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Epilepsia, 45(7):729–736, 2004 Blackwell Publishing, Inc.  C 2004 International League Against Epilepsy

Inhibitory Effect of Lamotrigine on A-type Potassium Current in Hippocampal Neuron–Derived H19-7 Cells ∗ †Chin-Wei Huang, †‡Chao-Ching Huang, §Yen-Chin Liu, and Sheng-Nan Wu ∗ Department of Neurology, †Institute of Clinical Medicine, ‡Department of Pediatrics, Institute of Basic Medical Sciences, National // Cheng-Kung University Medical Center; and §Department of Anesthesiology, Kaohsiung Veterans General Hospital, Tainan, Taiwan

Summary: Purpose: We investigated the effects of lamotrigine (LTG) on the rapidly inactivating A-type K+ current (I A ) in embryonal hippocampal neurons. Methods: The whole-cell configuration of the patch-clamp technique was applied to investigate the ion currents in cultured hippocampal neuron–derived H19-7 cells in the presence of LTG. Effects of various related compounds on I A in H19-7 cells were compared. Results: LTG (30 µM–3 mM) caused a reversible reduction in the amplitude of I A . The median inhibitory concentration (IC50 ) value required for the inhibition of I A by LTG was 160 µM. 4-Aminopyridine (1 mM), quinidine (30 µM), and capsaicin (30 µM) were effective in suppressing the amplitude of I A , whereas tetraethylammonium chloride (1 mM) and gabapentin (100 µM) had no effect on it. The time course for the inactivation of I A was changed to the biexponential process during cell expo-

sure to LTG (100 µM). LTG (300 µM) could shift the steady-state inactivation of I A to a more negative membrane potential by approximately −10 mV, although it had no effect on the slope of the inactivation curve. Moreover, LTG (100 µM) produced a significant prolongation in the recovery of I A inactivation. Therefore in addition to the inhibition of voltage-dependent Na+ channels, LTG could interact with the A-type K+ channels to suppress the amplitude of I A . The blockade of I A by LTG does not simply reduce current magnitude, but alters current kinetics, suggesting a state-dependent blockade. LTG might have a higher affinity to the inactivated state than to the resting state of the I A channel. Conclusions: This study suggests that in hippocampal neurons, during exposure to LTG, the LTG-mediated inhibition of these K+ channels could be one of the ionic mechanisms underlying the increased neuronal excitability. Key Words: Lamotrigine—A-type K+ current—Hippocampal neurons.

Lamotrigine (LTG), a phenyltriazine derivative, is a clinically useful antiepileptic drug (AED) and is administered primarily as an add-on medication to other AEDs in therapy-resistant epilepsy. Although consensus is found on the general mode of action by LTG on voltagedependent Na+ current (1,2), still little consistency appears concerning the effects of LTG on other types of ion currents. For example, LTG was reported to suppress Ca2+ -sensing cation current in cultured hippocampal neurons (3). It has been demonstrated that LTG could alter 5-hydroxytryptamine receptor–mediated response in rat brain (4). Moreover, LTG was previously reported to increase the hyperpolarization-activated cation current in the dendrites of pyramidal neurons (5). More interestingly, this drug has been shown to enhance transient K+ outward current in CA1 pyramidal cells and in neocortical cells (6–8).

Several lines of evidence show that LTG is likely to aggravate preexisting seizures and trigger new seizure types (9–13). However, the biologic mechanisms involved are unknown. In addition, LTG gains increasing interest as a potential mood stabilizer and antimanic drug in psychiatry (14,15). However, the mechanism of action underlying its efficacy on mood disorders also is poorly understood. The I A , prominent in hippocampal neurons (16,17) is an important factor in repolarizing the membrane potential, determining the level of neuronal excitability, and controlling the interspike interval during repetitive firing. This current has been reported to be regulated by the mitogenactivated protein kinase ERK (18). As compared with that in young neurons, the magnitude of this current has been recently found to be reduced in old rat hippocampal neurons (17). The H19-7 cell line is known to possess the characteristics of embryonic hippocampal neurons (19,20). This cell line could potentially be used for studies of development, plasticity, and commitment in hippocampal neurons. However, no studies concerning its electrophysiologic properties have been thus far reported. Therefore the

