Neuronal nicotinic acetylcholine receptors (nAChRs) with putative alpha3 beta4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of alpha3 beta4-selective ligands. In this report, we describe the purification and characterization of an alpha3 beta4 nAChR antagonist, alpha-conotoxin AuIB, from the venom of the "court cone," Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. alpha-Conotoxin AuIB blocks alpha3 beta4 nAChRs expressed in Xenopus oocytes with an IC50 of 0.75 microM, a kon of 1.4 x 10(6) min-1 M-1, a koff of 0.48 min-1, and a Kd of 0.5 microM. Furthermore, alpha-conotoxin AuIB blocks the alpha3 beta4 receptor with >100-fold higher potency than other receptor subunit combinations, including alpha2 beta2, alpha2 beta4, alpha3 beta2, alpha4 beta2, alpha4 beta4, and alpha1 beta1 gamma delta. Thus, AuIB is a novel, selective probe for alpha3 beta4 nAChRs. AuIB (1-5 microM) blocks 20-35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the alpha3 beta2-specific alpha-conotoxin MII (100 nM) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, alpha3 beta4-containing nAChRs mediate norepinephrine release, whereas alpha3 beta2-containing receptors mediate dopamine release.

We report the characterization of a new sodium channel blocker, mu-conotoxin PIIIA(mu-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: [sequence: see text] where Z = pyroglutamate and O = 4-trans-hydroxyproline. We demonstrate that Arginine-14 (Arg14) is a key residue; substitution by alanine significantly decreases affinity and results in a toxin unable to block channel conductance completely. Thus, like all toxins that block at Site I, mu-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized mu-conotoxin GIIIA (mu-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of mu-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to mu-PIIIA and mu-conotoxin GIIIA, (2) sensitive to mu-PIIIA but not to mu-GIIIA, and (3) resistant to mu-PIIIA and mu-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, mu-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.

-Conotoxin PVIIA (-PVIIA), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. -Conotoxin PVIIA has the following structure. STRUCTURE IThis is the first Conus peptide known to target K ؉ channels. Although the Shaker K ؉ channel is sensitive to -PVIIA, the rat brain Kv1.1 subtype is resistant. Chimeras between Shaker and the Kv1.1 K ؉ channels were constructed and expressed in Xenopus oocytes. Only channels containing the putative pore-forming region between the fifth and sixth transmembrane domains of Shaker retained toxin sensitivity, indicating that the toxin target site is in this region of the channel. Evidence is presented that -PVIIA interacts with the external tetraethyl-ammonium binding site on the Shaker channel.Although both -PVIIA and charybdotoxin inhibit the Shaker channel, they must interact differently. The F425G Shaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes -PVIIA sensitivity.The precursor sequence of -PVIIA was deduced from a cDNA clone, revealing a prepropeptide comprising 72 amino acids. The N-terminal region of the -PVIIA prepropeptide exhibits striking homology to the -, O-, and ␦-conotoxins. Thus, at least four pharmacologically distinct superfamilies of Conus peptides belong to the same "O" superfamily, with the -and -conotoxins forming one branch, and the ␦-and O-conotoxins forming a second major branch.The venoms of the 500 species of predatory marine snails belonging to the genus Conus have proven to be a rich source of peptidic ligands with high affinity and specificity. The conotoxins are relatively small, disulfide-bridged peptides (12-30 amino acids) that can readily be chemically synthesized. A large number of peptides that target voltage-gated calcium and sodium channels, as well as the acetylcholine receptor, have been characterized. However, the peptides that target potassium channels in Conus venoms are relatively unexplored; recently, we described an initial characterization of -PVIIA, 1 a peptide from the fish-hunting snail Conus purpurascens, which inhibited the Shaker K ϩ channel but had no effects on any voltage-gated Ca 2ϩ or Na ϩ channel tested (1). The sequence of -PVIIA bears little apparent homology to the dendrotoxins or to scorpion and spider toxins, which block the Shaker K ϩ channels. The Shaker potassium channel is one of the most intensively investigated neuronal signaling macromolecules. Several polypeptide toxins, notably charybdotoxin and the agitoxins (2), have been used to probe this channel; this approach has made the outer vestibule of the Shaker potassium channel the most well mapped region of any ion channel complex. A novel toxin based on a different structural framework from existing ligands could potentially interact with unmapped extracellular regions of the ion channel complex.In this report, we describe the successful chemical synthesis of -PVIIA ...

