Alanine Scan of α-Conotoxin RegIIA Reveals a Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist

Activation of the α3β4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective α3β4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by α3β4, α3β2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3β4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3β4 than the α3β2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3β4 nAChR antagonist (IC₅₀ of 370 nm) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A,N12A]RegIIA bound to α3β4 and α3β2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3β2 (α3-Tyr⁹², Ser¹⁴⁹, Tyr¹⁸⁹, Cys¹⁹², and Tyr¹⁹⁶; β2-Trp⁵⁷, Arg⁸¹, and Phe¹¹⁹) may form the molecular basis for the selectivity shift.     

Ketamine Self-Administration Elevates αCaMKII Autophosphorylation in Mood and Reward-Related Brain Regions in Rats

Modulation of αCaMKII expression and phosphorylation is a feature shared by drugs of abuse with different mechanisms of action. Accordingly, we investigated whether αCaMKII expression and activation could be altered by self-administration of ketamine, a non-competitive antagonist of the NMDA glutamate receptor, with antidepressant and psychotomimetic as well as reinforcing properties. Rats self-administered ketamine at a sub-anesthetic dose for 43 days and were sacrificed 24 h after the last drug exposure; reward-related brain regions, such as medial prefrontal cortex (PFC), ventral striatum (vS), and hippocampus (Hip), were used for the measurement of αCaMKII-mediated signaling. αCaMKII phosphorylation was increased in these brain regions suggesting that ketamine, similarly to other reinforcers, activates this kinase. We next measured the two main targets of αCaMKII, i.e., GluN2B (S1303) and GluA1 (S831), and found increased activation of GluN2B (S1303) together with reduced phosphorylation of GluA1 (S831). Since GluN2B, via inhibition of ERK, regulates the membrane expression of GluA1, we measured ERK2 phosphorylation in the crude synaptosomal fraction of these brain regions, which was significantly reduced suggesting that ketamine-induced phosphorylation of αCaMKII promotes GluN2B (S1303) phosphorylation that, in turn, inhibits ERK 2 signaling, an effect that results in reduced membrane expression and phosphorylation of GluA1. Taken together, our findings point to αCaMKII autophosphorylation as a critical signature of ketamine self-administration providing an intracellular mechanism to explain the different effects caused by αCaMKII autophosphorylation on the post-synaptic GluN2B- and GluA1-mediated functions. These data add ketamine to the list of drugs of abuse converging on αCaMKII to sustain their addictive properties.     

The Role of α7 Nicotinic Acetylcholine Receptor in Modulation of Heart Rate Dynamics in Endotoxemic Rats

Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in modulation of heart rate dynamics during systemic inflammation.     

Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC₅₀ ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC₅₀ ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH₄C_l cells, but does not compete with ¹²⁵I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.     

Selective Expression of KCNS3 Potassium Channel α-Subunit in Parvalbumin-Containing GABA Neurons in the Human Prefrontal Cortex

The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV) or somatostatin (SST). Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K(+) channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K(+) channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with (35)S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.     

Distribution of α-Tocopherol Stereoisomers in Rats

The distribution of α-tocopherol (α-Toc) stereoisomers in the tissues of rats fed on a diet containing all-rac-α-tocopheryl acetate was investigated by a newly revised HPLC. The concentrations of 2R-isomers of α-Toc in blood and tissues of the rats were significantly higher than those of 2S-isomers. In most tissues, the levels of 2S-isomers were in order SRS> (SSS +SSR)/2 > SRR.     

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe²²³ residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.     

Changes of the Level of G Protein α-Subunit mRNA by Tolerance to and Withdrawal from Pentobarbital in Rats

Pentobarbital was continuously infused intracerebroventricularly (i.c.v.) at the rate of 300 g/10 l/h for 7 days, and withdrawal from pentobarbital was rendered 24 h after the stopping of the infusion. To eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. Little is known about the functional modulation of the G protein -subunits at the molecular level. The effects of continuous infusion of pentobarbital on the modulation of G protein -subunits mRNA were investigated by using in situ hybridization study. In situ hybridization showed that the level of Gs mRNA was increased in the septum and brainstem, and the level of Go mRNA was elevated in the cortex during the pentobarbital withdrawal. The level of Gi mRNA was significantly elevated in almost all area of brain during the pentobarbital withdrawal. These results suggest that region-specific changes of G protein -subunit mRNA were involved in the withdrawal from pentobarbital, whereas -subunit is not so highly involved in the pentobarbital tolerance.     

Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin α-Subunit,a Unique TGFβ Superfamily Antagonist

Transforming growth factor-beta (TGFβ) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFβ superfamily that share a common β-subunit. The evolution of the inhibin α-subunit created the only antagonist within the TGFβ superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin α-subunit appear to reflect specific insertion-deletion changes within the inhibin β-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFβ superfamily.     

