We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.

Blockade of Ca2+ channels by ai-conotoxin GVIA, a 27 amino acid peptide from the venom of the marine snail Conus geographus, was investigated with patch-clamp recordings ofwhole-cell and unitary currents in a variety ofcell types. In dorsal root ganglion neurons, the toxin produces persistent block of L-and N-type Cab channels but only transiently inhibits T-type channels. Its actions appear to be neuron-specific, since it blocks high-threshold Ca2' channels in sensory, sympathetic, and hippocampal neurons of vertebrates but not in cardiac, skeletal, or smooth muscle cells. Block occurs through direct interaction of the toxin with an external site closely associated with the Ca2+ channel, without apparent involvement of a second messenger or dependence on channel gating. The tissue and channel-type specificity and the directness and slow reversibility of the block are features that favor use of co-conotoxin as a tool for purifying particular neuronal Ca2+ channels and defining their physiological function.

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 nonopioid analgesics, such as subtype-selective sodium channel blockers. -Conotoxin KIIIA is representative of -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 ϳ20% 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 (IC 50 8 M), Na V 1.5 (IC 50 284 M), and Na V 1.4 (IC 50 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 -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.Venoms are a rich source of neuroactive compounds that target various ion channels and receptors with exquisite potency and selectivity (1-4). There is a continuing need for more subtype-selective pharmacological agents against sodium channels (5), and cone snail venoms provide a unique pharmacopoeia of diverse sodium channel-targeting toxins, including channel blockers as well as inhibitors of channel inactivation (6 -18). -Conotoxins are short peptides that potently block sodium channels (Table 1). The first -conotoxins to be discovered from venom of Conus snails, GIIIA, GIIIB, GIIIC, and PIIIA, were paralytic in fish and potently inhibited skeletal muscle sodium channels in amphibian and mammalian systems.Recently, a second group of -conotoxins has been identified that, in contrast to previously characterized peptides that targeted the skeletal muscle sodium channels, inhibited TTX-resistant (TTX-r) 4 sodium channels when screened on amphibian neuronal preparations (19 -21). This group of conotoxins includes -conotoxin SmIIIA from Conus stercusmuscarum and -conotoxin KIIIA from Conus kinoshitai (Fig. 1). Structural and functional studies on peptides in this group to date suggest that amino acid residues in the C-terminal region of these peptides, including Trp and His (see Table 1), are important for function (19,22).It ...

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.

SUMMARY1. Post-synaptic responses to acetylcholine (ACh) released from nerve terminals and from iontophoretic micropipettes were investigated in skeletal muscle fibres of the snake. Each fibre has a compact end-plate consisting of fifty to seventy synaptic boutons. The fibres were voltage clamped, and synaptic currents were recorded from visually identified end-plates.2. When acetylcholinesterase (AChE) is inhibited, a potentiating interaction is observed between two or more quanta that are released close to each other from a synaptic bouton and act upon partially overlapping postsynaptic areas. The potentiation is expressed as a prolongation of the synaptic current. This potentiation also occurs under normal conditions of release when about 300 quanta are distributed over the entire end-plate, so that the presynaptic release sites are separated by an average of 2 ,um. An analogous potentiating interaction is observed when micropipettes, closely apposed to the subsynaptic membrane, substitute for quantal release sites. ACh from one pipette potentiates the response to ACh from another pipette less than 2 /um away.3. In contrast, with AChE fully active no post-synaptic potentiation is seen when the normal complement of quanta is released over the entire end-plate. The time course of the synaptic currents in response to a single quantum or to 300 quanta is similar. It is concluded that functionally the quanta act independently of each other, because AChE isolates each quantum from its neighbours by limiting the lifetime of ACh and its lateral diffusion in the synaptic cleft. The estimated area over which a quantum normally acts is less than 2 JUm2. H. CRIS5 HARTZELL AND OTHERS produced by an appropriate background concentration of ACh from a pipette. This conclusion is supported by the observation that upon inhibition of AChE the peak amplitude of the quantal current response increases by about 20 % with no change in its time to peak. 5. It is suggested that post-synaptic potentiation between quanta may play a role in signalling at synapses in which non-linear dose-response characteristics have been observed and where transmitter is not as rapidly inactivated as at the neuromuscular synapse.