Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Among voltage-dependent Ca²⁺ channels, T-type channels, by virtue of their activation at relatively negative potentials, are believed to play a crucial role in the control of cell firing rates (Llinas, 1988; Huguenard, 1996). Major proposed roles for T-type channels in neurons include promotion of Ca²⁺-dependent burst firing, low-amplitude intrinsic neuronal oscillations, promotion of Ca²⁺ entry, and boosting of synaptic signals (Huguenard, 1996). Furthermore, T-type currents appear to play a role in seizure susceptibility and initiation (Chung et al., 1993; Huguenard and Prince, 1994; Tsakiridou et al., 1995).

T currents in different native tissues appear to share a number of common features that help distinguish them from high-voltage-activated Ca²⁺ currents. These include relative permeation by Ca²⁺ and Ba²⁺ (Carbone and Lux, 1987a,b), more negative activation voltages (Carbone and Lux, 1984), similarity in deactivation and inactivation kinetics, and generally a greater sensitivity to blockade by Ni²⁺ over Cd²⁺ (Fox et al., 1987). However, despite these similarities, differences in both kinetic behavior of some native T currents (Huguenard and Prince, 1992; Huguenard et al., 1993) and pharmacological properties (e.g., Herrington and Lingle, 1992; review by Huguenard, 1996; Todorovic and Lingle, 1998) have been observed. Diversity in native T-type Ca²⁺ channel function might arise from tissue-specific expression of the 1G, 1H, and 1I subunits now known to encode T-type Ca²⁺ channels (Perez-Reyes et al., 1998; Cribbs et al., 1998). For example, the 1H subunit appears to be the primary contributor to T-type current in sensory neurons (Talley et al., 1999). In addition, T-like Ca²⁺ currents in native cells might also arise from other mechanisms. For example, Meir and Dolphin (1998) reported that multiple subunits of high-voltage-activated (HVA) channels can under certain conditions give rise to T-type currents.

One difficulty in evaluating pharmacological studies of T currents in native cells is that for some agents the conditions under which a compound has been investigated have not been identical. Similarly, in many cases, full concentration-response curves for effects on T currents have not been defined. As a consequence, it has not been possible to determine the extent to which different T currents share a particular pharmacological profile or represent distinct current variants. Incomplete concentration-response information also makes it unclear whether effects seen in vitro correlate with the concentrations of a particular drug in plasma necessary to achieve therapeutic effects. In those cases where more complete information about the sensitivity of a T current to particular pharmacological agents is available, some clear differences in pharmacological properties are revealed. Two sets of agents for which quite disparate blocking effects on T currents among different cells have been reported include divalent cations, including Ni²⁺ and Cd²⁺ (Kaneda and Akaike, 1989; Herrington and Lingle, 1992; Ye and Akaike, 1993; Todorovic and Lingle, 1998) and anticonvulsants (Herrington and Lingle, 1992; Gross et al., 1997; Todorovic and Lingle, 1998). With the availability of the cloned 1G, 1H, and 1I subunits encoding T-type currents, it is now possible to assess directly the pharmacological sensitivities of the  subunits.

Toward this end, we examine here the sensitivity of 1G and 1H currents to anesthetics and anticonvulsants. Where the blocking effect of a compound is found to differ from previously described effects on T currents in dorsal root ganglion (DRG) neurons (Todorovic and Lingle, 1998), we have re-examined the blocking effect of that compound on DRG T current under identical ionic and recording conditions. We observed that, for the anesthetics examined, blocking effects are essentially identical for 1G, 1H, and native DRG T currents. However, for the anticonvulsants, phenytoin and -methyl--phenylsuccinimide (MPS), blockade of 1G current differed from blockade of native DRG T current. Specifically, in contrast to blockade of DRG T current where block was of higher affinity, but only partial, these agents blocked 1G current completely but with lower affinity. Furthermore, for 1H current, phenytoin produced two different kinds of responses, one similar to its effects on DRG T current and one similar to the effects on 1G current. No physiological differences in the 1H currents underlying these responses were observed. This raises the possibility that either post-translational modification of 1H subunits or an additional accessory subunit may contribute to the pharmacological sensitivity of native T-type currents.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Cell Preparation. HEK 293 cells were stably transfected with either 1G or 1H constructs as described previously (Lee et al., 1999). Cells were typically used 1 to 3 days after plating. Average cell capacitance (C_m) was 20.9 ± 5.7 pF, and the average series resistance (R_s) was 5.29 ± 2.08 (n = 34). For DRG neurons, 100- to 300-g male rats (Sprague-Dawley) were used as we described elsewhere (Todorovic and Lingle, 1998). Eight to ten DRG from thoracic and upper lumbar regions were dissected out and incubated at 36°C for 60 to 90 min in Tyrode's solution (140 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 10 mM glucose, 10 mM HEPES, adjusted to pH 7.4 with NaOH) containing 5 mg/ml collagenase (type I, Sigma Chemical Co., St. Louis, MO) and 5 mg/ml dispase II (Boehringer-Manheim, Indianapolis, IN). Single neuronal cell bodies were obtained by trituration in Tyrode's solution at room temperature. Cells were kept at room temperature and used for electrophysiology within 4 to 6 h from dissociation. For recordings, neuronal cell bodies were plated onto a glass cover-slip and placed in a culture dish that was perfused with external solution. All data were obtained from smaller diameter DRG neurons (21-27 µm) without visible processes. The average C_m for DRG cells was 14.6 ± 2.5 pF, and the average R_s was 6.4 ± 1 (n = 12).