Intranigral Dopamine Toxicity and α-Synuclein Response in Rats

There is increasing evidence that, in addition to its function as the main neurotransmitter in the nigrostriatal pathway, dopamine (DA) may be neurotoxic in certain conditions. In this study, the toxicity of DA was assessed by direct injection into the substantia nigra of anaesthetised rats, and its effects were compared with those of 6-hydroxydopamine. Brains were removed 1, 2 and 3 weeks after the lesion for histological or neurochemical analysis. DA caused a significant loss of 35% of tyrosine hydroxylase-positive neurons in the pars compacta of substantia nigra and a 40% reduction of striatal DA content. Cells with signs compatible with both apoptosis and autophagy were observed. GADD153, a parameter of endoplasmic reticulum stress, was strongly induced by 6-hydroxydopamine but not by DA. DA increased the α-synuclein content 1 week after the lesion (but not at the later times analyzed) in tyrosine hydroxylase-positive and in non-dopaminergic fibers of pars reticulata. The α-synuclein increase may be a physiological temporal response to DA accumulation and/or to cell damage, but the simultaneous presence of α-synuclein and DA in the cell cytoplasm at concentration higher than normal is not exempt from risk. In fact, their incubation in a free cell system gives a stable dimerized form of α-synuclein that has been described as the critical rate-limiting step for its abnormal fibrillation.     

The Duplicated α7 Subunits Assemble and Form Functional Nicotinic Receptors with the Full-length α7

The α7 nicotinic acetylcholine receptor gene (CHRNA7) is linked to schizophrenia. A partial duplication of CHRNA7 (CHRFAM7A) is found in humans on 15q13–14. Exon 6 of CHRFAM7A harbors a 2-bp deletion polymorphism, CHRFAM7AΔ2bp, which is also associated with schizophrenia. To understand the effects of the duplicated subunits on α7 receptors, we fused α7, dupα7, and dupΔα7 subunits with various fluorescent proteins. The duplicated subunits co-localized with full-length α7 subunits in mouse neuroblastoma cells (Neuro2a) as well as rat hippocampal neurons. We investigated the interaction between the duplicated subunits and full-length α7 by measuring Förster resonance energy transfer using donor recovery after photobleaching and fluorescence lifetime imaging microscopy. The results revealed that the duplicated proteins co-assemble with α7. In electrophysiological studies, Leu at the 9′-position in the M2 membrane-spanning segment was replaced with Cys in dupα7 or dupΔα7, and constructs were co-transfected with full-length α7 in Neuro2a cells. Exposure to ethylammonium methanethiosulfonate inhibited acetylcholine-induced currents, showing that the assembled functional nicotinic acetylcholine receptors (nAChRs) included the duplicated subunit. Incorporation of dupα7 and dupΔα7 subunits modestly changes the sensitivity of receptors to choline and varenicline. Thus, the duplicated proteins are assembled and transported to the cell membrane together with full-length α7 subunits and alter the function of the nAChRs. The characterization of dupα7 and dupΔα7 as well as their influence on α7 nAChRs may help explain the pathophysiology of schizophrenia and may suggest therapeutic strategies.     

The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein α subunit confers functional specificity to G-proteins. Vertebrate and insect Gα-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Gα genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Gα subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Gα gene classes, C. elegans and other nematodes also possess two lineage-specific Gα gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Gα genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution. [Reviewing Editor: Dr. John Oakeshott] Damien M. O’Halloran and David A. Fitzpatrick contributed equally to this work.     

Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites.Heterotrimeric G proteins undergo cycles of GTP-dependent conformational rearrangements and alterations of their oligomeric αβγ form to convey receptor signals to downstream effectors that control diverse cellular processes ranging from movement to division and differentiation. Interaction with activated receptor promotes the exchange of GDP for GTP on the G protein α subunit (Gα) and its separation from its βγ subunit partners (Gβγ). Both isolated Gα and Gβγ then interact with downstream effectors. GTP hydrolysis deactivates Gα, which reassociates with Gβγ, becoming ready to restart the cycle. Each of these stages has been subjected to extensive crystallographic studies with high-resolution structures of Gα in complex with GDP, GTP analog, Gβγ, and, most recently, the G-protein-coupled receptors now available. These studies have provided extensive mechanistic insight. However, a number of important questions remain, including:

• How do the distinct conformations evident in the accumulated structures interconvert?
• How do disease-associated mutations affect the fidelity of these transitions?
• And, critically, how do distal functional sites responsible for nucleotide and protein partner binding allosterically coordinate their activities?

