PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Conserved structures of ESCRT-III superfamily members across domains of life.

Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.

Long-term application of glycine transporter inhibitors acts antineuropathic and modulates spinal N-methyl-D-aspartate receptor subunit NR-1 expression in rats.

BACKGROUND: Dysfunction of spinal glycinergic neurotransmission is a major pathogenetic factor in neuropathic pain. The synaptic glycine concentration is controlled by the two glycine transporters (GlyT) 1 and 2. GlyT inhibitors act antinociceptive in various animal pain models when applied as bolus. Yet, in some studies, severe neuromotor side effects were reported. The aim of the current study was to elucidate whether continuous inhibition of GlyT ameliorates neuropathic pain without side effects and whether protein expression of GlyT1, GlyT2, or N-methyl-D-aspartate receptor subunit NR-1 in the spinal cord is affected. METHODS: In the chronic constriction injury model of neuropathic pain, male Wistar rats received specific GlyT1 and GlyT2 inhibitors (ALX5407 and ALX1393; Sigma-Aldrich, St. Louis, MO) or vehicle for 14 days via subcutaneous osmotic infusion pumps (n = 6). Mechanical allodynia and thermal hyperalgesia were assessed before, after chronic constriction injury, and every 2 days during substance application. At the end of behavioral assessment, the expression of GlyT1, GlyT2, and NR-1 in the spinal cord was determined by Western blot analysis. RESULTS: Both ALX5407 and ALX1393 ameliorated thermal hyperalgesia and mechanical allodynia in a time- and dose-dependent manner. Respiratory or neuromotor side effects were not observed. NR-1 expression in the ipsilateral spinal cord was significantly reduced by ALX5407, but not by ALX1393. The expression of GlyT1 and GlyT2 remained unchanged. CONCLUSIONS: Continuous systemic inhibition of GlyT significantly ameliorates neuropathic pain in rats. Thus, GlyT represent promising targets in pain research. Modulation of N-methyl-D-aspartate receptor expression might represent a novel mechanism for the antinociceptive action of GyT1 inhibitors.

Lidocaine metabolites inhibit glycine transporter 1: a novel mechanism for the analgesic action of systemic lidocaine?

BACKGROUND: Lidocaine exerts antinociceptive effects when applied systemically. The mechanisms are not fully understood but glycinergic mechanisms might be involved. The synaptic glycine concentration is controlled by glycine transporters. Whereas neurons express two types of glycine transporters, astrocytes specifically express glycine transporter 1 (GlyT1). This study focuses on effects of lidocaine and its major metabolites on GlyT1 function. METHODS: The effects of lidocaine and its metabolites monoethylglycinexylidide (MEGX), glycinexylidide, and N-ethylglycine on GlyT1 function were investigated in uptake experiments with [¹4C]-labeled glycine in primary rat astrocytes. Furthermore, the effect of lidocaine and its metabolites on glycine-induced currents were investigated in GlyT1-expressing Xenopus laevis oocytes. RESULTS: Lidocaine reduced glycine uptake only at toxic concentrations. The metabolites MEGX, glycinexylidide, and N-ethylglycine, however, significantly reduced glycine uptake (P < 0.05). Inhibition of glycine uptake by a combination of lidocaine with its metabolites at a clinically relevant concentration was diminished with increasing extracellular glycine concentrations. Detailed analysis revealed that MEGX inhibits GlyT1 function (P < 0.05), whereas N-ethylglycine was identified as an alternative GlyT1 substrate (EC50 = 55 µM). CONCLUSIONS: Although lidocaine does not function directly on GlyT1, its metabolites MEGX and N-ethylglycine [corrected] were shown to inhibit GlyT1-mediated glycine uptake by at least two different mechanisms. Whereas N-ethylglycine [corrected] was demonstrated to be an alternative GlyT1 substrate, MEGX was shown to inhibit GlyT1 activity in both primary astrocytes and in GlyT1-expressing Xenopuslaevis oocytes at clinically relevant concentrations. These findings provide new insights into the possible mechanisms for the antinociceptive effect of systemic lidocaine.

Binding and/or hydrolysis of purine-based nucleotides is not required for IM30 ring formation.

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.

GTP hydrolysis by Synechocystis IM30 does not decisively affect its membrane remodeling activity.

The function of IM30 (also known as Vipp1) is linked to protection and/or remodeling of the thylakoid membrane system in chloroplasts and cyanobacteria. Recently, it has been revealed that the Arabidopsis IM30 protein exhibits GTP hydrolyzing activity in vitro, which was unexpected, as IM30 does not show any classical GTPase features. In the present study, we addressed the question, whether an apparent GTPase activity is conserved in IM30 proteins and can also be observed for IM30 of the cyanobacterium Synechocystis sp. PCC 6803. We show that Synechocystis IM30 is indeed able to bind and hydrolyze GTP followed by the release of Pi. Yet, the apparent GTPase activity of Synechocystis IM30 does not depend on Mg2+, which, together with the lack of classical GTPase features, renders IM30 an atypical GTPase. To elucidate the impact of this cryptic GTPase activity on the membrane remodeling activity of IM30, we tested whether GTP hydrolysis influences IM30 membrane binding and/or IM30-mediated membrane fusion. We show that membrane remodeling by Synechocystis IM30 is slightly affected by nucleotides. Yet, despite IM30 clearly catalyzing GTP hydrolysis, this does not seem to be vital for its membrane remodeling function.

