αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish.

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 µM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis,C. auratus, and D. rerio. (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

α-Conotoxins Identify the α3ß4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

Ligands that selectively inhibit human α3ß2 and α6ß2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3ß4 and α6ß4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3ß2 than α3ß4 and 165-fold more potent on human α6/α3ß2ß3 than α6/α3ß4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with ß2 ligand-binding sites. In contrast, the ß4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained ß4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3ß4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, ß2, and ß4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.

Probing the Redox States of Sodium Channel Cysteines at the Binding Site of µO§-Conotoxin GVIIJ.

µO§-Conotoxin GVIIJ is a 35-amino acid peptide that readily blocks six of eight tested NaV1 subunit isoforms of voltage-gated sodium channels. µO§-GVIIJ is unusual in having an S-cysteinylated cysteine (at residue 24). A proposed reaction scheme involves the peptide-channel complex stabilized by a disulfide bond formed via thiol-disulfide exchange between Cys24 of the peptide and a Cys residue at neurotoxin receptor site 8 in the pore module of the channel (specifically, Cys910 of rat NaV1.2). To examine this model, we synthesized seven derivatives of µO§-GVIIJ in which Cys24 was disulfide-bonded to various thiols (or SR groups) and tested them on voltage-clamped Xenopus laevis oocytes expressing NaV1.2. In the proposed model, the SR moiety is a leaving group that is no longer present in the final peptide-channel complex; thus, the same koff value should be obtained regardless of the SR group. We observed that all seven derivatives, whose kon values varied over a 30-fold range, had the same koff value. Concordant results were observed with NaV1.6, for which the koff was 17-fold larger. Additionally, we tested two µO§-GVIIJ derivatives (where SR was glutathione or a free thiol) on two NaV1.2 Cys replacement mutants (NaV1.2[C912A] and NaV1.2[C918A]) without and with reduction of channel disulfides by dithiothreitol. The results indicate that Cys910 in wild-type NaV1.2 has a free thiol and conversely suggest that in NaV1.2[C912A] and NaV1.2[C918A], Cys910 is disulfide-bonded to Cys918 and Cys912, respectively. Redox states of extracellular cysteines of sodium channels have hitherto received scant attention, and further experiments with GVIIJ may help fill this void.

Molecular interaction of α-conotoxin RgIA with the rat α9α10 nicotinic acetylcholine receptor.

The α9α10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that α-conotoxin (α-CTx) RgIA, one of the few α9α10 selective ligands identified to date, is 300-fold less potent on human versus rat α9α10 nAChR. This species difference was conferred by only one residue in the (-), rather than (+), binding region of the α9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with α-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the α9(+) face and two residues on the α10(-) face of the α9α10 nAChR. However, mutations of these residues had little or no effect on toxin block of the α9α10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (α10 Ε197, P200 and α9 T61, D121) resulted in 19- to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human α9 nAChR, we modeled the rat α9α10 ECD and its complexes with α-CTx RgIA and acetylcholine. Our data support the interaction of α-CTx RgIA at the α10/α9 rather than the α9/α10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.

Α- and ß-subunit composition of voltage-gated sodium channels investigated with µ-conotoxins and the recently discovered µO§-conotoxin GVIIJ.

