Solution structure of alpha-conotoxin PIA, a novel antagonist of alpha6 subunit containing nicotinic acetylcholine receptors

alpha-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing alpha6 and alpha3 subunits. alpha-conotoxin PIA displays 75-fold higher affinity for rat alpha6/alpha3beta2beta3 nAChRs than for rat alpha3beta2 nAChRs. We have determined the three-dimensional structure of alpha-conotoxin PIA by nuclear magnetic resonance spectroscopy. The alpha-conotoxin PIA has an "omega-shaped" overall topology as other alpha4/7 subfamily conotoxins. Yet, unlike other neuronally targeted alpha4/7-conotoxins, its N-terminal tail Arg1-Asp2-Pro3 protrudes out of its main molecular body because Asp2-Pro3-Cys4-Cys5 forms a stable type I beta-turn. In addition, a kink introduced by Pro15 in the second loop of this toxin provides a distinct steric and electrostatic environment from those in alpha-conotoxins MII and GIC. By comparing the structure of alpha-conotoxin PIA with other functionally related alpha-conotoxins we suggest structural features in alpha-conotoxin PIA that may be associated with its unique receptor recognition profile.     

Alpha-conotoxin GIC from Conus geographus,a novel peptide antagonist of nicotinic acetylcholine receptors

Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, alpha-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. alpha-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 microm, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC(50) approximately 1.1 nm) for the human alpha3beta2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although alpha-conotoxin GIC shares some sequence similarity with alpha-conotoxin MII, which is also a potent alpha3beta2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the alpha3beta2 nAChR subtype (possibly inhibiting the fight-or-flight response).     

Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha 3- and chimeric alpha 6-containing nAChRs; BuIA blocks alpha 6/alpha 3 beta 2 nAChRs with a 40,000-fold lower IC(50) than alpha 4 beta 2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta 4 subunit have very slow off-times, compared with the corresponding beta 2 subunit-containing nAChR. In each instance, rat alpha x beta 4 may be distinguished from rat alpha x beta 2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha 3 beta 2 to mouse alpha 3 beta 4, and human alpha 3 beta2 to human alpha 3 beta 4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta 2- and beta 4-containing nAChRs.     

Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha-subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 versus alpha6beta2, and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2, and alpha1beta1deltaepsilon nAChRs.     

5-Iodo-A-85380 binds to alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) as well as alpha4beta2* subtypes

Recent work suggests that 5-iodo-A-85380, a radioiodinated analog of the 3-pyridyl ether A-85380, represents a promising imaging agent for non-invasive, in vivo studies of alphaAbeta2* nicotinic acetylcholine receptors (nAChRs; *denotes receptors containing the indicated subunits), because of its low non-specific binding, low in vivo toxicity and high selectivity for alpha4beta2* nAChRs. As an approach to elucidate nAChR subtypes expressed in striatum, we carried out competitive autoradiography in monkey and rat brain using 5-[125I]iodo-A-85380 ([125I]A-85380) and [125I]alpha-conotoxin MII, a ligand that binds with high affinity to alpha6* and alpha3* nAChRs, but not to alpha4beta2* nAChRs. Although A-85380 is reported to be selective for alpha4beta2* nAChRs, we observed that A-85380 completely inhibited [125I]alpha-conotoxin MII binding in rat striatum and that A-85380 blocked >90% of [125I] alpha-conotoxin MII sites in monkey caudate and putamen. These results suggest that A-85380 binds to non-alpha4beta2* nAChRs, including putative alpha6* nAChRs. Experiments to determine the percentage of [125I]A-85380 sites that contain alpha-conotoxin MII-sensitive (alpha6beta2*) nAChRs indicate that they represent about 10% of [125I]A-85380 sites in rodent striatum and about 30% of sites in monkey caudate and putamen. These data are important for identifying alterations in nicotinic receptor subtypes in Parkinson's disease and other basal ganglia disorders both in in vitro and in in vivo imaging studies.     

Nuclear magnetic resonance solution conformation of alpha-conotoxin AuIB, an alpha(3)beta(4) subtype-selective neuronal nicotinic acetylcholine receptor antagonist

The neuronal nicotinic acetylcholine receptors constitute a highly diverse group, with subtypes consisting of pentameric combinations of alpha and beta subunits. alpha-Conotoxins are a homologous series of small peptides that antagonize these receptors. We present the three-dimensional solution structure of alpha-conotoxin AuIB, the first 15-residue alpha-conotoxin known to selectively block the alpha(3)beta(4) nicotinic acetylcholine receptor subtype. The pairwise backbone and heavy-atom root mean square deviation for an ensemble of 20 structures are 0.269 and 0.720 A, respectively. The overall fold of alpha-conotoxin AuIB closely resembles that of the alpha4/7 subfamily alpha-conotoxins. However, the absence of Tyr(15), normally present in other alpha4/7 members, results in tight bending of the backbone at the C terminus and effectively renders Asp(14) to assume the spatial location of Tyr(15) present in other neuronal alpha4/7 alpha-conotoxins. Structural comparison of alpha-conotoxin AuIB with the alpha(3)beta(2) subtype-specific alpha-conotoxin MII shows different electrostatic surface charge distributions, which may be important in differential receptor subtype recognition.     

Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR

The alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits assemble to form the alpha9alpha10 nAChR subtype. This receptor is believed to mediate cholinergic synaptic transmission between efferent olivocochlear fibers and the hair cells of the cochlea. In addition alpha9 and/or alpha10 expression has been described in dorsal root ganglion neurons, lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary. Specific antagonists that selectively block the alpha9alpha10 channel could be valuable tools for elucidating its role in these diverse tissues. This study describes a novel alpha-conotoxin from the Western Atlantic species Conus regius, alpha-conotoxin RgIA (alpha-RgIA), that is a subtype specific blocker of the alpha9alpha10 nAChR. alpha-RgIA belongs to the alpha4/3 subfamily of the alpha-conotoxin family; sequence and subtype specificity comparisons between alpha-RgIA and previously characterized alpha4/3 toxins indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR subtype.     

Isolation,structure, and activity of GID,a novel alpha 4/7-conotoxin with an extended N-terminal sequence

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3 beta 2 nAChRs with IC(50) values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha 1 beta 1 gamma delta, alpha 3 beta 4, and alpha 4 beta 4 combinations. GID also potently inhibits the alpha 4 beta 2 subtype (IC(50) of 150 nm). Deletion of the N-terminal sequence (GID Delta 1-4) significantly decreased activity at the alpha 4 beta 2 nAChR but hardly affected potency at alpha 3 beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha 4 beta 2 and alpha 7 activity but not to alpha 3 beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha 7 versus alpha 3 beta 2 selectivity.     

Alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT3 chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs.     

Subtype-selective conopeptides targeted to nicotinic receptors: Concerted discovery and biomedical applications

Conus peptides that are selectively targeted to different molecular isoforms of nicotinic acetylcholine receptors (nAChRs) have been identified and characterized; several have recently been shown to have significant biomedical potential. An emerging strategy for the discovery from animal biodiversity of subtype-specific ligands for ion channel families is described in this review. Characterization of the gene family encoding a set of related ligands is required for discovery using a molecular genetics approach; when discovery is guided by a knowledge of the phylogeny of the biodiverse animal lineage being used as a source of ligands, a rational, efficient scan of the library of putative ligands becomes feasible. Together, these constitute an approach to uncover subtype-specific ligands, called "concerted discovery"; this was applied to the alpha-conotoxins, a family of Conus peptides generally targeted to nAChRs. Subtype-specific alpha-conotoxins were developed that target two groups of nAChRs, alpha(6)* and alpha(9)*. alpha-conotoxin MII has become the defining ligand for identifying the alpha(6)* nAChR subtype. A synthetic analog, MII [E11A], further subdivides alpha(6)* nAChRs into those that contain an alpha(4) subunit and those that do not. Importantly, these two subtypes are differentially affected by nigrostriatal damage, findings of likely relevance to the pathopysiology of Parkinson's disease. In contrast, alpha-conotoxins that target alpha(9) nAChR subtypes have potential as analgesics for the treatment of neuropathic pain that develops after nerve injury. The discovery of alpha-conotoxin RgIA enabled the identification of a novel role for alpha(9)* nAChRs. Use of alpha(9)* nAChR antagonists is associated with reversal of inflammation caused by the nerve injury. Thus, subtype-specific alpha-conotoxins targeted to particular nAChR isoforms are not only useful for understanding the physiological role of these receptors, but can have important diagnostic and therapeutic applications as well.     

Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist

Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11L,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11L,V16D] is the most selective ligand yet reported for alpha7 nAChRs.     

Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors

The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. Alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M(-1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.     

Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors

Alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.     

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists.     

Identification of a novel class of nicotinic receptor antagonists: dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus vexillum

The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, alphaD-VxXIIA, alphaD-VxXIIB, and alphaD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an approximately 11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that alphaD-VxXIIA, alphaD-VxXIIB, and alphaD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for alpha7 and beta2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.     

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes

The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.     

Synthesis of fluorescent analogs of alpha-conotoxin MII

Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.     

Single-residue alteration in alpha-conotoxin PnIA switches its nAChR subtype selectivity.

alpha-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different alpha-conotoxins are able to discriminate among different subtypes of mammalian nAChRs. In this report, the activity of two peptides from the venom of Conus pennaceus, alpha-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks alpha3beta2 nAChRs, whereas PnIB prefers the alpha7 subtype. Point mutation chimeras of these alpha-conotoxins were synthesized and their activities assessed on Xenopus oocytes expressing specific nAChRs. Change of a single residue, Ala10 to Leu, in PnIA (to form PnIA [A10L]) converts the parent peptide from alpha3beta2-preferring to alpha7-preferring; furthermore, PnIA [A10L] blocks the alpha7 receptor with an IC(50) (12.6 nM) that is lower than that of either parent peptide. Kinetic analysis indicates that differences in affinity among the analogues are primarily due to differences in off-rate, with PnIA [A10L]'s interaction with alpha7 having the smallest off-rate (k(off) = 0.17 min(-)(1)). Thermodynamic analysis indicates that Leu10 enhances the peptide's interaction with alpha7, but not alpha3beta2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both alpha7 and alpha3beta2 nAChRs.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.