No abstract

Carbamazepine and phenytoin, two of the most commonly prescribed antiepileptic drugs, have been proposed to share a similar mechanism of action by use-dependent inhibition of Na+ channels. The proposed similar mechanism of action, however, cannot explain the common clinical experiences that the two drugs are different; in some patients, one drug may be more effective than the other. This may occur even when optimal therapeutic concentrations are reached with both medications in plasma or the cerebrospinal fluid. In this study, we show that the action of the two drugs on Na+ channels are quantitatively very different. The affinity between inactivated Na+ channels and carbamazepine (apparent dissociation constant approximately 25 microM) is approximately 3 times lower than that of phenytoin, yet the binding rate constant of carbamazepine onto the inactivated Na+ channels is approximately 38,000 M(-1)/sec(-1), or approximately 5 times faster than that of phenytoin. It is speculated that carbamazepine may be more effective than phenytoin in treating seizures whose ictal depolarization shift is relatively short, whereas a better response to phenytoin may imply abnormal discharges characterized by more prolonged depolarization.

1 Lamotrigine (LTG), a new antiepileptic drug, requires long depolarizations to inhibit Na + currents. This suggests either slow binding of LTG to the fast inactivated state or selective binding of LTG to the slow inactivated state of Na + channels. To di erentiate between these possibilities and to characterize further the action of LTG, we studied the a nity and kinetics of LTG binding to the Na + channels in acutely dissociated hippocampal neurones of the rat. 2 LTG inhibited more Na + currents at more depolarized holding potentials. The inhibitory e ect at various holding potentials could be described by one-to-one binding curves, which yielded an apparent dissociation constant of *7 mM for LTG binding to the inactivated channels (K I ), and a dissociation constant more than 200 times larger for LTG binding to the resting channels. A similar value of K I (*9 mM) was also derived from the LTG concentration-dependent shift of the inactivation curve. 3 The recovery of LTG-bound inactivated Na + channels was faster than the recovery of normal (drugfree) slow inactivated channels. Moreover, the binding kinetics of LTG onto the inactivated channels were faster than the development of the slow inactivated state, and were linearly correlated with LTG concentrations, with a binding rate constant of *10,000 M 71 s 71 . These ®ndings suggest that LTG chie¯y binds to the fast inactivated state rather than the slow inactivated state. 4 We conclude that LTG, in therapeutic concentrations and at relatively depolarized membrane potentials, may potently inhibit Na + currents by slow binding to the fast inactivated state of Na + channels. Like phenytoin, the slow binding rates may explain why LTG e ectively inhibits seizure discharges, yet spares most normal neuronal activities.

An increase in neuronal burst activities in the subthalamic nucleus (STN) is a well-documented electrophysiological feature of Parkinson disease (PD). However, the causal relationship between subthalamic bursts and PD symptoms and the ionic mechanisms underlying the bursts remain to be established. Here, we have shown that T-type Ca 2+ channels are necessary for subthalamic burst firing and that pharmacological blockade of T-type Ca 2+ channels reduces motor deficits in a rat model of PD. Ni 2+ , mibefradil, NNC 55-0396, and efonidipine, which inhibited T-type Ca 2+ currents in acutely dissociated STN neurons, but not Cd 2+ and nifedipine, which preferentially inhibited L-type or the other non-T-type Ca 2+ currents, effectively diminished burst activity in STN slices. Topical administration of inhibitors of T-type Ca 2+ channels decreased in vivo STN burst activity and dramatically reduced the locomotor deficits in a rat model of PD. Cd 2+ and nifedipine showed no such electrophysiological and behavioral effects. While low-frequency deep brain stimulation (DBS) has been considered ineffective in PD, we found that lengthening the duration of the low-frequency depolarizing pulse effectively improved behavioral measures of locomotion in the rat model of PD, presumably by decreasing the availability of T-type Ca 2+ channels. We therefore conclude that modulation of subthalamic T-type Ca 2+ currents and consequent burst discharges may provide new strategies for the treatment of PD.

