Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.

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-terminally-amidated 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 >100 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.

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 ␣4␤2 nAChRs. It has been reported that dFBr is selective for the ␣4␤2 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 Ͻ10 M and inhibition of responses at concentrations Ͼ10 M. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human ␣4␤2 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 pIC 50 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 ␣4␤2-containing synapses.

Two ␣4␤2 nicotinic acetylcholine receptor (␣4␤2-nAChR) isoforms exist with (␣4) 2 (␤2) 3 and (␣4) 3 (␤2) 2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of ␣4(؉)/(؊)␤2 agonist-binding sites. The LS isoform also contains a unique ␣4(؉)/ (؊)␣4 site with lower agonist affinity than the ␣4(؉)/(؊)␤2 sites. However, the relative roles of the conserved ␣4(؉)/(؊)␤2 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 ␣4␤2*-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 ␤2 subunit (؊)-face E-loop residues to their non-conserved ␣4 subunit counterparts or vice versa (␤2HQT and ␣4VFL, 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 125 I-mAb 295 binding and was used to define function/nAChR. If the ␣4(؉)/(؊)␤2 sites contribute equally to function, making identical ␤2HQT 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, ␣4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent ␣4(؉)/(؊)␤2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

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’ 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.