Accepted February 29, 2004. Address correspondence and reprint requests to Dr. S-N. Wu at Institute of Basic Medical Sciences, National Cheng-Kung University Medical Center, No. 1, University Road, Tainan, Taiwan. E-mail: [email protected]

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main objective of this study was to address the question of whether LTG could affect the amplitude and kinetics of I A in these cells. We provide evidence that LTG can have a depressant effect on I A in a concentration- and state-dependent fashion. MATERIALS AND METHODS Cell preparation The H19-7 cell line, originally derived from hippocampi dissected from embryonic day 17 (E17) Holtzman rat embryos and immortalized by retroviral transduction of temperature-sensitive tsA58 SV40 large T antigen, was obtained from American Type Culture Collection [(CRL2526), Manassas, VA, U.S.A.] (19). H19-7 cells were maintained in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 10% fetal bovine serum, 200 µg/ml G418, and 1 µg/ml puromycin in flasks coated with 15 µg/ml poly-L-lysine. They were equilibrated in a humidified atmosphere of 5% CO2 /95% air at a temperature of 34◦ C. The experiments were performed after 5 days of subcultivation (60–80% confluence) with cells obtained from passages 2 and 4. Electrophysiologic measurements Immediately before each experiment, cells were dissociated, and an aliquot of cell suspension was transferred to a recording chamber positioned on the stage of an inverted microscope (DM IL; Leica Microsystems, Wetzlar, Germany). Cells were bathed at room temperature (20–25◦ C) in normal Tyrode’s solution containing 1.8 mM CaCl2 . The recording pipettes were pulled from thin-walled borosilicate glass capillaries (Kimax-51; Kimble Glass, Vineland, NJ, U.S.A.) by using a two-stage microelectrode puller (PP-830; Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83; Narishige). When the pipettes were filled with pipette solution, their resistance ranged between 3 and 5 M. Ion currents were recorded in the whole-cell configuration of the patch-clamp technique, by using an RK-400 patchclamp amplifier (Bio-Logic, Claix, France) (21). All potentials were corrected for liquid junction potential, a value that would always develop at the tip of the pipette when the composition of the pipette solution was different from that in the bath. Data recording and analysis The signals were displayed on an analog/digital oscilloscope (HM 507; Hameg Inc., East Meadow, NY, U.S.A.) and on a liquid crystal projector (PJ550-2; ViewSonic Corporation, Walnut, CA, U.S.A.). The data were stored online in a Pentium III-grade laptop computer (Slimnote VX3; Lemel, Taipei, Taiwan) via a universal serial bus port at 10 kHz through a high-speed/low-noise analog/digital interface (Digidata 1322A; Axon Instruments, Union City, Epilepsia, Vol. 45, No. 7, 2004

