Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

PR55��, a Regulatory Subunit of PP2A, Specifically Regulates PP2A-mediated ��-Catenin Dephosphorylation

A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/β-catenin signaling plays essential roles in development and tumorigenesis (1–3). Our previous work found that β-catenin is sequentially phosphorylated by CKIα⁴ and GSK3 (4), which creates a binding site for β-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in β-catenin or APC genes that prevent β-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/β-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (8–10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11–21). Toward the goal of understanding the mechanism of β-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates β-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate β-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/B′, PR/B″, or PR/B‴). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55α, a regulatory subunit of PP2A, specifically regulates β-catenin phosphorylation and degradation. Mechanistically, we found that PR55α directly interacts with β-catenin and regulates PP2A-mediated β-catenin dephosphorylation in Wnt signaling.     

The regulatory C-terminal domain of subunit ε of F₀F₁ ATP synthase is dispensable for growth and survival of Escherichia coli

The C-terminal domain of subunit ε of the bacterial F_oF₁ ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.F_oF₁ ATP synthases are ubiquitous enzymes that synthesize ATP using a transmembrane electrochemical potential of protons or proton motive force (PMF) generated by the respiratory chain across the cytoplasmic membrane of bacteria, the thylakoid membrane of chloroplasts, or the mitochondrial inner membrane (4, 5, 37). The enzyme consists of two parts: membrane-embedded F_o subcomplex (a complex of subunits a, b, and c in bacteria) and hydrophilic F₁ subcomplex (composed of subunits α, β, γ, δ, and ε). The enzyme is also known as a molecular motor, which is composed of the stator subcomplex (α, β, δ, a, and b) and the rotor subcomplex (γ, ε, and c), and its rotation is coupled to ATP synthesis and proton flow across the membrane (20, 31, 52). The reaction of the enzyme is reversible; ATP is hydrolyzed into ADP and inorganic phosphate, the rotor subcomplex rotates in reverse, and protons are extruded to the periplasmic side, resulting in the generation of PMF. Although some bacteria utilize the reverse reaction under particular conditions, the primary function of F_oF₁ ATP synthase is generation of ATP from the PMF. Therefore, the direction of the activity of F_oF₁ ATP synthase is regulated to avoid wasteful ATP hydrolysis.Subunit ε in bacterial F_oF₁ has been known to be an intrinsic inhibitor of F₁ and F_oF₁ complex (18, 21, 23) and is proposed to have a regulatory function (10, 11, 42). Although the inhibitory effects of subunit ε vary among species, in general, ε inhibits ATP hydrolysis activity while repressing ATP synthesis activity to a lesser degree (14, 27). This regulatory function of the ε subunit is mediated almost exclusively by the C-terminal region of ε, which is comprised of two antiparallel α-helices (18, 49, 50). Biochemical and crystallographic studies have revealed that the C-terminal helices can adopt two different conformations (34, 46, 47, 48). In the retracted conformation, the α-helices form a hairpin-like structure and sit on the N-terminal β-sandwich domain of the ε subunit. When the ε subunit exhibits an inhibitory effect, it adopts a more extended conformation in which the C-terminal α-helices extend along the γ subunit, which composes the central stalk. It has also been shown that basic, positively charged residues on the second α-helix of the ε subunit interact with negatively charged residues in the DELSEED segment of subunit β to exert the inhibitory effect (12).Escherichia coli mutants deleted in the entire ε subunit exhibit a reduced growth rate and growth yield, and this effect is proposed to be a result of a deficiency