Here we describe a comprehensive analysis of the accumulated Gα crystallographic structures supplemented with extensive conventional (cMD) and accelerated molecular dynamics (aMD) simulations (1) that together map the structural and dynamical features of Gα in different nucleotide states. These enhanced sampling simulations reveal the spontaneous interconversion between GDP and GTP conformations and also characterize large-scale opening motions of the α-helical domain (HD) that were not accessible to previous simulation studies (2–5). Furthermore, the current simulations results reveal a distinctive pattern of collective motions that provide evidence for a nucleotide-dependent network of dynamic communication between the active site and the receptor and effector binding sites.Principal component analysis of 53 Gα experimental structures homologous to transducin (Gαt) reveals that the major variation in accumulated structures is the concerted association/disassociation of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII). An additional small-scale (<10°) rotation of the HD relative to the main catalytic Ras-like domain (RasD) is also apparent (see Fig. S1 in the Supporting Material). The distinct conformation of SI–SIII regions gives rise to nucleotide-associated segregation of GDP- and GTP-analog-bound experimental structures along the PC1-PC2 plane. Interestingly, both GDP- and GTP-bound structures display a skewed distribution along the PC1-PC2 plane that arises from HD rotation. In comparison, the distribution of the GTP-bound structures becomes more restricted and the skew decreases when the mapping is based on a principle component analysis that excludes the HD region (see Fig. S1).Recently, the HD region of Gαs (the α-subunit of the stimulatory G protein for adenyl cyclase) was shown to adopt a dramatically more open conformation in a crystal structure complex with the β2 adrenergic receptor (β2AR) (6). This clam-shell-like 127° opening in the absence of nucleotide and presence of receptor is consistent with electron microscopy (7) and double electron-electron resonance analysis (8). These results, together with recent hydrogen-deuterium exchange mass spectrometry data (9), indicate that there may be additional functional motions and inherent flexibility in the ensemble of native states beyond those apparent in the accumulated crystal structures of Gαt (9). To address this question, we performed multiple 100-ns aMD simulations of nucleotide-free Gαt. These simulations reveal a spontaneous large-scale opening and closing motion of larger magnitude (>60°) than those evident in the distribution of crystallographic structures (Fig. 1 A and see Fig. S2). In addition, the trajectory reveals two dominant modes of HD opening: an out-of-plane shifting (PC1 in Fig. S3) and an in-plane rotation (PC2 in Fig. S3). It is also notable that nucleotide-free aMD simulations sample both active (GTP-like) and inactive (GDP-like) structures (see Fig. S2) in an analogous manner to the spontaneous GDP to GTP interconversion sampled for Ras and Rho small G proteins with similar methods (10–12).Open in a separate windowFigure 1Nucleotide-associated differences in flexibility and dynamic coupling. (A) Mapping aMD simulation trajectories (blue points) onto the principal components obtained from analysis of Gα crystallographic GDP-bound (green) and GTP-analog bound (red) experimental structures. (Orange) Open β2AR-Gαs complex structure. (B) Results of dynamic coupling analysis mapped onto the average structure for each nucleotide state. (Spheres) Nodes for the nucleotide; the protein cartoon is colored by community structure. (C) Community network graph. (Circles) Communities, colored as in panel B. Radius of the circle indicates the number of residues in the community. Thickness of linking lines is determined by the maximum betweenness of the respective intercommunity edges (see the Supporting Material). (Red, blue, and green edges) Major topological difference between states.The low sequence identity between Gαt and Gαs (44.5%), as well as the absence of the receptor and Gβγ in the simulations, may explain the difference between the predicted ∼60° Gαt-HD rotation and that displayed in the β2AR-Gαs crystallographic structure (see Fig. S3). It is notable that, although the amplitude is much smaller, aMD simulations with bound nucleotide display similar dominant HD motions to those observed in the nucleotide-free simulations (see Fig. S4). This suggests that the interdomain flexibility of RasD and HD is likely an intrinsic feature of Gαt regardless of nucleotide state.The transition between distinct conformations (structural clusters; see Fig. S5) was observed to correspond to significant dynamical changes in side-chain contacts (see Fig. S6). Specifically, we found sequential contacts breaking during the HD in-plane rotation motion starting from the region between HD helix αD and RasD helix αG toward that between HD helix αE and RasD SIII and the P-loop. In comparison, for the out-of-plane shift, we found simultaneous breaking and formation of contacts in the region containing the loop between helices αB and αC, the N-terminus of αA, αE, and αF of HD; α1, SI, and the loop between strand β6 and helix α5 of RasD. Interactions highlighted in these regions as potentially important for the conformational transitions include D137::K276, S140::K273, S140::D227, Q143::R238, N145::E39, and D146::K266, the effect of which can be further evaluated by mutagenesis experiments and simulations.Dynamic network analysis methods developed by Sethi et al. (13) were used to examine whether the motions of one residue were correlated to the motions of another (distant) residue. In this approach, a weighted graph is constructed where each residue represents a node and the weight of the connection between nodes represents their respective correlation value. A clustering of edges is then used to define local communities of highly correlated residues that represent substructures that are highly intraconnected, but loosely interconnected. Applying this approach to multiple 40-ns cMD simulations initiated from GTP-, GDP-, and aMD-derived APO conformations revealed a consistent community composition as well as a distinct pattern of intercommunity connection between nucleotide states (Fig. 1, B and C).The dynamics of the RasD region can be decomposed into two main communities that stem from the nucleotide base and phosphate regions in GDP and GTP states: The first community is composed of residues from the P-loop, helix α1, strands β1–β3, and the phosphates of the nucleotide (orange in Fig. 1, B and C). The second community comprises residues from helix αG, strands β4–β6, and the nucleotide base region (tan in Fig. 1, B and C). This dynamic partitioning of the central β-sheet and central role of the nucleotide is consistent with the bilobal structure and dynamics previously reported for Ras (14). In the presence of GTP, the first community includes or is dynamically coupled to SI, SII, and SIII regions (see the orange node and the red edge in Fig. 1 C). Removal of the γ-phosphate of GTP disrupts this region, leading to decoupling of the switch regions from the nucleotide. Also evident for GDP states is an apparent tighter coupling of RasD and HD regions (blue edges in Fig. 1 C). We note that these findings are robust to the choice of initial simulation conditions and are observed in both cMD and aMD simulations (see Fig. S7 and Fig. S8). Nucleotide-free Gαt simulations display an altered dynamical network with respect to those of nucleotide bound states. In particular, RasD and HD regions lose connecting edges consistent with the large-scale opening of these domains (e.g., SIII-HD green edges in Fig. 1 C).A number of residues highlighted here as potentially important for mediating the coupling between prominent communities (see Table S1 in the Supporting Material) have been shown by previous mutagenesis studies to affect GDP release. For example, the double mutation A322S/R174M was found to significantly enhance the rate of GDP release (15). The current results indicate that these positions are involved in coupling the nucleotide and RasD. Also, mutations R144A and L232Q caused a faster basal GDP release rate in Gαi1 (16). The current analysis indicates that the equivalent positions in Gαt (S140 and M228) couple the RasD and HD, and suggests that their mutation could promote domain-domain motions. We also note the apparent coupling of α5 with the nucleotide base and Ploop-β1 with the phosphate regions of GDP. These direct connections of the receptor connecting N- and C-terminus to GDP are suggestive of potential routes for receptor-mediated GDP release. We expect further study of these sites and of receptor-bound dynamics to be informative in this regard.In conclusion, simulations suggest a flexible HD in Gαt similar to that found for Gαs. In particular, in the absence of nucleotide we observed the spontaneous large-scale opening and closing of HD relative to RasD, which was unseen in previous computational studies. Moreover, we found that the functional states of Gαt are associated with the distinct dynamical couplings of functional regions including SI–SIII, P-loop, α5, and the HD region. Finally, our results indicate that nucleotide may not directly induce large-scale conformational changes but, instead, act as a modulator of intrinsically accessible conformations and as a central participant in their associated dynamical couplings.     