Electrophysiological Methods. Recordings were made with the standard whole-cell voltage-clamp technique (Hamill et al., 1981). Electrodes were prepared from microcapillary tubes (Drummond Scientific Company, Broomall, PA), coated with Sylgard (Dow Corning, Midland, MI) and fire-polished. Pipette resistances were 2 to 5 M. Voltage commands and digitization of membrane currents were done with Clampex 5.5 of the pClamp software package (Axon Instruments, Foster City, CA) running on an IBM-compatible computer. Membrane currents were recorded with an Axopatch 200A patch-clamp amplifier (Axon Instruments). Typically, cells were held at 90 mV and depolarized to 35 mV every 20 s to evoke inward currents. Data were analyzed using Clampfit (Axon Instruments). Currents were filtered at 5 kHz. Reported series resistance values were taken from the reading of the amplifier. In all experiments, series resistance was compensated 60 to 80%. All reported membrane potentials are nominal values. All experiments were done at room temperature (20-23°C). In most experiments, leakage subtraction was achieved with a P/5 on-line subtraction protocol. Error bars indicate standard deviations of multiple determinations obtained from at least five different cells.

Analysis of Current Blockade. The percentage reduction in peak T current at a given blocker concentration was used to generate concentration-response curves. For each concentration-response curve, all points are averages of multiple determinations obtained from at least five different cells. On all plots, vertical bars indicate standard deviation. Mean values on concentration-response curves were fit to the following function:

(1)

where PB_max is the maximal percent block of peak T current, the IC₅₀ is the concentration that produces 50% of maximal inhibition, and h is the apparent Hill coefficient for blockade. In the case of isoflurane, because each application of blocker involved a separate determination of anesthetic concentration, all data points were fit with the above function. Fitted values are typically reported with 95% linear confidence limits. Fitting was done with Origin 3.7 (Microcal Software, Northhampton, MA).

Solution Exchange Procedures. The solution application system consisted of multiple, independently controlled glass capillary tubes, whereas solution was removed from the other end of the chamber with the use of constant suction. Manually controlled valves accomplished switching between solutions. Test solutions were maintained in closed, weighted all-glass syringes (to avoid saline evaporation and loss of volatile drugs) and allowed to fall by gravity. Changes in Ca²⁺ current amplitude in response to rapidly acting drugs or ionic changes were typically complete in 10 to 20 s. Switching between separate perfusion syringes, each containing control saline, was without effect on Ca²⁺ current amplitude. No dependence on the order of presentation or desensitization with repeated applications was observed for any of the pharmacological agents.

Solutions and Current Isolation Procedures. The standard extracellular saline for recording of Ca²⁺ current contained (in millimolar): 160 TEA-Cl, 10 HEPES, 2 CaCl₂, adjusted to pH 7.4 with TEA-OH, osmolarity 316 mOsm. Cells were generally maintained in a Tyrode's solution until seal formation, at which time the bath solution was switched to the Ca²⁺ saline. Internal solution consisted of (in millimolar) 110 Cs-methane sulfonate, 14 phosphocreatine, 10 HEPES, 9 EGTA, 5 Mg-ATP, and 0.3 Tris-GTP, pH adjusted to 7.15 to 7.20 with CsOH (standard osmolarity: 300 mOsm). When this internal saline was used for recording of T current in DRG cells, most of the HVA current in these cells was blocked by preincubating cells with 1 µM -CgTx-GVIA, 2 µM -CgTx-MVIIC and by including 5 µM nifedipine in the external solution to block N, P, Q, and L types of HVA current. Because in control experiments this effect was irreversible for up to 60 min, we routinely preincubated every slide with these toxins and recorded within this time frame. In most cells included in this study, blockade of L-, N-, P-, and Q-type currents was sufficient to allow investigation of T current in virtual isolation. Because of the possibility of some residual HVA current contamination, all measurements of T current amplitude in DRG cells were made from the peak of the inward current to the current remaining at the end of a 200-ms test step. Typically, the residual current at 200 ms was indistinguishable from leak current.

Drugs and Chemicals. -Conotoxin GVIA (-CgTx-GVIA), -conotoxin MVIIC (-CgTx-MVIIC) and tert-butylbicyclo[2.2.2]phosphorothionate (TBPS) were obtained from RBI (Natick, MA), and etomidate powder and isoflurane were obtained from Abbott (Abbott Park, IL). All other chemicals were obtained from Sigma or Aldrich Chemicals (Milwaukee, WI).