CA, U.S.A.). This device was controlled by commercially available software (pCLAMP 9.0; Axon Instruments). Cell-membrane capacitance of 17–38 pF (26.5 ± 4.8 pF; n = 26) was compensated. Series resistance, always in the range of 5–15 M, was electronically compensated. Currents were low-pass filtered at 1 or 3 kHz. In some experiments, the linear passive-leak currents were digitally subtracted by using a P/4 regimen. Ion currents recorded during whole-cell experiments were stored without leakage correction and analyzed subsequently by using the pCLAMP 9.0 software (Axon Instruments), the Origin 6.0 software (Microcal Software, Inc., Northampton, MA, U.S.A.), SigmaPlot 7.0 software (SPSS, Inc., Apex, NC, U.S.A.), or custom-made macros in Excel (Microsoft, Redmond, WA, U.S.A.). The pCLAMP-generated voltage-step protocols were used to examine the current– voltage (I-V) relations for ion currents. To calculate the percentage inhibition of LTG on I A , cells were bathed in Ca2+ -free Tyrode’s solution, and each cell was depolarized from –50 to +50 mV. The peak amplitudes in the presence of LTG were compared with those measured by a subsequent application of 4-aminopyridine (5 mM). 4-Aminopyridine is known to block the amplitude of I A . The concentration of LTG required to inhibit 50% of current amplitude was fitted to a Hill equation: y = (Emax × [C]nh )/(IC50 nh + [C]nh ), where [C] is the concentration of LTG; IC50 and nh are the concentration required for a 50% inhibition and the Hill coefficient, respectively; and Emax is the LTG-induced maximal inhibition in channel activity. The Solver subroutine built in Excel (Microsoft) was used to fit data by a least-squares minimization procedure. The averaged results are presented as the mean values ± SEM. The paired or unpaired t test and one-way analysis (ANOVA) with the least significance difference method for multiple comparisons were used for the statistical evaluation of differences among the mean values. Differences between values were considered significant when p < 0.05. Drugs and solutions LTG (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine) was a gift from GlaxoSmithKline Research and Development (Hertfordshire, NY, U.S.A.). Gabapentin (GBP) was a gift from Pfizer Pharmaceuticals (Vega Baja, Puerto Rico). Tetraethylammonium chloride, 4-aminopyridine, poly-L-lysine, puromycin, and quinidine were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). Capsaicin was obtained from Sigma/RBI (Natick, MA, U.S.A.), and tetrodotoxin was from Alomone Labs (Jerusalem, Israel). Tissue-culture media and trypsin/ethylenediaminetetraacetic acid (EDTA) were obtained from American Type Culture Collection (Manassas, VA, U.S.A.). L-Glutamine, penicillin–streptomycin, and amphotericin B (Fungizone) were obtained from

EFFECT OF LAMOTRIGINE ON K + CURRENT

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FIG. 1. Effect of lamotrigine (LTG) on the I-V relationships of I K in hippocampal H19-7 cells. A: Superimposed current traces obtained when the cell was depolarized from –50 mV to various potentials ranging from – 100 to +40 mV with 10-mV increments. The current traces shown in the upper part are control, and those in the lower part were obtained 1 min after application of 300 µM LTG. The uppermost part shown in A indicates the voltage protocol used. The averaged I-V relations for peak and steady-state components of I K in the absence (solid symbols) and presence (open symbols) of 300 µM LTG are plotted in B and C, respectively. Each point represents the mean ± SEM (n = 5–8).

Life Technologies (Grand Island, NY, U.S.A.). All other chemicals were obtained from regular commercial chemicals and were of reagent grade. The twice-distilled water that had been deionized through a Millipore-Q system (Millipore, Bedford, MA, U.S.A.) was used in all experiments. The composition of normal Tyrode’s solution was as follows (in mM): NaCl, 136.5; KCl, 5.4; CaCl2 , 1.8; MgCl2 , 0.53; glucose, 5.5; and HEPES-NaOH buffer, 5.5 (pH 7.4). To record K+ current, the patch pipette was filled with a solution (in mM): KCl, 140; MgCl2 , 1; Na2 ATP, 3; Na2 GTP, 0.1; EGTA, 0.1; and HEPES-KOH buffer, 5 (pH 7.2). The pipette solution was filtered on the day of use with a 0.22-µm pore size syringe filter (Millipore). RESULTS Effect of LTG on rapidly inactivating A-type K+ outward current (IA ) in H19-7 hippocampal neurons The whole-cell configuration of the patch-clamp technique was used to investigate the effect of LTG on ion currents in H19-7 cells. In these experiments, cells were bathed in Ca2+ -Tyrode’s solution containing tetrodotoxin (1 µM) and CdCl2 (0.5 mM). Figure 1 shows that in H197 cells, membrane currents were evoked at 0.1 Hz in response to various step pulses, with a duration of 300 ms in the absence and presence of LTG (300 µM). Under control conditions, when the cell was held at the level of –50 mV, the depolarizing pulses more positive to –20 mV elicited the outward currents, which inactivated within the first 100 ms of the step pulse (Fig. 1A). The amplitudes of these