Glycine transporter GlyT1, but not GlyT2, is expressed in rat dorsal root ganglion--Possible implications for neuropathic pain.

Glycinergic inhibitory neurotransmission plays a pivotal role in the development of neuropathic pain. The glycine concentration in the synaptic cleft is controlled by the glycine transporters GlyT1 and GlyT2. GlyT1 is expressed throughout the central nervous system, while GlyT2 is exclusively located in glycinergic neurons. Aim of the present study was to investigate whether GlyTs are also expressed in the peripheral sensory nervous system and whether their expression is modulated in experimental neuropathic pain. Neuropathic pain was induced in male Wistar rats by Chronic Constriction Injury (CCI) and verified by assessment of mechanical allodynia (von Frey method). Expression patterns of GlyTs and the glycine binding subunit NR1 of the N-methyl-d-aspartate (NMDA) receptor in the spinal cord and dorsal root ganglia (DRG) were analyzed by Western blot analysis, PCR and immunohistochemistry. While both GlyT1 and GlyT2 were detected in the spinal cord, only GlyT1, but not GlyT2, was detected in DRG. Immunofluorescence revealed a strictly neuronal localization of GlyT1 and a co-localization of GlyT1 and NR1 in DRG. Compared to sham procedure, spinal cord and DRG expression of GlyT1 was not altered and NR1 was unchanged in DRG 12 days after CCI. GlyT1, but not GlyT2, is expressed in the peripheral sensory nervous system. The co-expression of GlyT1 and NMDA receptors in DRG suggests that GlyT1 regulates glycine concentration at the glycine binding site of the NMDA receptor. Differential regulation of GlyT1 expression in the spinal cord or DRG, however, does not seem to be associated with the development of neuropathic pain.

Expression of spinal cord microRNAs in a rat model of chronic neuropathic pain.

Neuropathic pain is accompanied by significant alterations of gene expression patterns in the somatosensory nervous system. The spinal cord is particularly prone to neuroplastic changes. Since the expression of microRNAs (miRNAs) has been linked to numerous pathophysiological processes, a contribution of miRNAs to the maladaptive plasticity of the spinal cord in neuropathic pain is possible. Aim of the present study therefore was to characterize the specific expression pattern of miRNAs in the rat spinal cord. Furthermore, we evaluated the time-dependent changes in expression patterns of spinal miRNAs in the chronic constriction injury (CCI) model of neuropathic pain in rats. Results from miRNA microarrays revealed a distinct expression pattern of miRNAs in the rat spinal cord. MiRNAs-494, -720, -690 and -668 showed the highest signal intensities. Members of the let-7 family as well as miR-124 belong to the group of the most highly expressed miRNAs. Induction of neuropathic pain by CCI did not lead to relevant differences in spinal miRNA expression levels compared to sham-operated animals at any studied time point. Therefore, modulation of miRNAs does not seem to contribute significantly to the changes in gene expression that cause neural plasticity in the spinal cord in this model of chronic neuropathic pain.

Effect of continuous posttraumatic intrathecal nocistatin on the development of mechanical allodynia.

BACKGROUND AND OBJECTIVES: The neuropeptide nocistatin has a variety of effects on nociception and other central nervous system functions. It has shown to exert diverging effects on nociceptive behavior in various experimental pain models depending on the dose administered. The inhibitory effect of spinal nocistatin on the release of glycine and γ-aminobutyric acid is thought to be responsible for pronociceptive effects, whereas the antinociceptive action of nocistatin can be attributed to diminished glycine-dependent N-methyl-D-aspartate receptor activation. So far, nocistatin has only been investigated in experimental models of already established pain and has been injected as a bolus. METHODS: In the present study, we investigated the effects of continuous intrathecal administration of nocistatin on the development of mechanical allodynia in the chronic constriction injury model of neuropathic pain in rats. The spinal infusion via intrathecal catheters connected to osmotic minipumps was started immediately after the surgical procedure and lasted 24 hrs. The development of mechanical allodynia was assessed with von Frey-type filaments for 2 weeks after chronic constriction injury. RESULTS: Despite a wide range of doses used, the continuous spinal application of nocistatin had no significant effect on the development of pathological nociceptive behavior at any time point, that is, mechanical allodynia developed equally in all groups in the injured paw, whereas nociceptive behavior was unchanged in comparison with baseline values in the uninjured paw in all groups. CONCLUSIONS: Because nocistatin has well-documented effects on established pathological pain, it is conceivable that its effect on nociception is only effective when spinal circuitry is pathologically altered.