Interactions between the rabies virus and nicotinic acetylcholine receptors: A potential role in rabies virus induced behavior modifications

Rabies causes approximately 60,000 casualties annually and has a case fatality rate approaching 100% once clinical signs occur. The glycoprotein on the surface of the virion is important for the host immune response and facilitates interaction of the virion with host cell receptors. Nicotinic acetylcholine receptors were the first receptors identified as a molecular target for the rabies virus. Additional targets, including neural cell adhesion molecule, p75 neurotrophin receptor, metabotropic glutamate receptor subtype 2, and integrin β1, have been added to the list, all of which can mediate viral entry into the cell. Multiple receptors and different subtypes of nicotinic acetylcholine receptors result in a complex picture of virus-receptor interactions. In addition, some data suggest that the rabies virus glycoprotein inhibits cell signalling events mediated by various nicotinic receptor subtypes that have been implicated in altering behaviour in unaffected animals. This review focuses on interactions between the rabies virus glycoprotein and nicotinic receptors and proposes possible functional consequences, including behavioural modifications and therapeutic approaches for future research.publishedVersio

Distinctive Single-Channel Properties Of α4β2-Nicotinic Acetylcholine Receptor Isoforms

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2) 2 β2- and LS-(α4β2) 2 α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2) 2 α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2) 2 β2-nAChR exhibit a single conductance state, whereas LS-(α4β2) 2 α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2) 2 α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2) 2 α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2) 2 α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings

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)2(β2)3 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

Interactions between the rabies virus and nicotinic acetylcholine receptors: A potential role in rabies virus induced behavior modifications

Rabies causes approximately 60,000 casualties annually and has a case fatality rate approaching 100% once clinical signs occur. The glycoprotein on the surface of the virion is important for the host immune response and facilitates interaction of the virion with host cell receptors. Nicotinic acetylcholine receptors were the first receptors identified as a molecular target for the rabies virus. Additional targets, including neural cell adhesion molecule, p75 neurotrophin receptor, metabotropic glutamate receptor subtype 2, and integrin β1, have been added to the list, all of which can mediate viral entry into the cell. Multiple receptors and different subtypes of nicotinic acetylcholine receptors result in a complex picture of virus-receptor interactions. In addition, some data suggest that the rabies virus glycoprotein inhibits cell signalling events mediated by various nicotinic receptor subtypes that have been implicated in altering behaviour in unaffected animals. This review focuses on interactions between the rabies virus glycoprotein and nicotinic receptors and proposes possible functional consequences, including behavioural modifications and therapeutic approaches for future research

Distinctive Effects Of Nicotinic Receptor Intracellular-Loop Mutations Associated With Nocturnal Frontal Lobe Epilepsy

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations\u27 effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [125I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology

Pharmacological Characterization of the Allosteric Modulator Desformylflustrabromine and Its Interaction with α4β2 Neuronal Nicotinic Acetylcholine Receptor Orthosteric Ligands

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. nAChRs are involved in modulating nicotinic-based signal transmission in the central nervous system and are implicated in a range of disorders. Desformylflustrabromine (dFBr) is a positive allosteric modulator that potentiates α4β2 nAChRs. It has been reported that dFBr is selective for the α4β2 receptor relative to other common nAChR subtypes (Neurosci Lett 373:144–149, 2005). Coapplication of dFBr with acetylcholine (ACh) produces a bell-shaped dose–response curve with a peak potentiation of more than 265% (Bioorg Med Chem Lett 17:4855–4860, 2007) at dFBr concentrations <10 μM and inhibition of responses at concentrations >10 μM. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human α4β2 nAChRs expressed in Xenopus laevis oocytes. Currents to both partial and full agonists were potentiated by dFBr. Responses to low-efficacy agonists were potentiated significantly more than responses to high-efficacy agonists. Antagonist pIC50 values were unaffected by coapplication of dFBr. In addition to its potentiating effects, dFBr was able to induce current spikes when applied to desensitized receptors, suggestive of a shift in equilibrium from the desensitized to open conformation. In contrast to potentiation, inhibition of ACh responses by dFBr depends on membrane potential and is probably the result of open-channel block by dFBr and ACh. Our data indicate distinct mechanisms for the potentiation and inhibition components of dFBr action. dFBr could prove useful for therapeutic enhancement of responses at α4β2-containing synapses

Sleep-related hypermotor epilepsy associated mutations uncover important kinetic roles of α4β2- nicotinic acetylcholine receptor intracellular structures.

