Genomic regions underlying the species-specific mating songs of green lacewings.

Rapid species radiations provide insight into the process of speciation and diversification. The radiation of Chrysoperla carnea-group lacewings seems to be driven, at least in part, by their species-specific pre-mating vibrational duets. We associated genetic markers from across the genome with courtship song period in the offspring of a laboratory cross between Chrysoperla plorabunda and Chrysoperla adamsi, two species primarily differentiated by their mating songs. Two genomic regions were strongly associated with the song period phenotype. Large regions of chromosomes one and two were associated with song phenotype, as fewer recombination events occurred on these chromosomes relative to the other autosomes. Candidate genes were identified by functional annotation of proteins from the C. carnea reference genome. The majority of genes that are associated with vibrational courtship signals in other insects were found within QTL for lacewing song phenotype. Together these findings suggest that decreased recombination may be acting to keep together loci important to reproductive isolation between these species. Using wild-caught individuals from both species, we identified signals of genomic divergence across the genome. We identified several candidate genes both in song-associated regions and near divergence outliers including nonA, fruitless, paralytic, period, and doublesex. Together these findings bring us one step closer to identifying the genomic basis of a mating song trait critical to the maintenance of species boundaries in green lacewings.

Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

Central nervous system pathology in preclinical MPS IIIB dogs reveals progressive changes in clinically relevant brain regions.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

19F Paramagnetic Relaxation-Based NMR for Quaternary Structural Restraints of Ion Channels.

Quaternary distance restraints are essential to define the three-dimensional structures of protein assemblies. These distances often fall within a range of 10-18 Å, which challenges the high and low measurement limits of conventional nuclear magnetic resonance (NMR) and double electron-electron resonance electron spin resonance spectroscopies. Here, we report the use of 19F paramagnetic relaxation enhancement (PRE) NMR in combination with 19F/paramagnetic labeling to equivalent sites in different subunits of a protein complex in micelles to determine intersubunit distances. The feasibility of this strategy was evaluated on a pentameric ligand-gated ion channel, for which we found excellent agreement of the 19F PRE NMR results with previous structural information. The study suggests that 19F PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.

Structural basis of neurosteroid anesthetic action on GABAA receptors.

Type A γ-aminobutyric acid receptors (GABAARs) are inhibitory pentameric ligand-gated ion channels in the brain. Many anesthetics and neurosteroids act through binding to the GABAAR transmembrane domain (TMD), but the structural basis of their actions is not well understood and no resting-state GABAAR structure has been determined. Here, we report crystal structures of apo and the neurosteroid anesthetic alphaxalone-bound desensitized chimeric α1GABAAR (ELIC-α1GABAAR). The chimera retains the functional and pharmacological properties of GABAARs, including potentiation, activation and desensitization by alphaxalone. The apo-state structure reveals an unconventional activation gate at the intracellular end of the pore. The desensitized structure illustrates molecular determinants for alphaxalone binding to an inter-subunit TMD site. These structures suggest a plausible signaling pathway from alphaxalone binding at the bottom of the TMD to the channel gate in the pore-lining TM2 through the TM1-TM2 linker. The study provides a framework to discover new GABAAR modulators with therapeutic potential.

Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites.

Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.

Ketamine Inhibition of the Pentameric Ligand-Gated Ion Channel GLIC.

Ketamine inhibits pentameric ligand-gated ion channels (pLGICs), including the bacterial pLGIC from Gloeobacter violaceus (GLIC). The crystal structure of GLIC shows R-ketamine bound to an extracellular intersubunit cavity. Here, we performed molecular dynamics simulations of GLIC in the absence and presence of R- or S-ketamine. No stable binding of S-ketamine in the original cavity was observed in the simulations, largely due to its unfavorable access to residue D154, which provides important electrostatic interactions to stabilize R-ketamine binding. Contrary to the symmetric binding shown in the crystal structure, R-ketamine moved away from some of the binding sites and was bound to GLIC asymmetrically at the end of simulations. The asymmetric binding is consistent with the experimentally measured negative cooperativity of ketamine binding to GLIC. In the presence of R-ketamine, all subunits showed changes in structure and dynamics, irrespective of binding stability; the extracellular intersubunit cavity expanded and intersubunit electrostatic interactions involved in channel activation were altered. R-ketamine binding promoted a conformational shift toward closed GLIC. Conformational changes near the ketamine-binding site were propagated to the interface between the extracellular and transmembrane domains, and further to the pore-lining TM2 through two pathways: pre-TM1 and the ß1-ß2 loop. Both signaling pathways have been predicted previously using the perturbation-based Markovian transmission model. The study provides a structural and dynamics basis for the inhibitory modulation of ketamine on pLGICs.

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC.

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1ß3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1ß3GABAAR, is potentiated by 2-bromoethanol and ethanol. The results suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures and functional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

Ensemble-based virtual screening for cannabinoid-like potentiators of the human glycine receptor α1 for the treatment of pain.

The human glycine receptors (hGlyRs) are chloride-selective ion channels that mediate inhibitory neurotransmission in the brain stem and spinal cord. They are also targets for compounds of potential use in analgesic therapies. Here, we develop a strategy to discover analgesic drugs via structure-based virtual screening based on the recently published NMR structure of the hGlyR-α1 transmembrane domain (PDB ID: 2M6I ) and the critical role of residue S296 in hGlyR-α1 potentiation by Δ(9)-tetrahydrocannabinol (THC). We screened 1549 FDA-approved drugs in the DrugBank database on an ensemble of 180 hGlyR-α1 structures generated from molecular dynamics simulations of the NMR structure of the hGlyR-α1 transmembrane domain in different lipid environments. Thirteen hit compounds from the screening were selected for functional validation in Xenopus laevis oocytes expressing hGlyR-α1. Only one compound showed no potentiation effects; seven potentiated hGlyR-α1 at a level greater than THC at 1 µM. Our virtual screening protocol is generally applicable to drug targets with lipid-facing binding sites.

Obligatory duetting behaviour in the Chrysoperla carnea-group of cryptic species (Neuroptera: Chrysopidae): its role in shaping evolutionary history.

An unusual system of communication has evolved in green lacewings of the Chrysoperla carnea-group, triggering rapid proliferation of numerous cryptic species across all of the Northern Hemisphere and large portions of Africa. The system is based on sexually monomorphic, substrate-borne vibrational signals, produced by abdominal oscillation. These low-frequency signals are exchanged between courting individuals in a precise duetting format. The song of each of the more than 20 described species exhibits a unique acoustical phenotype that reproductively isolates the taxon from all other species with which it might come into contact. Here, we review what is known about duetting behaviour in the carnea-group, emphasizing the dominant role that duetting has played in the evolution, ecology, and speciation of the complex. Included are descriptions and discussions of song diversity and its impact on reproductive isolation among species, the genetic basis of interspecific song differences, partitioning of acoustic space among sympatric species, parallel song evolution in allopatric species pairs, and modes of speciation within the complex. We also emphasize the importance of correctly identifying by song all species of the carnea-group that are to be used either in experimental studies or programs of biological control, while acknowledging the continuing relevance of morphology to carnea-group systematics.