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 ␣2␦-1 and ␣2␦-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 ␣2␦ subunits to enhance voltage-gated Ca 2؉ channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of ␣2␦ only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal rootganglion 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 ␣2␦ subunits, being prevented by mutations in either ␣2␦-1 or ␣2␦-2 that abolish GBP binding, and is not observed for ␣2␦-3, which does not bind GBP. Furthermore, the trafficking of ␣2␦-2 and CaV2 channels is disrupted both by GBP and by the mutation in ␣2␦-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of ␣2␦-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 ␣2␦ subunit function, thereby impairing the trafficking function of the ␣2␦ subunits to which it binds. V oltage-gated Ca 2ϩ channels (VGCCs) are heteromeric complexes. The Ca V 1 and Ca V 2 subfamilies are made up of a pore-forming ␣1 subunit, associated with a membraneanchored, predominantly extracellular, ␣ 2 ␦ subunit (for review see ref. 1) and an intracellular ␤ subunit (for review see ref.2). Mammalian genes encoding four ␣ 2 ␦ subunits have been identified (for reviews see refs. 2 and 3). The topology of the ␣ 2 ␦ protein was first determined for ␣ 2 ␦-1 and is thought to generalize to all ␣ 2 ␦ subunits (for reviews see refs. 1 and 4). They are type I transmembrane proteins, the exofacial ␣ 2 subunit being disulfide-bonded to a transmembrane ␦ subunit, formed by posttranslational cleavage of the ␣ 2 ␦ preprotein (5).The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) itself was originally developed as an analog of ␥-amino-butyric acid (GABA), but is now believed to have no effect on GABA receptors or transporters (for review see ref. 6). The first key to understanding the mechanism of action of GBP came from purification of the GBP-binding protein from porcine brain (7), which was identified as the ␣ 2 ␦-1 auxiliary subunit of VGCCs. It is now known that GBP binds to an exofacial epitope present in both the ␣ 2 ␦-1 and ␣ 2 ␦-2 subunits (for reviews see refs. 1 and 8). However, although it was originally reported that GBP application results in a...

SignificanceCardiolipins are a unique class of phospholipids in mitochondrial membranes that are crucial for cellular bioenergetics as they stabilize respiratory chain complexes. In contrast to most other phospholipids, cardiolipins are substituted with four, rather than only two fatty acyl side chains. Consequently, this opens up a vast number of different theoretically possible molecular lipid species. Experimentally assessing the molecular diversity of cardiolipin species is analytically challenging. In this study we successfully combine tandem mass spectrometry with a mathematical structural modeling approach, to achieve the comprehensive characterization of complex biological cardiolipin compositions.

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Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes. EC 1.14.16.5 | glyceryl-ether monooxygenase | nitric oxide synthase | phenylalanine hydroxylase | transmembrane protein 195

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer’s and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1′-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1′-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of theTMEM189gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivatedTmem189gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign theTMEM189gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype ofTmem189-deficient mice may be caused by a lack of plasmalogens.

We present an experimental method aimed at measuring mass densities of solids at ambient pressure. The principle of the method is flotation in a mixture of liquid nitrogen and liquid argon, where the mixing ratio is varied until the solid hovers in the liquid mixture. The temperature of such mixtures is in the range of 77-87 K, and therefore, the main advantage of the method is the possibility of determining densities of solid samples, which are instable above 90 K. The accessible density range (~0.81-1.40 g cm(-3)) is perfectly suitable for the study of crystalline ice polymorphs and amorphous ices. As a benchmark, we here determine densities of crystalline polymorphs (ices I(h), I(c), II, IV, V, VI, IX, and XII) by flotation and compare them with crystallographic densities. The reproducibility of the method is about ±0.005 g cm(-3), and in general, the agreement with crystallographic densities is very good. Furthermore, we show measurements on a range of amorphous ice samples and correlate the density with the d spacing of the first broad halo peak in diffraction experiments. Finally, we discuss the influence of microstructure, in particular voids, on the density for the case of hyperquenched glassy water and cubic ice samples prepared by deposition of micrometer-sized liquid droplets.

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 Ca v 2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Ca v 2.1 channel activity, the molecular mechanism underlying dominant inheritance remains unclear. Here, we show that EA2 mutants as well as a truncated form (D I-II ) of the Ca v 3.2 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 Ca v subunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Ca v mutants is likely to occur in EA2 and in other inherited dominant channelopathies.

Co-stimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig up-regulates expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. Here we studied if 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 (DST) showed indefinite graft survival [>100 days] without signs of chronic rejection or donor specific antibody formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared to rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-DL-tryptophan, either at transplant or at POD 50, abrogated CTLA4Ig+DST-induced long-term graft survival. Importantly, IDO1 knock-out recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of 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 co-stimulatory blocker.