currents were increased with greater depolarization. When the cells were held at –50 mV, the measured potentials at which instantaneous current crossed the zero-current level with 5.4, 50, and 100 mM extracellular K+ were –44 ± 3, –10 ± 3, and –2 ± 1 mV (n = 5), respectively. The results indicate the dependence of changes of membrane currents on the extracellular K+ concentrations. Because evoked currents were voltage dependent, activating at potentials more positive to –10 mV and cumulatively inactivating during repetitive depolarization, these have thus been believed to be A-type K+ outward current (I A ). When the cells were exposed to LTG (300 µM), the amplitude of I A was suppressed throughout the entire voltage clamp step (Fig. 1A). After LTG was removed, I A returned almost to the control level. The averaged I-V relations for the amplitude of I A in the absence and presence of LTG are shown in Figure 1B and C. At +40 mV, LTG (300 µM) reduced the peak and steady-state amplitudes of I K by 75% and 43%, respectively. The effect of 4-aminopyridine, a prototypical blocker of I A , on the amplitude of IA in these cells also was examined. As shown in Figure 2, during the exposure to 5 mM 4-aminopyridine, the peak amplitude of I A was fully suppressed. However, the steady-state component of I A was relatively resistant to inhibition by 4aminopyridine. The majority of the steady-state component of outward current could be due to the noninactivating K+ current, because a subsequent application of tetraethylammonium chloride (10 mM) in continued presence of 4-aminopyridine (5 mM) abolished the steady-state amplitude of I A . Epilepsia, Vol. 45, No. 7, 2004

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FIG. 2. Effect of 4-aminopyridine on I A in H19-7 cells. The cell was depolarized from –50 to +50 mV with a duration of 300 ms. Lower panel: (a) is control, and (b) was obtained after cell exposure to 5 mM 4-aminopyridine. Upper panel: The voltage protocol used. Notably, 4-aminopyridine reduces the peak amplitude of I A .

During the exposure to LTG, in addition to the decreased amplitude of I A , the time to peak of I A tended to be shortened, and the time course of current inactivation was changed to the biexponential process, as shown in Fig. 3. For example, when the depolarizing pulse from –50 to +50 mV was evoked, the inactivation of I A was well fit to a single exponential process with a mean time constant of 47 ± 5 ms (n = 9); however, in the presence of LTG (100 µM), the fast and slow time constants of I A

FIG. 3. The inactivation time course of I A in the absence and presence of lamotrigine (LTG). The cell was depolarized from a holding potential of –50 mV to +50 mV. Control, 1; obtained in the presence of LTG (100 µM), 2. The curves were nonlinear least squares fits of either one or two exponentials to the data.

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FIG. 4. Concentration-dependent inhibition of I A by lamotrigine (LTG) in H19-7 cells. A: Superimposed current traces obtained in the absence and presence of LTG. Cells were bathed in Ca2+ free Tyrode’s solution containing tetrodotoxin (1 µM) and CdCl2 (0.5 mM). Each cell was depolarized from –50 to +50 mV with a duration of 300 ms. 1, control; 2, 3, and 4, obtained after application of 30, 100, and 300 µM LTG, respectively; 4, after the addition of 4-aminopyridine (5 mM), but in the continued presence of LTG (300 µM). The upper part in A indicates the voltage protocol used. B: Concentration–response relation for LTG-induced inhibition of I A (i.e., 4-aminopyridine–sensitive current). Each point represents the mean ± SEM (n = 5–8). Smooth line, The best fit to the Hill equation. The IC50 value, maximally inhibited percentage of 4-aminopyridine-sensitive current, and Hill coefficient were 160 µM, 99%, and 1.1, respectively.

