The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr2+) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr2+ (50 mM shifted G(V) by +21.7 mV), and Kv2.1 to be the least sensitive (+7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr2+-induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was ∼20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr2+ acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

Local anaesthetics (LAs) are generally assumed to block action potentials by binding to Nav channels, preferentially when in inactivated and/or open state. Recently, it has been suggested that they, in addition or preferentially, bind to Nav channels when in intermediate closed states. This is based on the finding that LAs reduce the peak current more at low voltage steps than at high in voltage clamp experiments. In previous studies we have concluded that LAs preferentially block Kv channels by binding to exclusively open channels. In the present study we have reanalysed the effect, with special reference to the new findings of closed state binding. We analysed the effects of bupivacaine on Kv3.1 channels expressed in Xenopus oocytes. In contrast to the results from the Na studies, bupivacaine reduced the early current more at higher voltages than at lower. Nevertheless, analysing kinetic models we found that the results are explained by binding preferentially to open channels. We thus conclude that bupivacaine block K channels mainly in the open state. We also conclude that a time and voltage-dependent block, similar to that reported for Na channels, does not necessarily imply binding of channels in different closed states. Furthermore, the results stress the general fact that a block of the early current in voltage clamp experiments does not necessarily imply that LAs bind to the channel when in closed state, contrary to a widely held view.

Local anaesthetics (LAs) block action potentials mainly by blocking Na channels. They are generally assumed to preferentially bind to channels in inactivated and/or open state. Recently it has been suggested that they mainly bind to channels in intermediate closed states. This is based on the finding that LAs affect the currents time and voltage-dependently in voltage clamped channels; that they reduce the peak current more at low voltage steps than at high. In previous studies on inactivating K channels we have concluded that LAs preferentially bind to channels in open state. In the present study we have reanalysed the effects of LAs on K channels with special reference to the new findings of closed state binding. We analysed the effects of bupivacaine and benzocaine on Kv3.1 and Shaker channels expressed in Xenopus oocytes. As shown previously bupivacaine induces a peaked current in both channel types. In accordance with the results on the Na currents bupivacaine reduced the peak less at +60 mV than at lower potentials. Nevertheless, a modelling analysis suggested that the results are explained by binding preferentially to open channels. In contrast, benzocaine did not induce a peak at any potential, but the early current was reduced more at low potentials than at high. The modelling analysis suggested that the effect is caused by binding to closed and open channels. We thus conclude that bupivacaine and benzocaine blocks K channels differently; bupivaciane open state-dependently and benzocaine both open and closed state-dependently. We also conclude that a time and voltage-dependent block, similar to that reported for Na channels, with less inhibition of the peak current at high potentials than at low potentials, does not necessarily imply binding of channels in a closed state.

Das Hertie-Institut für klinische Hirnforschung (HIH) in Tübingen wurde im Jahr 2000 gegründet. Es ist eine Initiative der Gemeinnützigen Hertie-Stiftung, der Universität Tübingen und des Universitätsklinikums Tübingen. Die Ziele sind, ein Institut zu schaffen, das international zu den führenden Forschungseinrichtungen der klinischen Hirnforschung gehört und das ein Reformmodell für universitäre Institute darstellt.

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K+ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K+ channel ligands can expand in the subunit interface whose residues are less conserved between K+ channels and hence may be targets for design of highly desirable subtype-specific K+ channel drugs.

Febrile seizures are a very common form of pathologic brain activity in children. This review summarizes current knowledge on the mechanisms and consequences of febrile seizures, which has been obtained from animal models in experimental epilepsy research. It is shown that an increase of body temperature alone is able to induce epileptiform seizures in animals and that fever-specific factors, e.g., production of cytokines, and compensatory reactions, e.g., increased breathing, can contribute significantly. Concerning the question of epileptogenesis caused by febrile seizures, long-lasting changes of functional and morphological parameters that appear after seizures caused by hyperthermia in animal brains are described. Finally, the relevance of the results from animal models for our understanding of human febrile seizures is discussed.

Fieberkrämpfe gehören zu den häufigsten Formen pathologischer Hirnaktivität bei Kindern. In diesem Übersichtsbeitrag wird das aktuelle Wissen über die Mechanismen und Folgen von Fieberkrämpfen zusammengefasst, das aus der Arbeit mit Tiermodellen in der experimentellen Epilepsieforschung gewonnen werden konnte. Es wird gezeigt, dass die alleinige Erhöhung der Körpertemperatur epileptiforme Anfälle im Tierversuch auslösen kann und dass fieberspezifische Faktoren, wie Zytokine, oder kompensatorische Reaktionen auf Fieber, wie Steigerung der Atmung, signifikant dazu beitragen können. Hinsichtlich der Frage nach der Entstehung von Epilepsien aufgrund von Fieberkrämpfen werden die Befunde zu den lang überdauernden funktionellen und morphologischen Veränderungen im Gehirn der Versuchstiere beschrieben, die nach Hyperthermieanfällen aufgetreten sind. Abschließend wird die Bedeutung der tierexperimentellen Ergebnisse für das Verständnis der Fieberkrämpfe des Menschen diskutiert.

Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and Kv3.4) and noninactivating (Shaker-IR and Kv3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280μM for Shaker and Shaker-IR, and 60 and 90μM for Kv3.4 and Kv3.2). The similar magnitudes of Kd, as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed.

The aim of this study was to investigate the effects of monomethyltin trichloride (MMT) and dimethyltin dichloride (DMT) on various neuronal ion channels heterologously expressed in Xenopus oocytes and on synaptic transmission in hippocampal slices of young (14–21 days old) and adult (2–4 months old) rats. The Xenopus oocyte expression system was chosen to allow direct assessment of the effects of MMT and DMT both on glutamate receptors sensitive to AMPA and NMDA and on various voltage-operated potassium and sodium channels. Hippocampal slices were used to analyze the effects of MMT and DMT on synaptic potentials generated by the important excitatory Schaffer collateral-CA1 synapse. In general, MMT and DMT were found to have no effect either on voltage-operated sodium and potassium channels or on the metabotropic glutamate receptor but they did differentially affect the functions of ionotropic glutamate receptors and glutamatergic synaptic transmission. MMT (100 μM) significantly reduced NMDA-mediated ion currents by up to 32%, but had no effect on ion currents through AMPA receptors. In slices of adult rats, MMT had no effect on the amplitudes of evoked fEPSPs and brought about a 35% reduction in the LTP amplitudes. In contrast, in slices of young rats MMT evoked a reversible 30% increase in the amplitudes of fEPSPs but had no effect on LTP induction. DMT (100 μM) reduced ion currents through NMDA-receptor ion channels by up to 29% and those through AMPA-receptor ion channels by up to 7%. In hippocampal slices 100 μM DMT reduced the amplitudes of fEPSPs (adults: 50%; young rats: 70%) and LTP (adults: 40%; young rats: 55%). Neither of the organotins affected the paired-pulse facilitation at this synapse, indicating that the organotins exert their effects at the postsynaptic site. The action of MMT and DMT may contribute to the organotin-induced impairment of behavior patterns in connection with learning and memory.

Pentavalent and trivalent organoarsenic compounds belong to the major metabolites of inorganic arsenicals detected in humans. Recently, the question was raised whether the organic arsenicals represent metabolites of a detoxification process or methylated species with deleterious biological effects. In this study, the effects of trivalent arsenite (AsO3 3−; iAIII), the pentavalent organoarsenic compounds monomethylarsonic acid (CH3AsO(OH)2; MMAV) and dimethylarsinic acid ((CH3)2AsO(OH); DMAV) and the trivalent compounds monomethylarsonous acid (CH3As(OH)2, MMAIII) and dimethylarsinous acid ((CH3)2As(OH); DMAIII) were tested on glutamate receptors and on voltage-operated potassium and sodium channels heterologously expressed in Xenopus oocytes. Membrane currents of ion channels were measured by conventional two-electrode voltage-clamp techniques. The effects of arsenite were tested in concentrations of 1–1,000 μmol/l and the organic arsenical compounds were tested in concentrations of 0.1–100 μmol/l. We found no significant effects on voltage-operated ion channels; however, the arsenicals exert different effects on glutamate receptors. While MMAV and MMAIII significantly enhanced ion currents through N-methyl-d-aspartate (NMDA) receptor ion channels with threshold concentrations <10 μmol/l, DMAV and DMAIII significantly reduced NMDA-receptor mediated responses with threshold concentrations <0.1 μmol/l; iAIII had no effects on glutamate receptors of the NMDA type. MMAIII and DMAV significantly reduced ion currents through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor ion channels with threshold concentrations <10 μmol/l (MMAIII) and <1 μmol/l (DMAV). MMAV and iAIII had no significant effects on glutamate receptors of the AMPA type. The effects of MMAV, MMAIII, DMAV and DMAIII on glutamate receptors point to a neurotoxic potential of these substances.

Organotin compounds such as trimethyltin chloride (TMT) are among the most toxic of the organometallics. As their main target for toxicity is the central nervous system, the aim of the present study was to investigate the effects of TMT on receptor channels involved in various processes of synaptic transmission. The Xenopus oocyte expression system was chosen for direct assessment of TMT effects on voltage‐operated potassium channels and glutamatergic and GABAergic receptors, and hippocampal slices from rat brain for analyzing TMT effects on identified synaptic sites. TMT was found to be ineffective, at 100 μ mol l −1 , against several potassium‐ and sodium‐operated ion channel functions as well as the metabotropic glutamate receptor. The functions of the ionotropic glutamate and the GABA A receptor channels were inhibited by TMT in micromolar concentrations. Thus, at a maximum concentration of 100 μ mol l −1 , around 20–30% of the α ‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid and GABA A receptor‐mediated ion currents and 35% of the N ‐methyl‐ D ‐aspartate receptor‐mediated ion currents were blocked. In the hippocampal slice model, the inhibitory effects of TMT were much stronger than expected from the results on the ion channels. Bath application of TMT significantly reduced the amplitudes of evoked excitatory postsynaptic field potentials in a concentration‐dependent and nonreversible manner. Induction of long‐term potentiation, recorded from the CA1 dendritic region, was inhibited by TMT and failed completely at a concentration of 10 μ mol l −1 . In general, TMT affects the excitatory and inhibitory synaptic processes in a receptor specific manner and is able to disturb the activity within a neuronal network. British Journal of Pharmacology (2005) 144 , 283–292. doi: 10.1038/sj.bjp.0706083