in assembly of the F_o and F₁ complexes (21). The N-terminal β-sandwich domain of the ε subunit is responsible for the assembly of F_o and F₁ and is therefore important for efficient coupling between proton translocation through F_o and ATP synthesis/hydrolysis in F₁ (15, 39). Deletion of the ε subunit leads to dissociation of the F_oF₁ complex and wasteful ATP hydrolysis by free (cytoplasmic) F₁ and dissipation of PMF through free F_o (21, 22, 51).While the importance of the entire ε subunit in the whole-cell physiology of E. coli is fairly well established, the role of the regulatory C-terminal region of ε has received little attention and warrants investigation to determine if the regulatory functions (e.g., inhibition of ATP hydrolysis) observed in vitro are manifested in the physiology of E. coli under various growth conditions. To address this question, we constructed isogenic E. coli mutants that were deleted in the C-terminal region of ε subunit (ε^DC) and used these strains to compare physiological properties of wild-type versus ε^DC cells under a wide range of environmental conditions and stressors.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (���¦æ�)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhK''s activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.In the cascade activation of glycogenolysis in skeletal muscle, phosphorylase kinase (PhK),¹ upon becoming activated through phosphorylation, subsequently phosphorylates glycogen phosphorylase in a Ca²⁺-dependent reaction. This phosphorylation of glycogen phosphorylase activates its phosphorolysis of glycogen, leading to energy production (1). The 1.3 MDa (αβγδ)₄ PhK complex was the first protein kinase to be characterized and is among the largest and most complex enzymes known (2). As such, the intact complex has proved to be refractory to high resolution x-ray crystallographic or NMR techniques; however, low resolution structures of the nonactivated and Ca²⁺-saturated conformers of PhK have been deduced through modeling (3) and solved by means of three-dimensional electron microscopic (EM) reconstruction (4–7), and they show that the complex is a bilobal structure with interconnecting bridges. Approximate locations of small regions of each subunit in the complex are known (8–10) and show that the subunits pack head-to-head as apparent αβγδ protomers that form two octameric (αβγδ)₂ lobes associating in D₂ symmetry (11), although direct evidence that the αβγδ protomers are discrete, functional subcomplexes has been lacking until now.Approximately 90% of the mass of the PhK complex is involved in its regulation. Its kinase activity is carried out by the catalytic core of the γ subunit (44.7 kDa), with the k_cat being enhanced up to 100-fold by multiple metabolic, hormonal, and neural stimuli that are integrated through allosteric sites on PhK''s three regulatory subunits, α, β, and δ (12). The small δ subunit (16.7 kDa), which is tightly bound integral calmodulin (13), binds to at least the C-terminal regulatory domain of the γ subunit (γCRD) (14, 15), thereby mediating activation of the catalytic subunit by the obligate activator Ca²⁺ (16). The α and β subunits, as deduced from DNA sequencing, are polypeptides of 1237 and 1092 amino acids, respectively, with calculated masses prior to post-translational modifications of 138.4 and 125.2 kDa (17, 18). Both subunits can be phosphorylated by numerous protein kinases, including cAMP-dependent protein kinase and PhK itself (2). The α and β subunits are also homologous (38% identity and 61% similarity); however, each subunit has unique phosphorylatable regions that contain nearly all the phosphorylation sites found in these subunits (17, 18).The regulation of PhK activity by both Ca²⁺ (19–23) and phosphorylation has been studied extensively (reviewed in Ref. 24); however, only the structural effects induced by Ca²⁺ are well characterized (25), primarily through comparison of the non-activated and Ca²⁺-activated conformers using three-dimensional EM reconstructions (4), small angle