Sleep-related hypermotor epilepsy (SHE) is a group of seizure disorders prominently associated with mutations in nicotinic acetylcholine receptors (nAChR). The most prevalent central nervous system nAChR subtype contains α4 and β2 subunits, in two ratios. (α4β2)2β2-nAChR have high agonist sensitivity (HS-isoform), whereas (α4β2)2α4-nAChR agonist responses exhibit a small high-sensitivity, and a predominant low-sensitivity, phase of function (LS-isoform). Multiple non-synonymous mutations in the second and third transmembrane domains of α4 and β2 subunits are associated with SHE. We recently demonstrated that two additional, SHE-associated, missense mutations in the major cytoplasmic loops of these subunits [α4(R336H) and β2(V337G)] cause increased macroscopic function-per receptor. Here, we use single-channel patch-clamp electrophysiology to show that these mutations influence single-channel amplitudes and open- and closed-state kinetics. Pure populations of HS- or LS-isoform α4β2-nAChR were expressed by injecting either 1:10 or 30:1 α4:β2 cRNA ratios, respectively, into Xenopus laevis oocytes. Functional properties of the resulting mutant α4β2-nAChR isoforms were compared to their wildtype counterparts. α4(R336H) subunit incorporation minimally affected single-channel amplitudes, whereas β2(V337G) subunit incorporation reduced them significantly in both isoforms. However, for both mutant subunits, increased function-per-receptor was predominantly caused by altered single channel kinetics. The α4(R336H) mutation primarily destabilizes desensitized states between openings. By contrast, the β2(V337G) mutation principally stabilizes receptor open states. The use of naturally-occurring and physiologically-impactful mutations has allowed us to define valuable new insights regarding the functional roles of nAChR intracellular domains. Further mechanistic context is provided by intracellular-domain structures recently published for other members of the Cys-loop receptor superfamily (α3β4-nAChR and 5-HT3AR)

Distinctive single-channel properties of α4β2-nicotinic acetylcholine receptor isoforms.

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site's role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings

A Novel α4/7-Conotoxin Lvia From Conus Lividus That Selectively Blocks α3β2 Vs. α6/α3β2β3 Nicotinic Acetylcholine Receptors

This study was performed to discover and characterize the first potent α3β2-subtype-selective nicotinic acetylcholine receptor (nAChR) ligand. A novel α4/7-conotoxin, α-CTxLvIA, was cloned from Conus lividus. Its pharmacological profile at Xenopus laevis oocyte-expressed rat nAChR subtypes was determined by 2-electrode voltage-clamp electrophysiology, and its 3-dimensional (3D) structure was determined by NMR spectroscopy. α-CTx LvIA is a 16-aa C-terminallyamidated peptide with 2-disulfide bridges. Using rat subunits expressed in Xenopus oocytes, we found the highest affinity of α-CTxLvIA was for α3β2 nAChRs (IC50 8.7 nM), where blockade was reversible within 2 min. IC50 values were \u3e100 nM at α6/α3β2β3, α6/ α3β4, and α3β4 nAChRs, and ≥3 μM at all other subtypes tested. α3β2 vs. α6β2 subtype selectivity was confirmed for human-subunit nAChRs with much greater preference (300-fold) for α3β2 over α6β2 nAChRs. This is the first α-CTx reported to show high selectivity for human α3β2 vs. α6β2 nAChRs. α-CTxLvIA adopts two similarly populated conformations water: one (assumed to be bioactive) is highly structured, whereas the other is mostly random coil in nature. Selectivity differences with the similarly potent, but less selective, α3β2 nAChR antagonist α-CTx PeIA probably reside within the three residues, which differ in loop 2, given their otherwise similar 3D structures. α4/7-CTx LvIA is a new, potent, selective α3β2 nAChR antagonist, which will enable detailed studies of α3β2 nAChR structure, function, and physiological roles. © FASEB