SUMMARY1. Ca2+ inward currents evoked by membrane depolarization have been studied by the intracellular dialysis technique in the somatic membrane of isolated dorsal root ganglion neurones of new-born rats. In about 20 % of the investigated cells a hump has been detected on the descending branch of the current-voltage curve, indicating the presence of two populations of Ca2+ channels differing in their potential-dependent characteristics.2. An initial less regular component ofthe Ca2+ current was activated at membrane potentials from -75 to -70 mV. Its amplitude reached 02-{09 nA at 14-6 mMextracellular Ca2+. The activation kinetics of this component could be approximated by the Hodgkin-Huxley equation using the square of the m variable. Tm varied in the range from 8 to 1 ms at potentials between -60 and -25 mV ('fast' Ca2+ current).3. The second component of the Ca2+ current was activated at membrane depolarizations to between -55 and -50 mV. It could be recorded in all cells investigated and reached a maximum value of 1-7 nA at the same extracellular Ca2+ concentration. This component decreased rapidly during cell dialysis with saline solutions. The decrease could be slowed down by cooling and accelerated by warming the extracellular solution. Intracellular introduction of 3',5'-cAMP together with ATP and Mg2+ not only prevented the decrease but often restored the maximal current amplitude to its initial level. The activation kinetics of this component could also be approximated by a square function, Tm being in the range 16-2-5 ms at membrane potentials between -20 and + 3 mV ('slow' Ca2+ current).4. The fast Ca2+ current inactivated exponentially at sustained depolarizations in a potential-dependent manner, Th varying from 76 to 35 ms at potentials between -50 and -30 mV. The inactivation of the slow Ca2+ current studied in double-pulse experiments was current-dependent and developed very slowly (time constant of several hundreds of milliseconds). It slowed down even more at low temperature or after substitution of Ba2+ for Ca2+ in the extracellular solution. 5. Both currents could also be carried by Ba2+ and Sr2+, although the ion-selecting properties of the two types of channels showed quantitative differences.

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SUMMARY1. Characteristics of the transmembrane ionic currents under controlled changes in ionic composition of extra-and intracellular medium were studied in isolated neurones from the ganglia of molluscs, Helix pomatia, Limnea 8taJnali8 and Planorbis corneus. The neurones were investigated by a new technique which allows for dialysis of their interior and for clamping of the potential at the surface membrane without using micro-electrodes.2. Replacement of K ions by Tris inside the neurones eliminated the outward K current so that the actual time course of the inward current could be measured. The latter was separated into two additive components, one of which was carried by Na ions and the other one by Ca ions.3. Both inward currents were unaltered by tetrodotoxin (TTX); however, Ca current could be separately blocked by externally applied Cd ions (Kd = 7*2 x 10-5 M) and by the use of fluoride as an intracellular anion.4. No reversal of Na inward current could be achieved in neurones dialysed with Na-free solution, indicating the absence of outward current carrying ions through the corresponding channels. With 5 mM-Na inside the cell, the equilibrium potential was close to the value predicted by the Nernst equilibrium.5. A non-specific outward current could be detected in K-free cells at membrane potentials exceeding 20-40 mV. Its time course was proportional to 1-exp (-t/Tnr). Cd ions depressed this current. The presence of the non-specific outward current made an exact measurement of the equilibrium potential for the Ca inward current impossible.6. The kinetics of Na inward currents could be described by m3h and those of the Ca current by m2h law. The corresponding values for Vm = 0 are: rm(Na) = 1.1 + 05 msec, rm(Ca) = 2-4 + 1*0 msec, rh(Na) = 7-9 + 2-0 msec. The inactivation of Ca current included two first-order kinetic processes with Th1 = 50 + 10 msec and Th = 320 + 30 msec.21-2 P. G. KOSTYUK AND 0. A. KRISHTAL 7. The data presented are considered to be a proof of the existence of separate systems of Na and Ca ion-conducting channels in the nerve cell membrane.

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...

Summary. The slow inward current carried by Na + through potential-dependent calcium channels in conditions when divalent cations were removed from the extracellular solution by EDTA has been investigated on isolated internally perfused neurons of the snail Helix pomatia. The calcium channels also acquire the capability to pass monovalent cations if other caIcium-binding substances are added to the extracellular solution. Based on these facts the conclusion is made that the immediate reason for the modification of the channel selectivity is the absence of divalent cations in the extracellular medium. All potential-dependent characteristics of the modified calcium channel are shifted by 60 to 70 mV in the hyperpolarizing direction compared with those of the original calcium channel. The series of relative permeabilities for modified calcium channels The induced sodium current decreases immediately when the concentration of divalent cations in the extracellular solution is elevated. This decrease is not potential dependent and can be approximated by Langmuir~ isotherm with dissociation constants pKc~ :PKsr :pKB,:pK~g = 6.6:5.5:4.8:4.2. The conclusion is drawn that the calcium channels in the somatic membrane have two ion-selecting filters with different functionsan external one consisting, probably, of several carboxylic groups which bind divalent cations in a highly specific manner and determine the impermeability of the channel to monovalent cations in physiological conditions, and the channel ion-selecting filter including a single carboxylic group normally determining the channel selectivity for different divalent cations.