inactivation were 10.1 ± 1.1 and 69 ± 5 ms (n = 9), respectively. The fast, not slow, component was found to be concentration dependent. In other words, the time constant decreased when the LTG concentration was increased. After LTG was removed, I A returned almost to the control level. Figure 4 shows the relation between the concentration of LTG and the percentage inhibition of I A . The current amplitudes of I A in the presence of LTG were compared with those after a subsequent application of 4-aminopyridine (1 mM). Application of LTG (30 µM –3 mM) was found to suppress the amplitude of 4aminopyridine–sensitive I A in a concentration-dependent manner. Fitting the concentration–response curve with

EFFECT OF LAMOTRIGINE ON K + CURRENT

FIG. 5. Comparison between the effect of lamotrigine (LTG) and those of 4-aminopyridine, quinidine, capsaicin, tetraethylammonium chloride, and gabapentin on the amplitude of I A in H19-7 cells. Each cell was held at the level of –50 mV, and the voltage pulses to +50 mV (300 ms in duration) were applied at 0.1 Hz. The peak amplitude of I A in the control was considered to be 1.0, and the relative amplitude of I A after application of each agent was compared and plotted. The parentheses shown in each bar indicate the number of cells from which the data were taken. Mean ± SEM. ∗ Significantly different from control. LTG (100 µM); 4AP: 4-aminopyridine (1 mM); Quin: quinidine (30 µM); Cap, capsaicin (30 µM); TEA, tetraethylammonium chloride (1 mM); Gab, gabapentin (100 µM).

the Hill equation yielded a half-block concentration of 160 µM and a slope coefficient of 1.1. LTG (3 mM) nearly abolished current amplitude. These results indicate that LTG has a significant action on the inhibition of I A in H19-7 cells. Effects of various compounds on IA in H19-7 cells Effects of 4-aminopyridine, quinidine, capsaicin, tetraethammonium chloride, and GBP on I A in H19-7 cells also were examined and compared. As shown in Figure 5, 4-aminopyridine (1 mM) and quinidine (30 µM) decreased the peak amplitude of I A by ∼70 and 65%, respectively. Capsaicin (30 µM) also was effective in suppressing I A , although tetraethylammonium chloride (1 mM) and GBP (300 µM) had no effect on it. Capsaicin is known to be a blocker of transient outward K+ current present in heart cells (22), whereas GBP is an antiepileptic drug (AED). The data presented here indicate that the observed I A in H19-7 cells is sensitive to inhibition by 4-aminopyridine, quinidine, and capsaicin, yet not by tetraethylammonium chloride. Voltage dependence of LTG-induced inhibition of IA To characterize the inhibitory effects of LTG on I A , we further examined the voltage dependence of the effect of LTG on I A in H19-7 cells. Figure 6 shows the steady-state inactivation curve of I A obtained in the absence and presence of LTG (100 µM). In this series of experiments, the interval between two sets of voltage pulses was 60 s to avoid incomplete recovery of I A . As shown in Figure 6B,

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FIG. 6. Steady-state inactivation of I A in the absence and presence of lamotrigine (LTG) in H19-7 cells. With the aid of a doublepulse protocol, the steady-state inactivation parameters of I A were determined. A: Superimposed current traces obtained in the absence (upper) and presence (lower) of 100 µM LTG. The uppermost part shown in A indicates the voltage protocol used. Arrowheads, Zero current level. B: Normalized amplitude of I A (I/I max ) was constructed against the conditioning potential, and the smooth curves were well fit by the Boltzmann equation (see text for details). Each point represents the mean ± SEM (n = 5–6). •, control; ◦, LTG (100 µM).