We investigated the identities of the isoforms of the α (NaV1)- and ß (NaVß)-subunits of voltage-gated sodium channels, including those responsible for action potentials in rodent sciatic nerves. To examine α-subunits, we used seven µ-conotoxins, which target site 1 of the channel. With the use of exogenously expressed channels, we show that two of the µ-conotoxins, µ-BuIIIB and µ-SxIIIA, are 50-fold more potent in blocking NaV1.6 from mouse than that from rat. Furthermore, we observed that µ-BuIIIB and µ-SxIIIA are potent blockers of large, myelinated A-fiber compound action potentials (A-CAPs) [but not small, unmyelinated C-fiber CAPs (C-CAPs)] in the sciatic nerve of the mouse (unlike A-CAPs of the rat, previously shown to be insensitive to these toxins). To investigate ß-subunits, we used two synthetic derivatives of the recently discovered µO§-conotoxin GVIIJ that define site 8 of the channel, as previously characterized with cloned rat NaV1- and NaVß-subunits expressed in Xenopus laevis oocytes, where it was shown that µO§-GVIIJ is a potent inhibitor of several NaV1-isoforms and that coexpression of NaVß2 or -ß4 (but not NaVß1 or -ß3) totally protects against block by µO§-GVIIJ. We report here the effects of µO§-GVIIJ on 1) sodium currents of mouse NaV1.6 coexpressed with various combinations of NaVß-subunits in oocytes; 2) A- and C-CAPs of mouse and rat sciatic nerves; and 3) sodium currents of small and large neurons dissociated from rat dorsal root ganglia. Our overall results lead us to conclude that action potentials in A-fibers of the rodent sciatic nerve are mediated primarily by NaV1.6 associated with NaVß2 or NaVß4.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3ß2ß3 and α3ß2 nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) subtype α6ß2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6ß2* and α3ß2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6ß2* and α3ß2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3ß2ß3 and α3ß2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for α3ß2 but not for α6/α3ß2ß3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3ß2ß3 than α3ß2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu(152) of the α6 subunit and Lys(152) of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6ß2* nAChRs.

Co-expression of Na(V)ß subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking µ-conotoxins.

BACKGROUND AND PURPOSE: Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (NaV 1) and one or two accessory ß-subunits (NaV ßs). Neurons in mammals can express one or more of seven isoforms of NaV 1 and one or more of four isoforms of NaV ß. The peptide µ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in NaV 1. Hitherto, the effects of NaV ß-subunit co-expression on the activity of these toxins have not been comprehensively assessed. EXPERIMENTAL APPROACH: Four µ-conotoxins (µ-TIIIA, µ-PIIIA, µ-SmIIIA and µ-KIIIA), TTX and STX were tested against NaV 1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of NaV ß1, ß2, ß3 or ß4 and, for NaV 1.7, binary combinations of thereof. KEY RESULTS: Co-expression of NaV ß-subunits modifies the block by µ-conotoxins: in general, NaV ß1 or ß3 co-expression tended to increase kon (in the most extreme instance by ninefold), whereas NaV ß2 or ß4 co-expression decreased kon (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by NaV ß-subunit co-expression. Tests of NaV ß1 : ß2 chimeras co-expressed with NaV 1.7 suggest that the extracellular portion of the NaV ß subunit is largely responsible for altering µ-conotoxin kinetics. CONCLUSIONS AND IMPLICATIONS: These results are the first indication that NaV ß subunit co-expression can markedly influence µ-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. µ-Conotoxins could, in principle, be used to pharmacologically probe the NaV ß subunit composition of endogenously expressed VGSCs.

Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA.

The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain, and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the α9α10 nAChR. Here, we report a two order of magnitude species difference in the potency of α-conotoxin RgIA for the rat versus human α9α10 nAChR. We investigated the molecular mechanism of this difference. Heterologous expression of the rat α9 with the human α10 subunit in Xenopus oocytes resulted in a receptor that was blocked by RgIA with potency similar to that of the rat α9α10 nAChR. Conversely, expression of the human α9 with that of the rat α10 subunit resulted in a receptor that was blocked by RgIA with potency approaching that of the human α9α10 receptor. Systematic substitution of residues found in the human α9 subunit into the homologous position in the rat α9 subunit revealed that a single point mutation, Thr56 to Ile56, primarily accounts for this species difference. Remarkably, although the α9 nAChR subunit has previously been reported to provide the principal (+) binding face for binding of RgIA, Thr56 is located in the (-) complementary binding face.

