Biomedical subjects
M Fainzilber
Publications and source records attributed to M Fainzilber.
Novel omega-conotoxins block dihydropyridine-insensitive high voltage-activated calcium channels in molluscan neurons.
We have identified two novel peptide toxins from molluscivorous Conus species that discriminate subtypes of high voltage-activated (HVA) calcium currents in molluscan neurons. The toxins were purified using assays on HVA calcium currents in the caudodorsal cells (CDCs) of the snail Lymnaea stagnalis. The CDC HVA current consists of a rapidly inactivating, transient current that is relatively insensitive to dihydropyridines (DHPs) and a slowly inactivating, DHP-sensitive L-current. The novel toxins, designated omega-conotoxins PnVIA and PnVIB, completely and selectively block the transient HVA current in CDCs with little (PnVIA) or no (PnVIB) effect on the sustained L-type current. The block is rapid and completely reversible. It is noteworthy that both PnVIA and PnVIB reveal very steep dose dependences of the block, which may imply cooperativity in toxin action. The amino acid sequences of PnVIA (GCLEVDYFCGIPFANNGLCCSGNCVFVCTPQ) and of PnVIB (DDDCEPPGNFCGMIKIGPPCCSGWCFFACA) show very little homology to previously described omega-conotoxins, although both toxins share the typical omega-conotoxin cysteine framework but have an unusual high content of hydrophobic residues and net negative charge. These novel omega-conotoxins will facilitate selective analysis of the functions of HVA calcium channels and may enable the rational design of drugs that are selective for relevant subtypes.
Interactions of delta-conotoxins with alkaloid neurotoxins reveal differences between the silent and effective binding sites on voltage-sensitive sodium channels.
The delta-conotoxin-TxVIA from Conus textile (delta TxVIA) is a mollusk-specific conotoxin that slows sodium channel inactivation exclusively in mollusk neuronal membranes but reveals high-affinity binding to both mollusk (effective binding) and rat brain (silent binding) neuronal membranes, despite not having any toxic effect in vertebrates in vivo and in vitro. Using binding studies with radioactive delta TxVIA we demonstrate that a different mollusk-specific conotoxin, delta-conotoxin-GmVIA from the venom of Conus gloriamaris, possesses "silent" and effective binding properties in rat brain and mollusk sodium channels, respectively. Binding studies and electrophysiological tests with both vertebrate muscle and insect neuronal preparations have indicated that the silent binding sites of delta TxVIA are highly conserved in a wide range of distinct vertebrate and insect sodium channels. Direct probing of receptor site 2 by a tritiated derivative of batrachotoxin ([3H]BTX-B) revealed that [3H]BTX-B binding in mollusk sodium channels is of high affinity with no addition of enhancing ligands, unlike [3H]BTX-B binding in rat brain. In contrast to the negative allosteric modulation of delta TxVIA binding by veratridine, delta TxVIA is not able to affect the binding of [3H]BTX-B in mollusk neuronal membranes but reduces [3H]BTX-B binding in rat brain in the presence of alpha-scorpion toxins. The latter finding indicates the existence of a pharmacological distinction between the silent and effective binding sites of delta TxVIA and points out possible functionally important structural differences between molluscan and rat brain sodium channels.
A new cysteine framework in sodium channel blocking conotoxins.
Two novel sodium channel blocking peptides from the venom of the molluscivorous snail Conus pennaceus, muPnIVA and muPnIVB, are described. Elucidation of their amino acid sequences was complicated by a previously undescribed anomalous product of reduction and pyridylethylation, which occurs on N-terminal cysteine residues and gives a PTH derivative eluting at the same position as PTH-Trp in reverse-phase chromatography. The amino acid sequences of the toxins were determined by a combination of Edman degradation and mass-spectrometric techniques as CCKYGWTCLLGCSPCGC (PnIVA) and CCKYGWTCWLGCSPCGC (PnIVB). These toxins block sodium channels in molluscan neurons, but have no effect on sodium currents in bovine chromaffin cells or in rat brain synaptosomes. Although there is only one amino acid difference in the two sequences, PnIVB is approximately 6 times more potent than PnIVA in blockade of the sodium current in Lymnaea neurons. The PnIV sequences reveal a new cysteine residue framework for conotoxins (CC-----C---C--C-C). Strikingly, the only charged residue in PnIVA/B is Lys3. Iodination reaction experiments on the adjacent Tyr4 suggest that this region of the peptide must be solvent exposed and essential for activity. These structurally novel mu-conotoxins target a sodium channel subtype with low affinity for tetrodotoxin and therefore provide new probes for functional studies on sodium channels.
New sodium channel-blocking conotoxins also affect calcium currents in Lymnaea neurons.