x-ray scattering modeling (3), and biophysical (26–28) and chemical crosslinking methods (29–32). In contrast to the Ca²⁺-activated versus non-activated conformers, there are no reported structures of phosphorylated PhK to compare against the non-activated form. A very small amount of structural information for phospho-activated PhK derived from chemical crosslinking raises the possibility of phosphorylation-dependent communication between the β and γ subunits: Arg-18 in the N-terminal phosphorylatable region of β was found to be relatively near the γCRD (33). Several lines of evidence suggest that transduction of the activating phosphorylation signal in PhK occurs concomitantly with conformational changes in β (33) that are detected via various methods (10, 34), including chemical crosslinking (35). For example, crosslinking of only the phosphorylated conformer by the short-span crosslinker 1,5-difluoro-2,4-dinitrobenzene results in the formation of β homodimers (35). Correspondingly, more recent two-hybrid screens of the full length β subunit against itself yielded positive binding interactions only for point mutants in which the N-terminal phosphorylatable serine residues were mutated to phosphomimetic glutamates (33). It should be noted, however, that both chemical crosslinking and two-hybrid screening have potential drawbacks in the study of subunit interactions within a multisubunit complex. In the case of the latter, it is difficult when observing homodimeric two-hybrid interactions to determine whether they correspond to naturally occurring interactions between two like subunits within a complex or between two interacting regions within a single subunit of that complex. Studying subunit interactions in a complex through chemical crosslinking comes with its own inherent limitations. For example, an initial mono-derivatization can potentially cause a conformational change in one subunit that might affect the subsequent crosslinking reaction. This is particularly the case if the crosslinker contains a functionality, such as an aromatic group, that can unexpectedly direct it to a specific locus on the protein complex (36, 37). In addition, the spacer arms on many crosslinkers are sufficiently long to confound interpretation as to whether two subunits within a complex are actually in contact. Similarly, it should be proved that any observed crosslinked conjugate is formed from subunits within a complex, as opposed to between complexes (38, 39), a control that is often not run. Thus, it is prudent to analyze subunit interactions within a complex using a variety of approaches.To corroborate, complement, and expand the previous two-hybrid screening and chemical crosslinking studies of PhK''s subunit interactions and to investigate changes in the pattern of subunit interactions induced by phosphorylation, we carried out comparative MS analyses of both intact and partially denatured forms of nonactivated and phospho-activated PhK using mass spectrometers modified specifically to enhance the transmission of large noncovalently bound protein complexes (40–42). The array of subunit interactions detected for the nonactivated PhK complex largely replicated those reported in the crosslinking literature for this conformer, both corroborating those earlier studies and validating the use of these MS approaches to study subunit interactions within the PhK complex. Additionally, several novel subcomplexes of PhK were revealed, most notably an αβγδ protomer, which corroborates the observed packing of this subcomplex in the D₂ symmetrical (αβγδ)₄ native complex (9, 11). Moreover, we show herein that the array of subunit interactions detected for phospho-activated PhK differs significantly from that observed for the nonactivated conformer, with only the former showing extensive self-interactions between and among the regulatory β subunits. As is discussed, this suggests that activation through phosphorylation is associated with increased interprotomeric interactions in the bridged core of the PhK complex (33, 35).     