The α5 Subunit Regulates the Expression and Function of α4*-Containing Neuronal Nicotinic Acetylcholine Receptors in the Ventral-Tegmental Area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.     

The α6 integrin subunit in the developing mouse olfactory bulb

Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the α 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the α 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an α 6 knockout (α 6^?/?). Immunohistochemical analyses and confocal microscopy were used to examine α 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the α 6^?/? mouse. In CD-1 mice from E13 to E17, α 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; ～E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the α 6^?/? mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for α 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.     

A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats

In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.     

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β₃ integrins α_IIbβ₃ and α_Vβ₃, but a potential role of this interaction on stability of the metal ion at LIMBS in β₃ integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β₃ integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded α_IIbβ₃ versus α_Vβ₃, the result of stronger interactions of LIMBS with α_V, which reduce stability of the LIMBS metal ion in α_Vβ₃. Replacing the α_IIb-LIMBS interface residue Phe¹⁹¹ in α_IIb (equivalent to Trp¹⁷⁹ in α_V) with Trp strengthened this interface and destabilized the metal ion at LIMBS in α_IIbβ₃; a Trp¹⁷⁹ to Phe mutation in α_V produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular α_IIbβ₃ and a W179/F substitution in α_Vβ₃ reduced and increased, respectively, the apparent affinity of Mn²⁺ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in α_Vβ₃ structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.