Two new conotoxins that affect both sodium and calcium currents have been characterized from the venom of Conus marmoreus, using direct assays on voltage-gated currents in caudodorsal neurons (CDC) of the freshwater snail Lymnaea stagnalis. The designations and amino acid sequences of the new toxins are MrVIA, ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV, and MrVIB, ACSKKWEYCIVPILGFVYCCPGLICGPFVCV. Both toxins block voltage dependent sodium currents in snail neurons with ED50's of 0.1-0.2 microM. Effects are also observed on the fast-inactivating calcium current subtype in the CDC at > or = 1 microM. At concentrations of 1-5 microM, MrVIA acts as a calcium current agonist whereas MrVIB acts as a blocker. At higher doses both toxins block the fast-inactivating calcium current. Almost no effects of MrVIB are seen on the second (sustained kinetics) CDC calcium current subtype, while MrVIA also slightly blocks the sustained current. The calcium current block is rapidly reversible, whereas in contrast recovery of the sodium current requires extensive wash. MrVIA/B have the same cysteine framework as the omega- and delta-conotoxins and a high content of hydrophobic residues, in common with the delta-conotoxins. There is only one localized concentration of charged residues in MrVIA/B, in the first intercysteine loop. These two conotoxins provide unique probes for structure and function studies on voltage-gated sodium and L-type calcium channels. Their unusual cross-channel activity suggests they may represent an "intermediate" variant of conotoxin, in the diversification of one conotoxin structural family that selectively targets either sodium or calcium channels.
Electrophysiological characterization of a novel conotoxin that blocks molluscan sodium channels.
A novel peptide toxin, PnIVB, isolated from the venom of Conus pennaceus blocks voltage-gated sodium current in Aplysia neurons. Complete blockade is obtained at a PnIVB concentration of 80 +/- 2.2 nM and 50% blockade at 16 +/- 0.86 nM. The potency of PnIVB in blocking Aplysia sodium current is four orders of magnitude larger than that of tetrodotoxin. The toxin has no paralytic activity when injected into fish. The rapid blockade of sodium current by PnIVB is not associated with a change in the activation or inactivation kinetics of the current, or with the reversal potential. Sodium current blockade is reversible after a 30 min wash with 50 times the bath volume. The novel conotoxin PnIVB can be used as a powerful tool for mollusc neurobiological research and as a molecular probe to explore the structure-function relations of voltage-gated sodium channel subtypes.
A new conotoxin affecting sodium current inactivation interacts with the delta-conotoxin receptor site.
We describe a new peptide conotoxin affecting sodium current inactivation, that competes on binding with delta-conotoxin TxVIA (delta TxVIA). The amino acid sequence of the new toxin, designated conotoxin NgVIA (NgVIA), is SKCFSOGTFCGIKOGLCCSVRCFSLFCISFE (where O is trans-4-hydroxyproline). The primary structure of NgVIA has an identical cysteine framework and similar hydrophobicity as delta TxVIA but differs in its net charge. NgVIA competes with delta TxVIA on binding to rat brain synaptosomes and molluscan central nervous system and strongly inhibits sodium current inactivation in snail neurons, as does delta TxVIA. In contrast to delta TxVIA, NgVIA is a potent paralytic toxin in vertebrate systems, its binding appears to be voltage-dependent, and it synergically increases veratridine-induced sodium influx to rat brain synaptosomes. delta TxVIA acts as a partial antagonist to NgVIA in rat brain in vivo. NgVIA appears to act via a receptor site distinct from that of delta TxVIA but similar to that of Conus striatus toxin. This new toxin provides a lead for structure-function relationship studies in the delta-conotoxins and will enable analysis of the functional significance of this complex of receptor sites in gating mechanisms of sodium channels.
New mollusc-specific alpha-conotoxins block Aplysia neuronal acetylcholine receptors.
Two mollusc-specific neurotoxic peptides from the venom of the molluscivorous snail Conus pennaceus are described. These new toxins block acetylcholine receptors (AChR) of cultured Aplysia neurons. Bath application of 0.5-1 microM toxin induces 5-10-mV membrane depolarization, which recovers to the control level within 1-3 min in the presence of the toxin. This response is blocked by 1 mM hexamethonium. Concomitantly with the transient depolarization, the toxins block approximately 90% of the depolarizing responses evoked by brief iontophoretic application of acetylcholine. The pharmacology and amino acid sequences of the toxins (alpha PnIA, GCCSLPPCAANNPDYC-NH2; alpha PnIB, GCCSLPPCALSNPDYC-NH2) enable their classification as novel alpha-conotoxins. The sequences differ from those of previously described alpha-conotoxins in a number of features, the most striking of which is the presence of a single negatively charged residue in the C-terminal loop. This loop contains a positively charged residue in piscivorous venom alpha-conotoxins. In contrast to other alpha-conotoxins, which are selective for vertebrate skeletal muscle nicotinic ACh receptors, these Conus pennaceus toxins block neuronal ACh receptors in molluscs. As such they are new probes which can be used to define subtypes of ACh receptors, and they should be useful tools in the study of structure-function relationships in ACh receptors.
