Biomedical subjects

Heinrich Terlau

Publications and source records attributed to Heinrich Terlau.

18 recordsLinked to original sources

PubMed2006

Discovery and characterization of the short kappaA-conotoxins: a novel subfamily of excitatory conotoxins.

We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.

Amino Acid Sequence↗

PubMed2006

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins.

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Amino Acid Sequence↗

PubMed2006

Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity.

MuO-conotoxin MrVIB is a blocker of voltage-gated sodium channels, including TTX-sensitive and -resistant subtypes. A comprehensive characterization of this peptide has been hampered by the lack of sufficient synthetic material. Here, we describe the successful chemical synthesis and oxidative folding of MrVIB that has made an investigation of the pharmacological properties and therapeutic potential of the peptide feasible. We show for the first time that synthetic MrVIB blocks rat NaV1.8 sodium channels and has potent and long-lasting local anesthetic effects when tested in two pain assays in rats. Furthermore, MrVIB can block propagation of action potentials in A- and C-fibers in sciatic nerve as well as skeletal muscle in isolated preparations from rat. Our work provides the first example of analgesia produced by a conotoxin that blocks sodium channels. The emerging diversity of antinociceptive mechanisms targeted by different classes of conotoxins is discussed.

Amino Acid Sequence↗

PubMed2006

Production of recombinant Conkunitzin-S1 in Escherichia coli.

Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-S1 is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channels of the Shaker family. The peptide was expressed in insoluble form in fusion with an N-terminal intein. Refolding in the presence of glutathione followed by pH shift-induced cleavage of the fusion protein resulted in a functional toxin as demonstrated by voltage-clamp measurements.

Animals↗

PubMed2006

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3.

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Animals↗

PubMed2006

Molecular and Functional Differences between Heart mKv1.7 Channel Isoforms.

Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.

Animals↗

PubMed2005

Molecular interaction of delta-conotoxins with voltage-gated sodium channels.

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.

Amino Acid Sequence↗

PubMed2005

Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.

Amino Acid Sequence↗

PubMed2005

Identification of a novel pharmacophore for peptide toxins interacting with K+ channels.

KappaM-conotoxin RIIIK blocks TSha1 K+ channels from trout with high affinity by interacting with the ion channel pore. As opposed to many other peptides targeting K+ channels, kappaM-RIIIK does not possess a functional dyad. In this study we combine thermodynamic mutant cycle analysis and docking calculations to derive the binding mode of kappaM-conotoxin RIIIK to the TSha1 channel. The final model reveals a novel pharmacophore, where no positively charged side chain occludes the channel pore. Instead the positive-charged residues of the toxin form a basic ring; kappaM-RIIIK is anchored to the K+ channel via electrostatic interactions of this basic ring with the loop and pore helix residues of the channel. The channel amino acid Glu-354 is likely to be a fundamental determinant of the selectivity of kappaM-RIIIK for the TSha1 channel. The Cgamma-OH of Hyp-15 is in contact with the carbonyls of the selectivity filter, disturbing the charge distribution pattern necessary for the coordination of K+ ions. This novel, experimentally based pharmacophore model proves the existence of diverse binding modes of peptidic toxins to K+ channels and underlines the role of intermolecular electrostatic interactions involving channel loop side chains in determining the selectivity of toxins for specific K+ channel types.

Amino Acid Sequence↗

PubMed2004

KappaM-conotoxin RIIIK, structural and functional novelty in a K+ channel antagonist.

Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K(+) channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-Conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K(+) channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K(+) channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of mu-conotoxin GIIIA, a specific blocker of the skeletal muscle Na(+) channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K(+) channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K(+) channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K(+) channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.

Amino Acid Sequence↗

PubMed2004

Identification of a mammalian target of kappaM-conotoxin RIIIK.

Despite the great variability of the conus peptides characterized until now only relatively few have been identified that interact with K+ channels. kappaM-conotoxin RIIIK (kappaM-RIIIK) is a 24 amino acid peptide from Conus radiatus, which is structurally similar to micro-conotoxin GIIIA, a peptide known to block specifically skeletal muscle Na+ channels. Recently, it has been shown that kappaM-RIIIK does not interact with Na) channels, but inhibits Shaker potassium channels expressed in Xenopus oocytes. It was demonstrated that kappaM-RIIIK binds to the pore region of Shaker channels and a teleost homologue of the Shaker channel TSha1 was identified as a high affinity target of the toxin. In contrast the mammalian Shaker-homologues Kv1.1, Kv1.3, Kv1.4 are not affected by the toxin. In this study the activity of kappaM-RIIIK on other mammalian Kv1 K+ channels expressed in Xenopus oocytes was investigated. We demonstrate that kappaM-conotoxin RIIIK up to 5 microM exhibits no significant effect on Kv1.5 and Kv1.6 mediated currents, but the human Kv1.2 K+ channel is blocked by this peptide. The binding of kappaM-RIIIK to Kv1.2 channels is state dependent with an IC50 for the closed state of about 200 nM and for the open state of about 400 nM at a test potential of 0 mV. kappaM-conotoxin RIIIK is the first conotoxin described to block human Kv1.2 potassium channels.

Animals↗

PubMed2004

The binding of kappa-Conotoxin PVIIA and fast C-type inactivation of Shaker K+ channels are mutually exclusive.