We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conus species were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (--CC--CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conus species. These peptides are likely to be functionally diverse; although the peptides are small (11-17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCP-KQMRCCTL*; tx5a, ␥CC␥DGW ؉ CCT § AAO; and au5a, FC-CPFIRYCCW (where ␥ ‫؍‬ ␥-carboxyglutamate, W ؉ ‫؍‬ bromotryptophan, O ‫؍‬ hydroxyproline, T § ‫؍‬ glycosylated threonine, and * ‫؍‬ COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional ␥-carboxylation recognition signal in the ؊1 to ؊20 propeptide region, consistent with the presence of ␥-carboxyglutamate residues in this peptide.Cone snails (genus Conus) are perhaps the most successful genus of marine invertebrates, with over 500 species, all of which are venomous (1, 2). These predatory marine snails have evolved a highly sophisticated neuropharmacological strategy based on small peptides (10 -35 amino acids) in their venoms (3, 4). Most Conus peptides potently affect ion channel function; these are widely used pharmacological reagents in neuroscience, and several are being directly developed as diagnostic and therapeutic agents. Most Conus peptides are highly disulfide-rich; generically, Conus peptides with multiple disulfide cross-links have been referred to as conotoxins. It has become apparent in recent years that there are tens of thousands of different conotoxins in Conus venoms. Because of the remarkably rapid interspecific divergence of peptide sequences, each Conus species has its own distinct repertoire of between 50 and 200 different venom peptides (5).A major simplification in understanding this complex array of Conus venom peptides is that most of the ϳ50,000 different molecular forms can be grouped into just a few superfamilies. Peptides in the same superfamily share both a conserved pattern of disulfide connectivity and a highly conserved signal sequence (when prepropeptide precursor sequences of the peptides are compared) (5, 6). Three large superfamilies of conotoxins are well characterized: the O-superfamily, comprising several distinct pharmacological families including the -, -, ␦-, and O-conotoxins (7); the A-superfamily, to which the ␣-conotoxins belong (8); and the M-superfamily, to which the -conotoxins belong. In this paper, we describe the ...

-Conotoxin PVIIA (-PVIIA), a 27-amino acid peptide with three disulfide cross-links, isolated from the venom of Conus purpurascens, is the first conopeptide shown to inhibit the Shaker K ؉ channel (Terlau, H., Shon, K., Grilley, M., Stocker, M., Stü hmer, W., and Olivera, B. M. (1996) Nature 381, 148 -151). Recently, two groups independently determined the solution structure for -PVIIA using NMR; although the structures reported were similar, two mutually exclusive models for the interaction of the peptide with the Shaker channel were proposed. We carried out a structure/function analysis of -PVIIA, with alanine substitutions for all amino acids postulated to be key residues by both groups. Our data are consistent with the critical dyad model developed by Mé nez and co-workers (Dauplais, M.,