Repair of Nitric Oxide-damaged DNA in ��-Cells Requires JNK-dependent GADD45�� Expression

Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately β-cell death. Not only does nitric oxide cause β-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged β-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in β-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45α, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45α expression attenuates the repair of nitric oxide-induced β-cell DNA damage. These findings show that β-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45α mediate the p53-independent repair of this DNA damage.Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting pancreatic β-cells found in the islets of Langerhans (1). Cytokines, released from invading leukocytes during insulitis, are believed to participate in the initial destruction of β-cells, precipitating the autoimmune response (2, 3). Treatment of rat islets with the macrophage-derived cytokine interleukin-1 (IL-1)² results in the inhibition of glucose-stimulated insulin secretion and oxidative metabolism and in the induction of DNA damage that ultimately results in β-cell death (4–6). Nitric oxide, produced in micromolar levels following enhanced expression of the inducible nitric-oxide synthase in β-cells, mediates the damaging actions of cytokines on β-cell function (7–9). Nitric oxide inhibits insulin secretion by attenuating the oxidation of glucose to CO₂, reducing cellular levels of ATP and, thereby, attenuating ATP-inhibited K⁺ channel activity (10, 11). The net effect is the inhibition of β-cell depolarization, calcium entry, and calcium-dependent exocytosis. In addition to the inhibition of β-cell function, nitric oxide induces DNA damage in β-cells (4, 12, 13). Nitric oxide or the oxidation products N₂O₃ and ONOO⁻ induce DNA damage through direct strand breaks and base modification (14–16) and by inhibition of DNA repair enzymes, thereby enhancing the damaging actions of nitric oxide (17, 18).Recent studies have shown that β-cells maintain a limited ability to recover from cytokine-mediated damage (19, 20). The addition of a nitric-oxide synthase inhibitor to islets treated for 24 h with cytokine and continued culture with the nitric-oxide synthase inhibitor and cytokine results in a time-dependent restoration of insulin secretion, mitochondrial aconitase activity, and the repair of nitric oxide-damaged DNA (20, 21). Nitric oxide plays a dual role in modifying β-cell responses to cytokines. Nitric oxide induces β-cell damage and also activates a JNK-dependent recovery response that requires new gene expression (22). The ability of β-cells to recover from cytokine-mediated damage is temporally limited because cytokine-induced β-cell damage becomes irreversible following a 36-h incubation, and islets at this point are committed to degeneration (19).The purpose of this study was to determine the mechanisms by which β-cells repair nitric oxide-damaged DNA. Previous reports have shown that DNA damage induced by oxidizing agents, such as nitric oxide, is repaired through the base excision repair pathway (23), but how this pathway is activated in response to nitric oxide is unknown. Similar to the recovery of metabolic function, we now show that the activation of JNK by nitric oxide is required for repair of cytokine-induced DNA damage in β-cells. p53 is a logical candidate to mediate this repair because it plays a central role in DNA repair, is a target of JNK, and is activated by nitric oxide (24–27). However, we show that cytokines do not stimulate p53 phosphorylation, and nitric oxide fails to stimulate p53 accumulation and phosphorylation. Growth arrest and DNA damage (GADD) 45α is a DNA damage-inducible gene that can be regulated by both p53-dependent and p53-independent mechanisms (28–31). In contrast to p53, we show that cytokines stimulate GADD45α expression in a nitric oxide- and JNK-dependent manner and that siRNA-mediated knockdown of GADD45α results in an attenuation in the repair of nitric oxide-mediated DNA damage. These findings support a role for JNK in the regulation of GADD45α-dependent and p53-independent repair of nitric oxide-damaged β-cell DNA.     

Presynaptic Targeting of ��4��2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1��

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre- and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs)⁵ regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of α2–α10 and β2–β4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The α4β2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for α4β2 AChRs in nicotine addiction. α4β2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7, 8). Furthermore, β2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). α4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing α4β2 AChRs hypersensitive to nicotine demonstrate that α4β2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12). High resolution ultrastructural studies show that α4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13), and a sequence motif has been identified within the α4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14). However, the mechanisms underlying the targeting and clustering of α4β2 AChRs to presynaptic sites in neurons remain elusive.Recently, bi-directional interactions between neurexins and neuroligins have been shown to promote synapse assembly and maturation by fostering pre- and postsynaptic differentiation (reviewed in Refs. 15–17). The neurexins are encoded by three genes corresponding to neurexins I–III (18, 19), each encoding longer α-neurexins and shorter β-neurexins, because of differential promoter use. Neurexins recruit N- and P/Q-type calcium channels via scaffolding proteins, including calmodulin-associated serine/threonine kinase (20), to active zones of presynaptic terminals (21, 22). Recently, α-neurexins were shown to specifically induce GABAergic postsynaptic differentiation (23). Neuroligins, postsynaptic binding partners of neurexins, cluster N-methyl-d-aspartate receptors and GABA_A receptors by recruiting the scaffolding proteins PSD-95 (post-synaptic density 95) and gephyrin, respectively (24, 25). Interestingly, neurexins and neuroligins also modulate the postsynaptic clustering of α3-containing AChRs in chick ciliary ganglia (26, 27). In this study, using multiple experimental strategies, we provide evidence for the formation of complexes between neurexin-1β and α4β2 AChRs and a role for neurexin in the targeting of α4β2 AChRs to presynaptic terminals of neurons.     

Crystal Structure of the LG1-3 Region of the Laminin ��2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)² domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.     