Kappa-conotoxin PVIIA (kappa-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K+ channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with kappa-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of kappa-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like kappa-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K+ channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest.

Animals↗

PubMed2004

Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore.

The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.

Animals↗

PubMed2004

Conus venoms: a rich source of novel ion channel-targeted peptides.

The cone snails (genus Conus) are venomous marine molluscs that use small, structured peptide toxins (conotoxins) for prey capture, defense, and competitor deterrence. Each of the 500 Conus can express approximately 100 different conotoxins, with little overlap between species. An overwhelming majority of these peptides are probably targeted selectively to a specific ion channel. Because conotoxins discriminate between closely related subtypes of ion channels, they are widely used as pharmacological agents in ion channel research, and several have direct diagnostic and therapeutic potential. Large conotoxin families can comprise hundreds or thousands of different peptides; most families have a corresponding ion channel family target (i.e., omega-conotoxins and Ca channels, alpha-conotoxins and nicotinic receptors). Different conotoxin families may have different ligand binding sites on the same ion channel target (i.e., mu-conotoxins and delta-conotoxins to sites 1 and 6 of Na channels, respectively). The individual peptides in a conotoxin family are typically each selectively targeted to a diverse set of different molecular isoforms within the same ion channel family. This review focuses on the targeting specificity of conotoxins and their differential binding to different states of an ion channel.

Animals↗

PubMed2004

Sodium channel toxins--receptor targeting and therapeutic potential.

Sodium channels underlie propagated electrical signalling in most excitable cells, including neurons and the myocytes of skeletal muscle and heart. These proteins are targeted by a variety of current therapeutic drugs to combat such maladies as pain, myotonias, epilepsies and cardiac arrhythmias. Typically, these problems are associated with overactivity of sodium channels leading to hyperexcitability in the relevant tissue. More than ten distinct but closely related molecular isoforms of mammalian sodium channel are now known to be specifically expressed in different cell types and tissues. Therapeutic attenuation of sodium channel activity must be effected with great precision in both targeting and the degree of reduction in channel activity if a malfunction is to be corrected without introducing deleterious or even catastrophic side effects. Numerous natural toxins have evolved to target sodium channels, either by blocking current through the pore or by modifying channel gating. Among the well studied toxins, the peptide conotoxins from cone snail venoms show a remarkable ability to discriminate among closely related forms of sodium channel, as well as exhibiting a variety of modes of action. Here, we examine the molecular basis of action of different Na channel targeted conotoxins and explore their potential as models for the future design of more specifically targeted drugs.

Amino Acid Sequence↗

PubMed2002

A novel conus peptide ligand for K+ channels.

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.

Amino Acid Sequence↗

PubMed2002

Four new bromotryptamine derivatives from the marine bryozoan Flustra foliacea.

Ten brominated alkaloids, 6-bromo-2-(1,1-dimethyl-2-propenyl)-1H-indole-3-carbaldehyde (1), N-(2-[6-bromo-2-(1,1-dimethyl-2-propenyl)-1H-indol-3-yl]ethyl)-N-methylmethanesulfonamide (2), deformylflustrabromine (3), flustrabromine (4), (3aR,8aS)-6-bromo-3a-[(2E)-3,7-dimethyl-2,6-octadienyl]-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]-indol-7-ol (5), flustramine C (6), dihydroflustramine C (7), flustramine A (8), flustramine D (9), and flustraminol A (10), and the diterpene 4,6-bis(4-methylpent-3-en-1-yl)-6-methylcyclohexa-1,3-diene-carbaldehyde (11) were isolated from the dichloromethane extract of the North Sea bryozoan Flustra foliacea. Of the 10, four (1, 2, 3, and 5) represent new natural products. The structures of all isolates were elucidated by interpretation of their spectroscopic data (NMR, MS, UV, and IR). For compound 4 complete (13)C NMR data are reported for the first time. Compounds 3 and 6-8 were tested on voltage-activated potassium and sodium channels. Flustramine A (8) shows an unspecific blocking activity on Kv1.4 potassium-mediated currents.

Alkaloids↗

PubMed1990

Fibroblast Growth Factor Enhances Long-term Potentiation in the Hippocampal Slice.

Recently we reported that perfusion of hippocampal slices with epidermal growth factor (EGF) lead to enhancement of potentiated responses after tetanic stimulation. In the present study we report that basic fibroblast growth factor (FGF) can also lead to an enhancement of potentiated responses. FGF is a mitogen for several cell types and exhibits neurotrophic effects on neurons of the central nervous system (CNS). Rat hippocampal slices were perfused with FGF at a concentration of 10-9 M. During extra- and intracellular recordings in the CA1-region, the addition of FGF to the perfusing medium produced no change in evoked responses if single pulse or paired pulse stimulation was used. Furthermore FGF had no influence on the resting membrane potential and input resistance. However, after tetanic stimulation, FGF-treated slices showed an increase in the magnitude of potentiation compared to control slices. Taken together with the EGF data these results support the hypothesis that growth factors like FGF with neurotrophic potential on CNS-neurons can influence synaptic efficacy. Furthermore these results show that factors which are able to modulate developmental plasticity and regenerative plasticity can also modulate synaptic plasticity.

Journal Article↗