One of these enzymes with uncharacterised sequence is plasmanylethanolamine desaturase, a key enzyme for the biosynthesis of one of the most abundant phospholipids in our body, the plasmalogens. This review aims to briefly summarise known functions of ether lipids, give an overview on their metabolism including the most prominent members, platelet-activating factor and the plasmalogens. A special focus is set on the description of orphan enzymes in ether lipid metabolism and on the successful strategies how four previous orphans have recently been assigned a sequence. Only one of these four was characterised by classical protein purification and sequencing, whereas the other three required alternative strategies such as bioinformatic candidate gene selection and recombinant expression or development of an inhibitor and multidimensional metabolic profiling.

Functional properties and modulation of extracellular epitope-tagged Ca V 2.1 voltage-gated calcium channels

Depolarisation-induced Ca2+ influx into electrically excitable cells is determined by the density of voltage-gated Ca2+ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope tagged CaV2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the CaV2.1 α1 subunit which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising α1 subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescent studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for CaV2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artifical sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca2+ channel for their selective modulation.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome

Significance We have recently identified the sequence of the ether lipid-cleaving enzyme alkylglycerol monooxygenase. Like nitric oxide synthases and aromatic amino acid hydroxylases, alkylglycerol monooxygenase needs tetrahydrobiopterin as cofactor. Whereas the former enzymes have well-established roles in the cell, the physiology of alkylglycerol monooxygenase is still not clear. Here we show its regulation in murine macrophage differentiation and its dependence on the cofactor tetrahydrobiopterin in live murine macrophage-like RAW264.7 cells. Upon modulation of the activity of alkylglycerol monooxygenase and the key enzyme in tetrahydrobiopterin biosynthesis, we observe extensive changes in various lipid classes ranging from ether lipids to far more complex lipids. These findings point to an important role of tetrahydrobiopterin in cellular lipid homeostasis. Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and “alternatively activated” M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide–induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

A gatekeeper helix determines the substrate specificity of Sjögren–Larsson Syndrome enzyme fatty aldehyde dehydrogenase

Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren–Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a ‘gatekeeper’ helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence

Abstract Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases – comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases – and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N5-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.

Donor Pretreatment with Tetrahydrobiopterin Saves Pancreatic Isografts from Ischemia Reperfusion Injury in a Mouse Model

Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B‐substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 μM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI‐related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia‐induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.