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 α ₂ δ-1 and α ₂ δ-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 α ₂ δ 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 α ₂ δ 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 α ₂ δ subunits, being prevented by mutations in either α ₂ δ-1 or α ₂ δ-2 that abolish GBP binding, and is not observed for α ₂ δ-3, which does not bind GBP. Furthermore, the trafficking of α ₂ δ-2 and Ca _V 2 channels is disrupted both by GBP and by the mutation in α ₂ δ-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of α ₂ δ-2 and Ca _V 2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of α ₂ δ subunit function, thereby impairing the trafficking function of the α ₂ δ subunits to which it binds.

Significance: Although sequencing of the human genome was completed years ago, we still do not know about the physiological significance of thousands of predicted proteins, particularly of predicted membrane proteins. On the other hand, for approximately 100 human enzymes, no coding gene is known even though their enzymatic reaction has been well characterized. In this work, we assign one of those predicted membrane proteins (transmembrane protein 189; TMEM189) to one of the enzymatic reactions with an uncharacterized gene (plasmanylethanolamine desaturase). This enzyme catalyzes the final step in the biosynthesis of plasmalogens, an abundant class of glycerophospholipids that is depleted in such diseases as Alzheimer’s. Our findings enable interpretation of the previously characterized impaired growth phenotype of Tmem189 -deficient mice.

Cardiolipin (CL) is a phospholipid specific for mitochondrial membranes and crucial for many core tasks of this organelle. Its acyl chain configurations are tissue specific, functionally important, and generated via post-biosynthetic remodeling. However, this process lacks the necessary specificity to explain CL diversity, which is especially evident for highly specific CL compositions in mammalian tissues. To investigate the so far elusive regulatory origin of CL homeostasis in mice, we combine lipidomics, integrative transcriptomics, and data-driven machine learning. We demonstrate that not transcriptional regulation, but cellular phospholipid compositions are closely linked to the tissue specificity of CL patterns allowing artificial neural networks to precisely predict cross-tissue CL compositions in a consistent mechanistic specificity rationale. This is especially relevant for the interpretation of disease-related perturbations of CL homeostasis, by allowing differentiation between specific aberrations in CL metabolism and changes caused by global alterations in cellular (phospho-)lipid metabolism.

Significance: Cardiolipins 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.

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 intoXenopus laevisoocytes. 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.

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_v2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Ca_v2.1 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_v3.2 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_vsubunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Ca_vmutants is likely to occur in EA2 and in other inherited dominant channelopathies.