µ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na(V)1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na(V)1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 µ-conopeptides for their ability to block rodent Na(V)1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked Na(V)1.8, a TTX-resistant isoform, the resulting "activity matrix" revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A- and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A- and C-fibers were Na(V)1.6 and 1.7, respectively. Ruled out as major players in both fiber types were Na(V)1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive Na(V)1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of µ-conopeptides should be useful in identifying the functional contributions of Na(V)1 isoforms in other preparations.

Navß subunits modulate the inhibition of Nav1.8 by the analgesic gating modifier µO-conotoxin MrVIB.

Voltage-gated sodium channels (VGSCs) consist of a pore-forming α-subunit and regulatory ß-subunits. Several families of neuroactive peptides of Conus snails target VGSCs, including µO-conotoxins and µ-conotoxins. Unlike µ-conotoxins and the guanidinium alkaloid saxitoxin (STX), which are pore blockers, µO-conotoxins MrVIA and MrVIB inhibit VGSCs by modifying channel gating. µO-MrVIA/B can block Na(v)1.8 (a tetrodotoxin-resistant isoform of VGSCs) and have analgesic properties. The effect of Na(v)ß-subunit coexpression on susceptibility to block by µO-MrVIA/B and STX has, until now, not been reported. Here, we show that ß1-, ß2-, ß3-, and ß4-subunits, when individually coexpressed with Na(v)1.8 in Xenopus laevis oocytes, increased the k(on) of the block produced by µO-MrVIB (by 3-, 32-, 2-, and 7-fold, respectively) and modestly decreased the apparent k(off). Strong depolarizing prepulses markedly accelerated MrVIB washout with rates dependent on ß-subunit coexpression. Thus, coexpression of ß-subunits with Na(v)1.8 can strongly influence the affinity of the conopeptide for the channel. This observation is of particular interest because ß-subunit expression can be dynamic, e.g., ß2-expression is up-regulated after nerve injury (J Neurosci, 25:10970-10980, 2005); therefore, the effectiveness of a µO-conotoxin as a channel blocker could be enhanced by the conditions that may call for its use therapeutically. In contrast to MrVIB's action, the STX-induced block of Na(v)1.8 was only marginally, if at all, affected by coexpression of any of the ß-subunits. Our results raise the possibility that µO-conotoxins and perhaps other gating modifiers may provide a means to functionally assess the ß-subunit composition of VGSC complexes in neurons.

α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6ß4 and α6ß2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release.

α6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of α6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. α-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of α-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC(50) by 2800-fold at α6/α3ß2ß3 but only by 6-fold at α6/α3ß4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in α6-null mutant mice. α-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in ß4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain α6ß2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related α6* nAChRs; α6ß4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors.

Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the alpha-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain alpha-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson's disease and nociception. Structure/function characterization of alpha-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of alpha-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of alpha-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.

Synergistic and antagonistic interactions between tetrodotoxin and mu-conotoxin in blocking voltage-gated sodium channels.

Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, mu-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered mu-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain NaV1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded NaV complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in NaV pharmacology.

Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus.

Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus ("cone snails"). Cone snail biodiversity was exploited to identify three new mu-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus. BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous mu-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Na(v)1.4. Our preliminary results indicate that BuIIIB and BuIIIC are potent inhibitors of Na(v)1.4 (average block approximately 96%, at a 1muM concentration of peptide), displaying a very slow off-rate not seen in previously characterized mu-conotoxins that block Na(v)1.4. In addition, the three new C. bullatus mu-conopeptides help to define a new branch of the M-superfamily of conotoxins, namely M-5. The exogene strategy used to discover these Na channel-inhibiting peptides was based on both understanding the phylogeny of Conus, as well as the molecular genetics of venom mu-conotoxin peptides previously shown to generally target voltage-gated Na channels. The discovery of BuIIIA, BuIIIB and BuIIIC Na channel blockers expands the diversity of ligands useful in determining the structure-activity relationship of voltage-gated sodium channels.

Structure, dynamics, and selectivity of the sodium channel blocker mu-conotoxin SIIIA.

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.

Specificity, affinity and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated sodium channels Na(V)1.2, 1.6 and 1.7.

