Lynx1 Shifts α4β2 Nicotinic Receptor Subunit Stoichiometry by Affecting Assembly in the Endoplasmic Reticulum

Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and β2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)₃(β2)₂ pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.     

Redistribution of EGF Receptor and α5, β1 Integrins in Response to Infection of Epithelial Cells by Serratia proteamaculans

Cell and Tissue Biology - Eukaryotic cells use surface receptors to interact with the environment. Interacting with the cell receptors, pathogenic and opportunistic bacteria can invade cells that...     

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756–765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639–641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase α2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase α2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase α subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.     

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.     

Photoactivatable Analogues of α-Conotoxins GI and MI and Their Interaction with Nicotinic Acetylcholine Receptor

Two photoactivatable analogues of -conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, -conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N of Gly1 or N of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native -conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [¹²⁵I] Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR crosslinking. All the AChR subunits were found to be crosslinked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the -conotoxin molecule.     

Differential α4(+)/(?)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)₂(β2)₃ and (α4)₃(β2)₂ subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with ¹²⁵I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.     

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC₅₀. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC₅₀’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.     

Preventing Expression of the Nicotinic Receptor Subunit α7 in SH-SY5Y Cells with Interference RNA Indicates that this Receptor may Protect Against the Neurotoxicity of Aβ

The present aim was to characterize the influence of the α7 nicotinic acetylcholine receptor (nAChR) on BACE, the enzyme that cleaves the amyloid precursor protein (APP) at the β-site, as well as on the oxidative stress induced by amyloid-β peptide (Aβ). To this end, human neuroblastoma SH-SY5Y cells were transfected with siRNAs targeting the α7 nAChR subunit and/or exposed to Aβ_1–42. For α7 nAChR, BACE1 (cleaving at the β-site of APP) and BACE2 (cleaving within the Aβ domain), α-secretase (ADAM10), and the two components of γ-secretase, PS and NCT, the mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. The level of Aβ_1–42 in the cell culture medium was determined by an ELISA procedure. The extent of lipid peroxidation and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were assayed spectrophotometrically. In the transfected SH-SY5Y cells, expression of α7 nAChR was reduced; the level of BACE1 increased and that of BACE2 decreased; the amount of ADAM10 lowered; and the level of PS raised. Moreover, the level of Aβ_1–42 in the culture medium was elevated. Treatment of non-transfected cells with Aβ elevated the level of malondialdehyde (MDA) and lowered the activities of SOD and GSH-Px and these changes were potentiated by inhibiting expression of α7 nAChR. These results indicate that α7 nAChR plays a significant role in amyloidogenic metabolism of APP and the oxidative stress evoked by Aβ, suggesting that this receptor might help protect against the neurotoxicity of Aβ.     

Species- and Subtype-Specific Recognition by Antibody WF6 of a Sequence Segment Forming an α-Bungarotoxin Binding Site on the Nicotinic Acetylcholine Receptor α Subunit

Abstract

The monoclonal antibody WF6 competes with acetylcholine and α-bungarotoxin (α-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) α1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR α1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for α-BGT, to the sequence segment α1(181–200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and α-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the α1 subunits from the muscle nAChRs of different species, the rat brain α2, α3, α4 and α5 nAChR subtypes, and the chick brain α-BGT binding protein subunits, αBGTBP α1 and αBGTBP α2. Our results indicate that WF6 is able to cross-react with the muscle α1 subunits of different species by virtue of conservation of several critical amino acid residues between positions 190–198 of the α1 subunit. These studies further define the essential structural features of the sequence segment α1(181–200) required to form the epitope for WF6.     

Tolerance to Diazepam-Induced Motor Impairment: A Study with GABAA Receptor α6 Subunit Knockout Mice

Development of tolerance to motor-impairing effects of repeated administration of moderate diazepam doses (5.0–7.5 mg/kg; three times daily PO 3 weeks) was compared between mice deficient in the cerebellar granule cell–restricted GABA_A receptor 6 subunit and their wild-type controls. The 6–/– mice were more impaired by the initial challenge doses of diazepam (5 or 10 mg/kg) than their controls, but acquired partial tolerance by the second tests with the same doses 4–7 days later. Chronic treatment produced complete tolerance in both mouse lines. Ligand autoradiography revealed a significant reduction in baseline benzodiazepine and chloride channel site-bindings in various regions of the 6–/– brains, but the chronic diazepam treatment did not consistently alter baseline or benzodiazepine site agonist and inverse agonist-modulated binding in the 6–/– and wildtype mice. The results indicate that tolerance to motor-impairing actions of diazepam is independent of the diazepam-insensitive 6 subunit-containing receptors, which rules out the possibility that tolerance emerges as an increase in structurally benzodiazepine-insensitive receptor population.     