No abstract

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp5O bearing the yeast ARSI, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to.grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product. The CDC8 protein of Saccharomyces cerevisiae is essential for chromosomal replication in vivo and is required for cell division. When the temperature-sensitive cdc8 mutant is grown at the permissive temperature, 23°C, and then shifted to the restrictive temperature, 36°C, cells accumulate that contain a nucleus located at the isthmus between parent cell and bud, but which do not divide. The first wave of DNA synthesis after shift up does not occur (8). The product of the CDC8 gene is apparently required throughout the period of DNA synthesis, since synchronized cultures of cells defective in the gene cease nuclear DNA replication when shifted to 36°C within the S period (9). Mitochondrial DNA replication also ceases at the nonpermissive temperature in cdc8 mutants (22), and replication of the 2-,um circle plasmid is defective in cdc8 mutants (17).Recently the CDC8 protein has been purified to homogeneity, using in vitro replication systems (1, 15). The purified protein binds to singlestranded DNA and stimulates DNA polymerase I activity on single-stranded DNA templates. The CDC8 protein may be identical to protein C, discovered by Chang et al. (4).To carry out detailed biochemical and functional characterization of an enzyme involved in DNA replication, it is necessary to obtain large quantities of purified protein. In Escherichia coli, one way to overcome the problem of low yield of replication proteins is to clone the gene coding for a given protein into temperatureinducible bacteriophage lambda vectors or into a high-copy-number plasmid. Overproduction of the replication proteins DNA polymerase I (12), DNA ligase (23), and dnaC protein (13) has been achieved. Overproduction of the LEU2 gene product in yeast strains carrying the LEU2 gene on an autonomously replicating, high-copy-number plasmid has also been achieved (10). Therefore, the same approach used in E. coli is applicable in yeast.In this co...

The increased tendency of STN burst discharges may by itself serve as a direct cause of parkinsonian locomotor deficits, even in the absence of deranged dopaminergic innervation. Effective DBS therapy in PD very likely relies on adequate depolarization, and consequent modification of the relevant ionic currents and discharge patterns, of STN neurons.

Felbamate (FBM) is a potent nonsedative anticonvulsant whose clinical effect may be related to the inhibition of N-methyl-Daspartate (NMDA) currents, but the exact molecular action remains unclear. Using whole-cell patch-clamp recording in rat hippocampal neurons, we found that submillimolar FBM effectively modifies the gating process of NMDA channels. During a single high-concentration (1 mM) NMDA pulse, FBM significantly inhibits the late sustained current but not the early peak current. However, if the 1 mM NMDA pulse is preceded by a low-concentration (10 M) NMDA prepulse, then FBM significantly inhibits both the peak and the sustained currents in the 1 mM pulse. In sharp contrast, the NMDA currents elicited by micromolar NMDA are only negligibly inhibited or even enhanced by FBM. These findings indicate that the inhibitory effect of FBM on NMDA currents is stronger with both higher NMDA concentration and longer NMDA exposure, and is thus "use-dependent". FBM also slows recovery of the desensitized NMDA channel, and quantitative analyses of FBM effects on the activation kinetics and the desensitization curve of the NMDA currents further disclose dissociation constants of ϳ200, ϳ110, and ϳ55 M for FBM binding to the resting, activated, and desensitized NMDA channels, respectively. We conclude that therapeutic concentrations (50 -300 M) of FBM could bind to and modify a significant proportion of the resting NMDA channel even when NMDA or other glutamatergic ligand is not present and then decrease the NMDA currents at subsequent NMDA pulses by stabilization of the desensitized channels. Because the inhibitory effect is apparent only when there is excessive NMDA exposure, FBM may effectively inhibit many seizure discharges but preserve most normal neuronal firings.Felbamate (FBM; 2-phenyl-1,3-propanediol dicarbamate) is a potent new-generation anticonvulsant that is effective against many different types of epilepsy (Pellock and Brodie, 1997). In addition to clinical cases, the broad-spectrum antiepileptic effect is also well documented in experimental seizures. For example, FBM is effective against both supramaximal extension seizures induced by maximal electroshock and threshold seizures induced by pentylenetetrazol (Swinyard et al., 1986). Although serious complications such as aplastic anemia and hepatotoxicity have limited its use, FBM is an anticonvulsant which is too important to discard. With informed consent of the patients, FBM has remained as a useful anticonvulsant for Lennox-Gastaut syndrome in children and for a variety of complex partial seizures that are refractory to the other anticonvulsants in adults (Kaufman et al., 1997;Pellock, 1999).The intriguing pharmacological profile of FBM implies a unique mechanism of action. Just as for the other nonsedative anticonvulsants, any proposed mechanism underlying FBM action preferably should explain why seizure discharges are effectively inhibited but normal neuronal firings are relatively preserved. FBM has been reported to have multiple pharmac...