Heinrich Terlau

Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II.

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA‐derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single‐channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single‐channel conductance.

μ-Conotoxin PIIIA, a New Peptide for Discriminating among Tetrodotoxin-Sensitive Na Channel Subtypes

We report the characterization of a new sodium channel blocker, μ-conotoxin PIIIA (μ-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: where Z = pyroglutamate andO = 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, μ-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized μ-conotoxin GIIIA (μ-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of μ-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to μ-PIIIA and μ-conotoxin GIIIA, (2) sensitive to μ-PIIIA but not to μ-GIIIA, and (3) resistant to μ-PIIIA and μ-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, μ-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

Extracellular Mg2+ regulates activation of rat eag potassium channel

The rat homologue of Drosophila ether à gogo cDNA (rat eag) encodes voltage-activated potassium (K) channels with distinct activation properties. Using the Xenopus expression system, we examined the importance of extracellular Mg2+ on the activation of rat eag. Extracellular Mg2+ at physiological concentrations dramatically slowed the activation in a dose-and voltage-dependent manner. Other divalent cations exerted similar effects on the activation kinetics that correlated with their enthalpy of hydration. Lowering the external pH also resulted in a slowing of the activation. Protons competed with Mg2+ as the effect of Mg2+ was abolished at low pH. A kinetic model for rat eag activation was derived from the data indicating that all four channel subunits undergo a Mg2+-dependent conformational transition prior to final channel activation. The strong dependence of rat eag activation on both the resting potential and the extracellular Mg2+ concentration constitutes a system for fine-tuning K channel availability in neuronal cells.

Structure/Function Characterization of μ-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. μ-Conotoxin KIIIA is representative of μ-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 ∼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 NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-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.

Molecular basis for pharmacological differences between brain and cardiac sodium channels.

Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn2+ and Cd2+. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn2+ and Cd2+, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn2+ or Cd2+, but this mutation markedly reduces sensitivity to block by Ca2+ and Co2+. The double mutant channel (F385CN388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.

Molecular interaction of δ-conotoxins with voltage-gated sodium channels

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

Structure/function characterization of -conotoxin kiiia, an analgesic, nearly irreversible blocker of neuronal mammalian 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. μ-Conotoxin KIIIA is representative of μ-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 ∼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 NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-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.

AMINO TERMINAL-DEPENDENT GATING OF THE POTASSIUM CHANNEL RAT EAG IS COMPENSATED BY A MUTATION IN THE S4 SEGMENT

1 Rat eag potassium channels (r‐eag) were expressed in Xenopus oocytes. They gave rise to delayed rectifying K+ currents with a strong Cole‐Moore effect. 2 Deletions in the N‐terminal structure of r‐eag either shifted the activation threshold to more negative potentials and slowed the activation kinetics (Δ2–190, Δ2–12 and Δ7–12) or resulted in a shift to more positive potentials and faster activation kinetics (Δ150–162). 3 The impact of the deletion Δ7–12 was investigated in more detail: it almost abolished the Cole‐Moore effect and markedly slowed down channel deactivation. 4 Unlike wild‐type channels, the deletion mutants Δ7–12 exhibited a rapid inactivation which, in combination with the slow deactivation, resulted in current characteristics which were similar to those of the related potassium channel HERG. 5 Both the slowing of deactivation and the inactivation induced by the deletion Δ7–12 were compensated by a single histidine‐to‐arginine change in the S4 segment, while this mutation (H343R) only had minor effects on the gating kinetics of the full‐length r‐eag channel. 6 These results demonstrate a functional role of the N‐terminus in the voltage‐dependent gating of potassium channels which is presumably mediated by an interaction of the N‐terminal protein structure with the S4 motif during the gating process.

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.

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.