Blocking mPTP on Neural Stem Cells and Activating the Nicotinic Acetylcholine Receptor α7 Subunit on Microglia Attenuate Aβ-Induced Neurotoxicity on Neural Stem Cells

Neurochemical Research - β-Amyloid (Aβ) can stimulate microglia to release a variety of proinflammatory cytokines and induce neurotoxicity. Nicotine has been reported to inhibit...     

Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin

Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended.     

Nicotine Acts on Growth Plate Chondrocytes to Delay Skeletal Growth through the α7 Neuronal Nicotinic Acetylcholine Receptor

Background

Cigarette smoking adversely affects endochondral ossification during the course of skeletal growth. Among a plethora of cigarette chemicals, nicotine is one of the primary candidate compounds responsible for the cause of smoking-induced delayed skeletal growth. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR), a specific receptor of nicotine, has been widely detected in non-excitable cells. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth.

Methodology/Principal Findings

We investigated the effect of nicotine on human growth plate chondrocytes, a major component of endochondral ossification. The chondrocytes were derived from extra human fingers. Nicotine inhibited matrix synthesis and hypertrophic differentiation in human growth plate chondrocytes in suspension culture in a concentration-dependent manner. Both human and murine growth plate chondrocytes expressed alpha7 nAChR, which constitutes functional homopentameric receptors. Methyllycaconitine (MLA), a specific antagonist of alpha7 nAChR, reversed the inhibition of matrix synthesis and functional calcium signal by nicotine in human growth plate chondrocytes in vitro. To study the effect of nicotine on growth plate in vivo, ovulation-controlled pregnant alpha7 nAChR +/− mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. Maternal nicotine exposure resulted in delayed skeletal growth of alpha7 nAChR +/+ fetuses but not in alpha7 nAChR −/− fetuses, implying that skeletal growth retardation by nicotine is specifically mediated via fetal alpha7 nAChR.

Conclusions/Significance

These results suggest that nicotine, from cigarette smoking, acts directly on growth plate chondrocytes to decrease matrix synthesis, suppress hypertrophic differentiation via alpha7 nAChR, leading to delayed skeletal growth.     

Mouse–Torpedo Chimeric α-Subunit Used to Probe Channel-Gating Determinants on the Nicotinic Acetylcholine Receptor Primary Sequence

1. To determine if structural domains are important for nicotinic acetylcholine receptor (nAChr) channel function, six mouse–Torpedo chimeric -subunits were constructed (Fig. 2) and coexpressed with Torpedo californica -, -, and -subunits in Xenopus laevis oocytes.2. nAChRs containing a chimeric -subunit were examined by voltage- and patch-clamp methods to determine their functional characteristics. Dose–response curves from voltage-clamped oocytes were used to estimate EC₅₀'s and Hill coefficients. Whole-cell currents were normalized against the -bungarotoxin (-BTX) binding sites to obtain normalized responses to acetylcholine (ACh). Open time constants at 4 M ACh were used to examine single-channel behavior.3. The EC₅₀ for ACh was modulated by the N-terminal half of the -subunit. When the Torpedo subunit sequence between position 1 and position 268 was replaced by mouse sequence, the EC₅₀ shifted toward the value for the wild-type mouse subunit. Replacement of either the 1–159 or the 160–268 positions of the Torpedo sequence with the mouse sequence lowered the EC₅₀. This suggests that at least two regions play a role in determining the EC₅₀.4. When the primary sequence (160–268) of the Torpedo -subunit was introduced in the mouse -subunit (T160–268), the expressed chimeric receptor was nonfunctional. The inverse chimera (M160–268) was functional and the open time constant and EC₅₀ were similar to those of mouse but the normalized response was characteristic of Torpedo.5. The normalized macroscopic response to ACh (300 M) of the chimera containing the mouse -subunit showed a ninefold increase relative to the Torpedo wild type. Receptors which contain the C terminal of the mouse -subunit also show an increase in the maximum normalized current. Receptors with the -subunit which contain the Torpedo C-terminal sequence have a lower normalized response.6. The combined results suggest that AChR channel function is modulated by structural determinants within the primary sequence. These structural domains might modulate channel function through specific allosteric interactions. The lack of response of the T160–268 chimera suggests that a critical interaction essential for the coupling of agonist binding and channel gating was disrupted. This result suggests that the interaction of structural domains within the nAChR primary structure are essential for channel function and that these intractions could be very specific within different nAChR species.