The excitotoxic conopeptide iota-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that iota-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel Na(V)1.6 to a more hyperpolarized level. Here, we performed voltage-clamp experiments to examine its activity against rodent Na(V)1.1 through Na(V)1.7 co-expressed with the beta1 subunit in Xenopus oocytes and Na(V)1.8 in dissociated mouse DRG neurons. The order of sensitivity to iota-RXIA was Na(V)1.6 > 1.2 > 1.7, and the remaining subtypes were insensitive. The time course of iota-RXIA-activity on Na(V)1.6 during exposure to different peptide concentrations were well fit by single-exponential curves that provided k(obs). The plot of k(obs)versus [iota-RXIA] was linear, consistent with a bimolecular reaction with a K(d) of approximately 3 microM, close to the steady-state EC(50) of approximately 2 microM. iota-RXIA has an unusual residue, D-Phe, and the analog with an L-Phe instead, iota-RXIA[L-Phe44], had a two-fold lower affinity and two-fold faster off-rate than iota-RXIA on Na(V)1.6 and furthermore was inactive on Na(V)1.2. iota-RXIA induced repetitive action potentials in mouse sciatic nerve with conduction velocities of both A- and C-fibers, consistent with the presence of Na(V)1.6 at nodes of Ranvier as well as in unmyelinated axons. Sixteen peptides homologous to iota-RXIA have been identified from a single species of Conus, so these peptides represent a rich family of novel sodium channel-targeting ligands.

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.

Structure/function characterization of micro-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels.

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only approximately 20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na(V)1.2 and Na(V)1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Na(V)1.2 and only partially for Na(V)1.6. Other isoforms were blocked reversibly: Na(V)1.3 (IC50 8 microM), Na(V)1.5 (IC50 284 microM), and Na(V)1.4 (IC50 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na(V)1.2 and Na(V)1.4; replacement of Trp-8 resulted in reversible block of Na(V)1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na(V)1.4 than of Na(V)1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na(V)1.2 and that further engineering of micro-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Characterization of nicotinic acetylcholine receptors that modulate nicotine-evoked [3H]norepinephrine release from mouse hippocampal synaptosomes.

Nicotine's modulation of hippocampal noradrenergic neurotransmission may contribute to its mnemonic properties, but the nicotinic acetylcholine receptor (nAChR) subtypes that modulate terminal release of norepinephrine are unknown. In the present study, we used a number of subtype-selective alpha-conotoxins in combination with nicotinic receptor subunit-deficient mice to characterize nAChRs that modulate [3H]nore-pinephrine release from synaptosomes. The results indicate that at least two populations of nAChRs contribute to this release: a novel alpha6(alpha4)beta2beta3beta4 subtype and an alpha6(alpha4)beta2beta3 subtype. These are distinct from subtypes that modulate synaptosomal norepinephrine release in the rat hippocampus in which an alpha6/beta2 and/or alpha6/beta4 ligand binding interface is not present. Whereas alpha-conotoxin MII fully inhibits nicotine-evoked [3H]norepinephrine release in mouse, it is ineffective in blocking [3H]norepinephrine release in rat. Block of [3H]norepinephrine release by alpha-conotoxin BuIA, a toxin that kinetically distinguishes between beta2- and beta4-containing nAChRs, was partially reversible in mouse but irreversible in rat. This indicates that in contrast to rat, mouse nAChRs are made of both beta4 and non-beta4-containing populations. Results from beta2 and beta4 null mutant mice confirmed this conclusion, indicating the presence of the beta2 subunit in all nAChRs and the presence of the beta4 subunit in a subpopulation of nAChRs. In addition, both alpha4 and beta3 subunits are essential for the formation of functional nAChRs on mouse noradrenergic terminals. Cytisine, a ligand formerly believed to be beta4-selective, was a highly effective agonist for alpha6beta2-containing nAChRs. The sum of these results suggests a possible novel nAChR subtype that modulates nor-adrenergic neurotransmission within the mouse hippocampus.