the relation between conditioning potentials and the normalized amplitudes of I A in the absence and presence of LTG (100 µM) were fitted to a Boltzmann function by using a nonlinear least-squares regression analysis: I = I max /{1 + exp[(V – a)/b]}, where I max is the maximal activated I A , V is the membrane potential in mV, a is the membrane potential for a half-maximal inactivation, and b is the slope factor of the inactivation curve. In the absence of LTG, a equals –12.4 ± 0.7 mV and b equals 6.8 ± 0.3 mV (n = 6), whereas in the presence of LTG (100 µM), a equals –21.8 ± 1.0 mV, and b equals 6.9 ± 0.4 mV (n = 5). Therefore LTG did not simply reduce the maximal conductance of I A , but it also shifted the inactivation curve to a hyperpolarized potential (9.4 ± 1.2 mV, n = 5). However, we found no significant change in the slope of the curve in the presence of LTG. Taken together, the results clearly indicate that LTG can suppress the amplitude of I A in a voltage-dependent fashion in H19-7 cells. Effect of LTG on recovery of IA from inactivation The effect of LTG on the recovery of I A from inactivation also was studied by using a double-pulse protocol. A 100-ms conditioning step to +50 mV inactivated most of the I A , and the recovery of I A from inactivation at the holding potential of –50 mV was examined at different times with a test step (+50 mV, 100 ms), as shown in Figure 7. In control condition, the current amplitude of I A almost completely recovered from inactivation when the recovery time was 1 s. The time course of recovery from inactivation was fitted to a single exponential function with a time constant of 323 ± 15 ms (n = 6). However, during Epilepsia, Vol. 45, No. 7, 2004

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FIG. 7. Effect of lamotrigine (LTG) on the time course of recovery from the inactivation of I A after the cell was depolarized from –50 to +50 mV. Cells were depolarized from –50 to +50 mV with a duration of 100 ms, and various interpulse durations were applied. A: Original current traces in the absence and presence of LTG (300 µM). Arrowheads, Zero current level. The uppermost part shown in A indicates the voltage protocol used. B: Effect of LTG on the time course of recovery from the inactivation of I A when the cells were depolarized from –50 to +50 mV. •, control; ◦, LTG (300 µM). Each smooth line was fitted by a single-exponential function. Each point represents the mean ± SEM (n = 5–8). Of note, the abscissa is shown at a logarithmic scale.

cell exposure to LTG (300 µM), recovery from inactivation was significantly prolonged, with a time constant of 412 ± 14 ms (n = 6). In other words, after a 1-s interval, the amplitude of I A was found to recover completely from inactivation in control condition; however, in the presence of LTG, a substantial block of I A still was seen. It is thus apparent that LTG produces a significant prolongation of the recovery from inactivation of I A in H19-7 cells.

DISCUSSION The characteristics of the voltage-dependent I A in hippocampal H19-7 cells were described in this study. This current, which was elicited when the voltage step was at the potential more positive to –20 mV, activates rapidly within a few milliseconds and then inactivates within a few tens of milliseconds. The current was found to inactivate with the time course that can be well fit to a single exponential process. Interestingly, the application of LTG did not simply reduce the peak amplitude of I A . It caused a significant reduction in the time to peak of I A . The rate of current inactivation also was increased in the presence of LTG. In other words, the time course of current decay in response to the depolarizing step was changed to two different exponentials. These findings reflect that LTG is capable of blocking I A channels in a state-dependent fashion. Binding speed and affinity of the LTG molecule for a