A new neurotoxin receptor site on sodium channels is identified by a conotoxin that affects sodium channel inactivation in molluscs and acts as an antagonist in rat brain.
The peptide conotoxin TxVIA is selectively toxic to molluscs and slows sodium current inactivation in mollusc neurons. Here we show that TxVIA binds with high affinity to new sites on sodium channels in both mollusc and rat central nervous systems, despite its lack of toxicity to vertebrates. Furthermore, TxVIA protects from the toxic effects of Conus striatus toxin in rat brain. The TxVIA binding site differs from other neurotoxin receptor sites affecting sodium channel inactivation in that binding is not voltage-dependent and undergoes negative allosteric modulation by veratridine. TxVIA therefore represents a novel category of sodium channel probes, designated delta-conotoxins. TxVIA is shown to discriminate between sodium channels in different phyla by activity but not by binding, thus providing a lead for the study of structural elements affecting gating modes of sodium channels.
Alteration of sodium currents by new peptide toxins from the venom of a molluscivorous Conus snail.
TxIA and TxIB, peptides with 27-amino acid residues recently isolated from the molluscivorous marine snail Conus textile neovicarius, exhibit strong paralytic activity in molluscs, with no paralytic effects on athropods and vertebrates. At concentrations of 0.25-0.5 microM the toxins cause spontaneous repetitive firing and dramatic broadening of the action potential of cultured Aplysia neurons. The action potential duration partially recovers within 30 min in the presence of the toxins. Under these conditions a second toxin application does not change the spike duration. TxI-induced spike broadening occurs when potassium and calcium conductances are blocked. Voltage-clamp experiments revealed that the toxins alter the kinetics of the sodium current either by slowing down the rate of sodium current inactivation or by recruiting silent sodium channels with slower activation and inactivation kinetics. The toxins shift the voltage-dependent steady-state Na+ current inactivation curve to more positive values by 6 mV. These changes are not associated with alteration in the rate of sodium current activation, in the peak sodium current, or the sodium current reversal potential. TxI apparently represents a new class of conotoxins with an unusual phylogenic specificity and may therefore be useful as a probe for the study of molluscan neuronal sodium channels.
Chemical and electrophysiological characterization of new peptide neurotoxins from the venom of the molluscivorous snail Conus textile neovicarius: a review.
Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCIVLVCT (identical to the so-called 'King Kong peptide'); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYC gamma VDS gamma CCSDNCVRSYCT (gamma = gamma-carboxyglutamate). There is a similarity of the Cys framework of these toxins to that of the omega-conotoxins; however, their net negative charges, high content of hydrophobic residues, and uneven number of Cys residues in TxIIA are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced spontaneous repetitive firing. The TxI toxins also induced a marked prolongation of the action potential duration. Voltage clamp experiments revealed that the TxI toxins alter the kinetics of the sodium current either by slowing down the rate of sodium current inactivation, or by recruiting silent sodium channels with slower activation and inactivation kinetics. The toxins shift the voltage-dependent steady-state Na+ current inactivation curve to more positive values by 6 mV. These changes are not associated with alteration in the rate of INa+ activation, in the peak INa+, or the sodium current reversal potential. TxI represents a new class of conotoxins with an unusual phylogenic specificity and may therefore be useful as a probe for the study of voltage gated sodium channels. (This review summarizes previously published papers).
A new bioassay reveals mollusc-specific toxicity in molluscivorous Conus venoms.
Contraction of the foot pedal of a limpet snail is described as a new and quantifiable bioassay for mollusc paralysis. This bioassay was used for screening the venoms of seven different species of Conus snails. Comparison of the results of the limpet assay with those obtained from fish and blowflies shows a correlation between the feeding specificities and venom toxicities of these Conidae. The limpet bioassay should be useful for identification and monitoring of the purification of new toxins active on molluscan systems.
Mollusc-specific toxins from the venom of Conus textile neovicarius.
Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCI-VLVCT (identical to the so called 'King Kong peptide'); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYC gamma VDS gamma CCSDNCVRSYCT (gamma = gamma-carboxyglutamate). There is a similarity of the Cys framework of these toxins to that of the omega-conotoxins; however, their net negative charges, high content of hydrophobic residues and uneven number of Cys residues in TxIIA, are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced membrane depolarization and spontaneous repetitive firing. The TxI toxins also induce a marked prolongation of the action potential duration, which is sodium dependent. These effects differ significantly from the blocking activities of piscivorous venom conotoxins. These mollusc specific conotoxins may therefore serve as new and selective probes for ion-channel functions in molluscan neuronal systems.