Thalamic slice preparations, in which intrathalamic connectivity between the reticular nucleus and relay nuclei is maintained, are capable of sustaining rhythmic burst firing activity in rodents and ferret. These in vitro oscillations occur spontaneously in the ferret and have frequencies (6-10 Hz) within the range of sleep spindles observed in vivo. In the rat, mainly lower frequency (2-4 Hz) oscillations, evoked under conditions of low bath [Mg(2+)] and/or GABA(A) receptor blockade, have been described. Here we show that faster rhythms in the range of 4-9 Hz can be evoked in rat thalamic slices by electrical stimulation of the internal capsule and also occur spontaneously. When bath [Mg(2+)] was 2 mM, these spindle-like oscillations were most common in a brief developmental time window, peaking at postnatal day 12 (P12). The oscillations were almost completely blocked by the GABA(A) receptor antagonist picrotoxin, and, in some cases, the frequency of oscillations was increased by the GABA(B) receptor antagonist CGP-35348. The selective blockade of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the antagonists 2-amino-5-phosphonovaleric acid or 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), respectively, significantly shortened oscillations but did not completely block them. A combination of the two drugs was necessary to abolish oscillatory activity. The barbituate pentobarbital, which enhances GABA(A)R responses, initially slowed and synchronized oscillations before completely blocking them. When bath [Mg(2+)] was reduced from 2 to 0.65 mM, evoked oscillations became more robust and were often accompanied by spontaneously arising oscillations. Under these conditions, GABA(A) receptor blockade no longer inhibited oscillations, but instead converted them into the slow, synchronous rhythms that have been observed in other studies. The effects of GABA(B) or NMDA receptor blockade were more pronounced in 0.65 mM than in 2 mM external [Mg(2+)]. Thus spindle-like oscillations occur in rat thalamic slices in vitro, and we find that, in addition to the previously demonstrated contributions of GABA(A) and AMPA receptors to these oscillations, NMDA and GABA(B) receptors are also involved. The strong influence of external [Mg(2+)] on GABAergic pharmacology and a contribution of NMDA receptors during oscillations suggest a link between the excitability of NMDA receptors and the activation of GABA(B)R-mediated inhibitory postsynaptic currents.

Working memory (WM) refers to the temporary storage and processing of goal-relevant information. WM is thought to include domain-specific short-term memory stores and executive processes, such as coordination, that operate on the contents of WM. To examine the neural substrates of coordination, we acquired functional magnetic resonance imaging data while subjects performed a WM span test designed specifically to measure executive WM. Subjects performed two tasks (sentence reading and shortterm memory for five words) either separately or concurrently. Dual-task performance activated frontal-lobe areas to a greater extent than performance of either task in isolation, but no new area was activated beyond those activated by either component task. These findings support a resource theory of WM executive processes in the frontal lobes.T he process whereby information is temporarily maintained in memory for use in ongoing mental operations is referred to as working memory (WM). The WM system is thought to consist of verbal and spatial short-term stores and executive processes that operate on the contents of these stores (1-3). Executive WM processes such as multiple task coordination, set shifting, interference resolution, and memory updating are thought to be essential for high-level thought processes and to be subserved by prefrontal cortex (PFC) (2-6). Executive WM is likely to be comprised of a number of distinct processes, and functional neuroimaging may be useful in dissociating those processes to the extent that these processes rely on distinct neural substrates (7-18). The present experiments focus on the neural substrates of dual-task coordination during the concurrent performance of two tasks.Psychologists have developed test paradigms with the goal of measuring executive WM capacity. These WM span tests, which include reading span (19)(20), listening span (21), operation span (22), and counting span (23), share the common property that they require concurrent processing (such as reading sentences for comprehension) and short-term maintenance (such as remembering the last word in each sentence). Unlike single-task short-term memory measures such as digit span or word span, WM span tests are powerful predictors of performance on a wide variety of verbal (e.g., verbal Scholastic Aptitude Test, text comprehension) and nonverbal measures (e.g., mathematical and reasoning problems) (19)(20)(24)(25)(26)(27)(28). WM span tests are also sensitive to changes in cognitive ability throughout development (23, 29) and in old age (26), as well as in neurological diseases that compromise frontal-lobe functioning (30, 31). WM span tests yield a psychometrically robust measure of executive WM capacity, but the neural substrates of performance of such a task have not been yet been thoroughly explored (32).In the present experiments, we used functional magnetic resonance imaging to measure brain activity associated with performance of a WM span test. Performing two tasks concurrently instead of one requires additional mental re...