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particular state may determine the kinetic behavior of the I A in the presence of LTG. A left shift in the midpoint of the inactivation curve of I A was observed in the presence of LTG. Cell exposure to LTG also was found to slow the recovery of inactivation of I A in H19-7 cells. It is thus possible that LTG has a higher affinity toward the open-inactivated I A channels than toward the closed or resting channels in these cells, although the gating conformational changes of this channel in the presence of LTG must be further characterized. In our study, the voltage-gated Ca2+ channels were blocked by CdCl2 , and the existence of I A was independent of intracellular Ca2+ concentrations. It thus seems unlikely that LTG-mediated inhibition of I A is due to its direct suppression of Ca2+ current and the resultant decrease in intracellular Ca2+ concentrations. This study seems to be inconsistent with previous reports showing that LTG might increase the amplitude of I A (6–8). The reason for this discrepancy is currently unknown. However, the action of LTG seems to be dependent on cell type studied and the specific types of K+ channels present. This probably reflects changes in the location of the binding site produced by differences in the channel protein sequence. In addition, it remains to be determined whether cytoplasmic factors (e.g., arachidonic acid) may also mediate the effect of LTG on I A in different types of cells (23,24). It has been reported that Kv4.2, a Shal-type K+ channel that passes an A-type current, is localized in the hippocampus. The cytoplasmic domain of the Kv4.2 channel is a substrate for mitogen-activated protein kinase ERK (18). However, when the recording pipettes were filled with LTG (300 µM), no change in the amplitude of I A was seen in H19-7 cells (data not shown). Thus it is unlikely that the LTG-mediated inhibition of I A shown here is related to direct phosphorylation of the pore-forming α subunit. Conversely, it was recently reported that the auxillary K+ channel subunit Kvβ 1.1 could confer fast inactivation of the I A in hippocampal neurons (25). It will thus be of interest whether the inhibition of I A by LTG involves its binding to this subunit. At least two types of inactivation are present in KA channels. N-type inactivation results from binding of an inactivating particle to the internal pore mouth of the activated channel, whereas C-type inactivation involves conformational changes at the external mouth of the pore (26). When the recording pipettes were filled with LTG (300 µM), no change in the amplitude of I A was seen in this study. The binding of LTG to the inactivated state suggests that the conformation of the external pore mouth appears to be similar to the character of C-type inactivation. Indeed, the time constant of recovery in control is ∼235 ms, a value much larger than those previously described for the time constants of recovery from N-type inactivation (26,27).

EFFECT OF LAMOTRIGINE ON K + CURRENT Our results showed that the IC50 value for the LTGinduced inhibition of I A was ∼160 µM. The plasma and cerebrospinal fluid concentration of LTG is usually 10 to 40 µM under therapeutic conditions (28); however, it may be >100 µM in overdose (28). Moreover, the ability of LTG to shift the inactivation curve of I A to a hyperpolarized potential suggests that the sensitivity to LTG will depend on the preexisting resting potential, the firing rate of action potentials, or the concentration of LTG, if the LTG action in cells in vivo is the same as those in the H197 cells shown here. This phenomenon might reflect one of the possible mechanisms underlying the increased neuronal excitability in LTG-inducing aggravation of seizures observed clinically. In addition, some serious side effect of LTG may be linked to a mechanism related to its blockade of K+ channels (9,12). However, it has been reported that levetiracetam (LEV), a novel AED, could block delayed rectifier K+ current, thus leading to a decrease in the repetitive firing of action potentials (29). Therefore it remains to be determined to what extent AEDs may influence different types of K+ currents, thus leading to change in the firing of action potentials. The amplitude of I A observed in H19-7 cells is sensitive to inhibition by 4-aminopyridine, quinidine, and capsaicin, yet not by tetraethylammonium chloride. The A-type K+ channels presented here are thus likely to be coded by genes from the Kv4 subfamily (e.g., Kv4.2 and Kv4.3) (30–32). A previous report showed that heterotopic pyramidal neurons in the hippocampus from rats with the methylazoxymethanol exposure did not express Kv4.2 (Atype) channel subunits. Such a lack of A-type K+ channels was proposed to decrease the seizure threshold in brain malformation in methylazoxymethanol-exposed rats (33). Whether the inhibition of the I A channels has potential benefit in treating underlying cellular disturbances in bipolar disorder remains to be clarified. Acknowledgment: We thank Mei-Han Huang and Su-Rong Yang for technical assistance in the preparation of cultured cells. This study was aided by grants from National Science Council (NSC-91-2320B-006-106 and NSC-92-2320B-006041), Taiwan.

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