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I(i,st)) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I(i,st) were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I(i,st), suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP(3))-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I(i,st) in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I(i,st). The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP(3) concentration, in turn releasing calcium from InsP(3)-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.

To gain insights in the molecular mechanisms of anesthesia, we analyzed the effects of bupivacaine on a series of voltage-gated K+ channels (Kv1.1, -1.2, -1.5, -2.1, -3.1, and -3.2) and various mutant channels derived from Kv2.1, using Xenopus laevis oocytes. Two phenomenologically different blocking effects were seen at room temperature: a time-dependent block of Kv1 and Kv3 channels (Kd between 110 and 240 microM), and a time-independent block on Kv2.1 (Kd = 220 microM). At 32 degrees C, however, Kv2.1 also showed a time-dependent block. Swapping the S6 helix between Kv1.2 and Kv2.1 introduced Kv1.2 features in Kv2.1. Critical residues were located in the N-terminal end of S6, positions 395 and 398. The triple substitution of residues 372, 373, and 374 in the S5-S6 linker decreased the bupivacaine affinity by 5-fold (Kd increased from 220 to 1170 microM). The results suggest that bupivacaine blocks Kv channels by an open-state-dependent mechanism and that Kv2.1 deviates from the other channels in allowing a partial closure of the channel with bupivacaine bound. The results also suggest that the binding site is located in the internal vestibule and that residues in the descending P-loop and the upper part of S6 are critical for the binding, most likely by allosteric mechanisms. A simple mechanistic scenario that explains the observations is presented. Thermodynamic considerations suggest that the interaction between bupivacaine and the channels is hydrophobic.

The effects of the antiarrhythmic drug propafenone at Kv2.1 channels were studied with wild-type and mutated channels expressed in Xenopus laevis oocytes. Propafenone decreased the Kv2.1 currents in a time- and voltage-dependent manner (decrease of the time constants of current rise, increase of block with the duration of voltage steps starting from a block of less than 19%, increase of block with the amplitude of depolarization yielding a fractional electrical distance delta of 0.11 to 0.16). Block of Kv2.1 appeared with application to the intracellular, but not the extracellular, side of membrane patches. In mutagenesis experiments, all parts of the Kv2.1 channel were successively exchanged with those of the Kv1.2 channel, which is much more sensitive to propafenone. The intracellular amino and carboxyl terminus and the intracellular linker S4-S5 reduced the blocking effect of propafenone, whereas the linker S5-S6, as well as the segment S6 of the Kv1.2 channel, abolished it to the value of the Kv1.2 channel. In the linker S5-S6, this effect could be narrowed down to two groups of amino acids (groups 372 to 374 and 383 to 384), which also affected the sensitivity to tetraethylammonium. In segment S6, several amino acids in the intracellularly directed part of the helix significantly reduced propafenone sensitivity. The results suggest that propafenone blocks the Kv2.1 channel in the open state from the intracellular side by entering the inner vestibule of the channel. These results are consistent with a direct interaction of propafenone with the lower part of the pore helix and/or residues of segment S6.

Summary: Purpose: The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy.Methods: Slices were used to evaluate the effects of LEV on two different models of epilepsy: low‐Mg2+–induced untriggered and bicuculline‐evoked stimulus‐triggered epileptiform burst discharges and spontaneously appearing rhythmic sharp waves.Results: LEV (0.1–1 mM) did not influence spontaneously appearing rhythmic sharp waves or Mg2+‐free aCSF‐induced epileptiform field potentials. LEV affected neither the amplitudes or duration nor the repetition rates of burst discharges in these epilepsy models. However, LEV (100–500 μM) significantly suppressed the ictal‐like discharges elicited by the γ‐aminobutyric acid subtype A (GABAA)‐receptor antagonist bicuculline. A marked reduction of the amplitude and duration of bicuculline‐evoked field response in the presence of LEV was observed.Conclusions: The results indicate the potential for LEV to inhibit epileptiform burst discharges in human neocortical tissue, which is consistent with its effects in animal models of epilepsy. These results also support the seizure reduction observed in clinical trials and support that this may, in part, be related to the ability of LEV to inhibit epileptiform discharges.