Evidence for a Second, High Affinity G�¦� Binding Site on G��i1(GDP) Subunits

It is well known that Gα_i1(GDP) binds strongly to Gβγ subunits to form the Gα_i1(GDP)-Gβγ heterotrimer, and that activation to Gα_i1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gα_i1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gα_i1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gα_i1(GDP)-(Gβγ)₂ complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Gα_i1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gα_i1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.The plasma membranes of cells are organized as a series of protein-rich and lipid-rich domains (1–3). Many of the protein-rich domains, in particular those organized by caveolin proteins, are thought to be complexes of functionally related proteins that transduce extracellular signals (2). There is increasing evidence that heterotrimeric G proteins exist in pre-formed membrane complexes with their receptors and their intracellular effectors (4–8).The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9–12). The ligand-bound receptor will then catalyze the exchange of GTP for GDP on the Gα subunit in the G protein heterotrimer. In the basal state, Gα(GDP) binds strongly to Gβγ, but in the GTP-bound state this affinity is reduced, allowing Gα(GTP) and Gβγ subunits to individually bind to a host of specific intracellular enzymes and change their catalytic activity.Although the interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. For example, in pure or semi-pure systems, activation of Gα(GDP) sufficiently weakens its affinity for Gβγ resulting in dissociation (13). However, in cells separation of the heterotrimer is observed under some circumstances, but not others (7, 14–17). The reason for these differences in behavior is not clear. There are four families of Gα subunits that each contain several members, and, additionally, there are many subtypes of Gβγ subunits (18). It is possible that differences in dissociation behavior reflect differences in affinity between G protein subunit subtypes (19), the presence of various protein partners, and/or differences in post-synthetic modifications of the subunits (20).The mechanism that allows activated G proteins to remain bound is not apparent from the crystal structure (21, 22). If G protein subunits do not dissociate in cells, then their interaction must change in such a manner as to expose the effector interaction site(s). We have found that phospholipase Cβ1 (PLCβ1),⁴ an important effector of Gα_q (23), is bound to Gα_q prior to activation and throughout the activation cycle (6) implying that Gα_q(GDP) interacts with PLCβ1 in a non-functional manner.We have evidence that signaling complexes are stabilized by a series of secondary interactions. Using purified proteins and model membranes, we have found that membranes of the Gα_q-Gβγ/PLCβ1/RGS4 signaling system have secondary, weaker binding sites to members of this signaling system in addition to their high affinity site(s) to their functional partner(s). We speculate that secondary contacts allow for self-scaffolding of signaling proteins. To understand the nature of these secondary contacts, we have studied the ability of the Gα_i1(GDP)-Gβγ heterotrimer to remain complexed through the activation cycle (24). Here, we present evidence that Gα_i1(GDP) has two distinct Gβγ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We also find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding.     

