Summary: Purpose:We investigated the effect of the new antiepileptic drug (AED) levetiracetam (LEV) on different types of high-voltage-activated (HVA) Ca 2+ channels in freshly isolated CA1 hippocampal neurons of rats.Methods: Patch-clamp recordings of HVA Ca 2+ channel activity were obtained from isolated hippocampal CA1 neurons. LEV was applied by gravity flow from a pipette placed near the cell, and solution changes were made by electromicrovalves. Ca 2+ channel blockers were used for separation of the channel subtypes.Results: The currents were measured in controls and after application of 1-200 M LEV. LEV irreversibly inhibited the HVA calcium current by ∼18% on the average. With a prepulse stimulation protocol, which can eliminate direct inhibition of Ca 2+ channels by G proteins, we found that G proteins were not involved in the pathways underlying the LEV inhibitory effect. This suggested that the inhibitory effect arises from a direct action of LEV on the channel molecule. The blocking mechanism of LEV was not related to changes in steady-state activation or inactivation of Ca 2+ channels. LEV also did not influence the rundown of the HVA Ca 2+ current during experimental protocols lasting ∼10 min. Finally, LEV at the highest concentration used (200 M) did not influence the activity of L-, P-or Q-type Ca 2+ channels in CA1 neurons, while selectively influencing the activity of N-type calcium channels. The maximal effect on these channels separated from other channel types was ∼37%.Conclusions: Our results provide evidence that LEV selectively inhibits N-type Ca 2+ channels of CA1 pyramidal hippocampal neurons. These data suggest the existence of a subtype of N-type channels sensitive to LEV, which might be involved in the molecular basis of its antiepileptic action. Key Words: Levetiracetam-Antiepileptic drugs-Calcium channels-Hippocampal neurons-Epilepsy.Levetiracetam (LEV) is a new antiepileptic drug (AED) with a unique pharmacologic profile, exerting potent seizure suppression in kindling models of epilepsy (1-3). It substantially inhibits neuronal hypersynchronization in hippocampal slices induced by application of high potassium-low calcium perfusion solutions, without any intrinsic effects on normal electrophysiologic responses. Therefore it is of obvious importance to evaluate possible cellular mechanisms of the antiepileptic action of LEV that might be related to its specific interaction with molecular structures responsible for the generation of electrical activity in brain neurons.Previous investigations have failed to find any modulatory activity of levetiracetam on voltage-gated Na + and low-voltage-activated Ca 2+ currents in rat neocortical neurons (4,5). Therefore special attention was devoted to high-voltage-activated (HVA) Ca 2+ currents, which also can be responsible for changes in the firing pattern of corresponding neurons. Recently it was shown that LEV can inhibit HVA calcium channels in pyramidal hippocampal neurons (6,7). Therefore it was of special interest to evaluate whether L...

Bovine chromaffin cells possess several types of Ca2+ channels, and influx of Ca2+ is known to trigger secretion. However, discrepant information about the relative importance of the individual subtypes in secretion has been reported. We used whole-cell patch-clamp measurements in isolated cells in culture combined with fura-2 microfluorimetry and pharmacological manipulation to determine the dependence of secretion on different types of Ca2+ channels. We stimulated cells with relatively long depolarizing voltage-clamp pulses in a medium containing 60 mM CaCl2. We found that, within a certain range of pulse parameters, secretion as measured by membrane capacitance changes was mainly determined by the total cumulative charge of Ca2+ inflow and the basal [Ca2+] level preceding a stimulus. Blocking or reducing the contribution of specific types of Ca2+ channels using either 20 microM nifedipine plus 10 microM nimodipine or 1 microM omegaCTxGVIA (omega-conotoxin GVIA) or 2 microM omegaCTxMVIIC (omega-conotoxin MVIIC) reduced secretion in proportion to Ca2+ charge, irrespective of the toxin used. We conclude that for long-duration stimuli, which release a large fraction of the readily releasable pool of vesicles, it is not so important through which type of channels Ca2+ enters the cell. Release is determined by the total amount of Ca2+ entering and by the filling state of the readily releasable pool, which depends on basal [Ca2+] before the stimulus. This result does not preclude that other stimulation patterns may lead to responses in which subtype specificity of Ca2+ channels matters.

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We examined the relationship between calcineurin (protein phosphatase 2B (PP2B)) and voltage‐operated Ca2+ channels (VOCCs) in NG108–15 cells. PP2B expression in NG108–15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2Bβ3 in sense (CN‐15) and antisense (CN‐21) orientation. Confocal immunocytochemical localization showed that in wild‐type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells. To test the Ca2+ dependence of the VOCC, we used high‐frequency stimulation (HFS). The L‐ and N‐type VOCCs decreased by 37 and 52 %, respectively, whereas the T‐type current was only marginally sensitive to this procedure. FK‐506 (2 μM), a specific blocker of PP2B, reduced the inhibition of L‐ and N‐type VOCCs induced by HFS by 30 and 33 %, respectively. In CN‐15‐transfected cells overexpressing PP2B, total high‐voltage‐activated (HVA) VOCCs were suppressed by about 60 % at a test potential of +20 mV. Intracellular addition of EGTA or FK‐506 into CN‐15‐transfected cells induced an up to 5‐fold increase of HVA VOCCs. These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca2+‐dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect being on N‐type VOCCs.

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