No abstract

The density of voltage-gated sodium channels is high in several regions of the neuronal membrane. It is unclear if this density of channels represents a reserve for the neuron, or if it fulfils a special role in action potential firing. This problem was addressed by studying sodium currents and action potentials in acutely isolated hippocampal CA1 neurons whose number of active sodium channels was acutely changed by applying the sodium channel blocker tetrodotoxin (TTX) at different concentrations. The results show that more than a third of the sodium channels can fail without affecting the single action potential. Thus, the neurons have a remarkable surplus of sodium channels. The surplus, however, is necessary for repetitive action potential firing, as every decrease in the fraction of sodium channels reduces the maximal frequency of action potentials that can be generated by the neuron.

The effects of 17 commonly used antiarrhythmic drugs on the rapidly activating cardiac voltage-gated potassium channels (Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1 and Kv4.2) were studied in the expression system of the Xenopus oocyte. A systematic overview on basic properties was obtained using a simple and restricted experimental protocol (command potentials 10 mV and 50 mV positive to the threshold potential; concentration of 100 micromol/l each). The study revealed that 8 of 17 drugs yielded significant effects (changes >10% of control) on at least one type of potassium channel in the oocyte expression system. These drugs were ajmaline, diltiazem, flecainide, phenytoin, propafenone, propranolol, quinidine and verapamil, whereas the effects of adenosine, amiodarone, bretylium, disopyramide, lidocaine, mexiletine, procainamide, sotalol and tocainide were negligible. The drug effects were characterized by reductions of the potassium currents (except for quinidine and ajmaline). A voltage-dependence of drug effect was found for quinidine, verapamil and diltiazem. The different effect of the drugs was not related to the fast or slow current inactivation of the potassium channels (except for verapamil). Profiles of the individual drug effects at the different potassium channel types were identical for propafenone and flecainide and differed for all other substances. The study demonstrates marked differences in sensitivity to antiarrhythmic drugs within the group of voltage-operated cardiac potassium channel types. Taking the restrictions of the oocyte system into consideration, the findings suggest that several antiarrhythmic drugs exert significant effects at rapidly activating cardiac potassium channels.

Propafenone has been shown to affect the delayed-rectifier potassium currents in cardiomyocytes of different animal models. In this study we investigated effects and mechanisms of action of propafenone on HERG potassium channels in oocytes of Xenopus laevis with the two-electrode voltage-clamp technique. Propafenone decreased the currents during voltage steps and the tail currents. The block was voltage-dependent and increased with positive going potentials (from 18% block of tail current amplitude at -40 mV to 69% at +40 mV with 100 micromol/l propafenone). The voltage dependence of block could be fitted with the sum of a monoexponential and a linear function. The fractional electrical distance was estimated to be delta=0.20. The block of current during the voltage step increased with time starting from a level of 83% of the control current. Propafenone accelerated the increase of current during the voltage step as well as the decay of tail currents (time constants of monoexponential fits decreased by 65% for the currents during the voltage step and by 37% for the tail currents with 100 micromol/l propafenone). The threshold concentration of propafenone effect was around 1 micromol/l and the concentration of half-maximal block (IC50) ranged between 13 micromol/l and 15 micromol/l for both current components. With high extracellular potassium concentrations, the IC50 value rose to 80 degrees mol/l. Acidification of the extracellular solution to pH 6.0 increased the IC50 value to 123 micromol/l, alkalization to pH 8.0 reduced it to 10 micromol/l and coexpression of the beta-subunit minK had no statistically significant effect on the concentration dependence. In conclusion, propafenone has been found to block HERG potassium channels. The data suggest that propafenone affects the channels in the open state and give some hints for an intracellular site of action.

The effects of strontium (Sr2+; 7-50 mM) on five different cloned rat K channels (Kvl.1, Kvl.5, Kvl.6, Kv2.1, and Kv3.4), expressed in oocytes of Xenopus laevis, were investigated with a two-electrode voltage clamp technique. The main effect was a shift of the G~(V) curve along the potential axis, different in size for the different channels. Kvl.1 was shifted most and Kv3.4 least, 21 and 8 mV, respectively, at 50 raM. The effect was interpreted in terms of screening of fixed surface charges. The estimated charge densities ranged from -0.37 (Kvl.1) to -0.11 (Kv3.4) e nm -2 and showed good correlation with the total net charge of the extracellularly located amino acid residues of the channel as well as with the charge of a specific region (the loop between the $5 segment and the pore forming segment). The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per micron 2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mM isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per micron 2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per micron 2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins.

To gain insights in the molecular mechanisms of anesthesia, we analyzed the effects of bupivacaine on a series of voltage-gated K ϩ 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 (K d between 110 and 240 M), and a time-independent block on Kv2.1 (K d ϭ 220 M). At 32°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 (K d increased from 220 to 1170 M). 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.

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-Mg 2+ -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 Mg 2+ -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 M) significantly suppressed the ictal-like discharges elicited by the ␥-aminobutyric acid subtype A (GABA A )-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. Key Words: Seizure-Cortex-Intractable epilepsy-Anticonvulsant drugs.Levetiracetam (LEV; ucb L059) is a racemically pure S-enantiomer ␣-ethyl-2-oxo-1-pyrrolidine acetamide with a wide spectrum of activity against clinical and experimental seizures. This second-generation antiepileptic drug (AED) showed a clear antiepileptic effect in patients with photosensitive seizures (1). The antiepileptic efficacy of LEV as add-on therapy was established in patients with refractory partial seizures in several clinical studies (2-4). Furthermore, conversion to LEV monotherapy was shown to be effective in patients with intractable seizures who responded to LEV as adjunctive therapy (5). In addition to antiepileptic effects, it was pointed out that LEV could be a promising agent for treating posthypoxic and postencephalitic myoclonus as well as for myoclonic jerks in epilepsy patients (1,6) and as a neuroprotective compound (7).The anticonvulsant effects of LEV were studied by using different in vivo and in vitro epilepsy models. In the in vivo experimental models, LEV displayed a unique preclinical profile in rodents by exhibiting potent protection in a broad range of animal models of epilepsy including kindled and animals genetically predisposed to spontaneous, recurrent seizures (8). LEV also was reported to protect against single acute seizures induced by submaximal bolus doses of some chemoconvulsants in both rats and mice (9). This is contrasted with a lack of anticonvulsant effect in classic screening models, the maximal electroshock and subcutaneous pentylenetetrazol seizure tests. However, a weak anticonvulsant activity was observed in threshold...