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion ��-Toxins

The scorpion α-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na_vs) and is inactive at insect Na_vs. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa_v1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved “core domain” (residues of the loops connecting the secondary structure elements of the molecule core), and a variable “NC domain” (five-residue turn and the C-tail) as was reported for other scorpion α-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhαIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe¹⁵ of the core domain in toxin potency at rNa_v1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion α-toxins we were able to eliminate the activity of Lqh2 at rNa_v1.4 (skeletal muscle), hNa_v1.5 (cardiac), and rNa_v1.6 channels, with no hindrance of its activity at Na_v1.1–1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.The pivotal role of voltage-gated sodium channels (Na_vs)⁴ in excitability mark them as major targets for a large variety of toxins that bind at distinct receptor sites and modify their gating (1). These channels are large membrane proteins made of a pore-forming α-subunit of ∼260 kDa and auxiliary β-subunits of ∼30 kDa. The α-subunit is composed of four homologous domains (D1–D4), each consisting of six α-helical transmembrane segments (S1–S6) connected by intracellular and extracellular loops. A key feature in Na_vs function is their ability to rapidly activate and inactivate, leading to transient increase in Na⁺ conductance through the cell membrane. This mechanism is attributed to the ability of the positively charged S4 voltage sensors to move across the membrane in response to changes in membrane potential (1, 2).In mammals, at least nine genes encode a variety of Na_v subtypes (1, 3), whose expression varies greatly in different tissues (Na_v1.1–1.3 mainly in the central nervous system; Na_v1.6 in both central and peripheral neurons; Na_v1.7 in the peripheral nervous system; Na_v1.8 and Na_v1.9 in sensory neurons; Na_v1.4 and Na_v1.5 in skeletal and cardiac muscles, respectively). Na_v subtypes are distributed heterogeneously in the human brain and their expression is regulated under developmental and pathological conditions (1, 3–5). In addition, many disorders in humans result from abnormal function due to mutations in various Na_v genes (6–8). Thus, ligands that show specificity for Na_v subtypes may be used for their identification at various tissues and as leads for design of specific drugs. This requires that the bioactive surfaces of these ligands be resolved along with molecular details that determine their specificity.Among the wide range of Na_v modifiers, those derived from scorpion venoms play an important role in studying channel activation (β-toxins) and inactivation (α-toxins) (9–11). The channel site of interaction with scorpion α-toxins, named neurotoxin receptor site-3 (12), is shared also by structurally unrelated toxins from sea anemone and spider venoms (13, 14), which raises questions as to its architecture and boundaries. Based on the findings that site-3 toxins eliminate a gating charge component associated with the movement of D4/S4 (15, 16), and that this segment plays a critical role in coupling channel inactivation to activation (17), scorpion α-toxins were postulated to inhibit channel inactivation by hindering the outward movement of this segment during depolarization (9).Scorpion α-toxins constitute a class of structurally and functionally related 61–67-residue long polypeptides reticulated by four conserved disulfide bridges. Despite a common βαββ core (10, 18, 19) these toxins are highly diverse in sequence and preference for insect and mammalian Na_vs. Indeed, the α-toxin class is divided to pharmacological groups according to their toxicity in insects and mice brain and ability to compete on binding at insect and mammalian Na_vs (10) (supplemental Fig. S1): (i) classical anti-mammalian toxins, such as Aah2 (from Androctonus australis hector) and Lqh2 (from Leiurus quinquestriatus hebraeus), which bind with high affinity to Na_vs at rat brain synaptosomes and are practically non-toxic to insects; (ii) α-toxins, such as LqhαIT, which strongly affect insect Na_vs and are weak in mammalian brain; and (iii) α-like toxins, such as Lqh3 and BmKM1 (from Buthus martensii Karsch), which are active in both mammalian brain and insects.Efforts to identify α-toxin residues involved in the interaction with the Na_v receptor site-3 revealed a generally common bioactive surface divided to two topologically distinct domains: a conserved “core domain” formed by residues of the loops connecting the secondary structure elements of the molecule core, and a variable “NC domain” formed by the five-residue turn (residues 8–12) and the C-tail (20–23). These analyses raised the hypothesis that a protruding conformation of the NC domain correlates with high activity at insect Na_vs, whereas a flat conformation of this domain appears in α-toxins active at the brain channel rNa_v1.2a (21). The correlation of this structural difference with toxin preference for Na_v subtypes was corroborated by constructing the bioactive surface of LqhαIT on the scaffold of the anti-mammalian α-toxin Aah2 ending up with a chimera (Aah2^{LqhαIT(face)}) active on insects, whose NC domain is in the protruding conformation (21). Despite this result, the molecular requirements that enable high affinity binding of classical α-toxins to mammalian Na_vs have not been clarified, and only initial data about the channel region that constitutes receptor site-3 is available (Refs. 24–26; also see Ref. 10 for review).Lqh2 is a 64-residue long toxin from L. quinquestriatus hebraeus (Israeli yellow scorpion) (27) that is almost identical in sequence (96% identity) to the most active anti-mammalian toxin, Aah2, whose structure and action are documented (18, 28, 29). By functional expression and mutagenesis we uncovered residues on the Lqh2 exterior that are putatively involved in bioactivity. By construction of these residues on the scaffold of the anti-insect toxin LqhαIT we confirmed their bioactive role and differentiated those that determine toxin potency from those contributing to toxin efficacy. Comparison to other α-toxins was then instrumental for the design of an Lqh2 mutant that exhibits high specificity for the neuronal channels hNa_v1.1, rNa_v1.2a, and rNa_v1.3.