Drug Preparation. Stock solutions of propofol (50 mM), etomidate (300 mM), MPS (1 M), TBPS (50 mM), and phenytoin (100 mM) were prepared in dimethyl sulfoxide (DMSO) and kept at 4°C until use. 0.1% DMSO had no effect when tested alone in DRG cells or HEK cells with either 1G or 1H constructs.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Here, we have studied the effects of anesthetic and anticonvulsant compounds on 1G and 1H currents in HEK cells. All of the compounds have been previously systematically examined for effects on native T currents in adult rat DRG neurons (Todorovic and Lingle, 1998) and, to some extent, on T current in GH3 cells (Herrington and Lingle, 1992). Some information on the blocking effects of these agents on high-voltage-activated Ca²⁺ currents is also available, in particular, for blockade of 1E current (Nakashima et al., 1998). For MPS and phenytoin, we describe differences between effects on 1G current and previous results on DRG cells (Todorovic and Lingle, 1998). Because we were concerned that the observed differences could be due to the different charge carriers used (10 mM Ba²⁺ versus 2 mM Ca²⁺ in this study), we have also repeated pharmacological experiments on DRG neurons under ionic conditions identical with those used to study 1G and 1H currents.

Effects of Anticonvulsants on 1G and 1H Current. A number of studies have indicated that various anticonvulsants, including succinimides (Coulter et al., 1989, 1990) and phenytoin (Twombly et al., 1988) have blocking effects on T-type Ca²⁺ current. Work on thalamic relay neurons (Coulter et al., 1989, 1990) suggested that the anticonvulsant drug, ES, was a blocker of T current within clinically effective concentrations. This effect was believed to contribute to its efficacy in petit mal seizures. More recent work now suggests that T current in thalamic neurons is not blocked by ES (Leresche et al., 1998), and other studies have shown that effects of ES on various T currents only occur at concentrations well beyond those obtained clinically (Herrington and Lingle, 1992; Todorovic and Lingle, 1998). However, the anticonvulsants, phenytoin and MPS, do have some blocking effects on DRG T currents at clinically relevant concentrations (Todorovic and Lingle, 1998). Furthermore, because of the proposed role of thalamic T currents in the initiation of epileptic activity (Chung et al., 1993; Huguenard and Prince, 1994; Tsakiridou et al., 1995), the effects of possible effects of anticonvulsants on T current variants remains of interest. Here, we have therefore examined the effects of phenytoin, MPS, and ES on 1G and 1H currents.

Phenytoin Differentially Affects Native DRG T Current and 1G and 1H Current. The effect of phenytoin on 1G current is illustrated in Fig. 1. 10 µM phenytoin produced a small reduction in 1G current (left traces in Fig. 1A), whereas 300 µM phenytoin produced a more complete blockade (right traces in Fig. 1A). The effects of three different phenytoin concentrations on peak 1G current are illustrated in Fig. 1B. The concentration dependence of phenytoin block of 1G current is summarized in Fig. 1E, indicating that with sufficiently high concentrations of phenytoin a nearly complete block of 1G current can be achieved.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   The anticonvulsant phenytoin has differential effects on 1G current in HEK cells and T currents in native DRG cells. A, traces show block by 10 µM phenytoin (left) and 300 µM phenytoin (right) of 1G current in HEK293 cells. B, plot of a temporal record of the reduction of peak 1G current by multiple applications of phenytoin, indicating the substantial difference in block produced by 100 and 300 µM phenytoin. Note that 300 µM phenytoin almost completely blocks the 1G currents. C, traces show block of T currents in DRG currents under ionic conditions identical with those used in A. Left traces show effect of 10 µM phenytoin, whereas right trace shows blocking effect of 300 µM phenytoin from the same cell. Note that 10 µM phenytoin blocked a larger percentage of DRG T current, and 300 µM phenytoin blocked less, in comparison to the 1G current. D, peak T current amplitude during the course of an experiment on a DRG neuron shows that 300 and 600 µM phenytoin produce a similar and partial block. Horizontal bars indicate time of application. E, the concentration dependencies of phenytoin inhibition of DRG T current (filled symbols) and 1G current (open symbols) are displayed. Vertical lines indicate SE and solid lines are best fits of eq. 1. For DRG T current, the fitted maximal block is 58% with little difference observed between 30 and 600 µM phenytoin. For 1G current, nearly 100% block is achieved with 600 µM phenytoin with an IC50 of 124.2 ± 9.1 µM (h = 1.4; 7 cells). However, the IC50 for block of DRG T current (IC50 of 8.0 ± 2.9 µM; h =1.9; 8 neurons) is more than 10-fold less than for block of 1G current.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   1H currents in HEK293 cells exhibit either of two types of sensitivities to phenytoin. A, traces show effects of 10 µM (left) and 300 µM (right) phenytoin on 1H current in a cell in which blockade by phenytoin increased substantially over the range of 100 to 300 µM. B, the temporal record of the inhibition of peak 1H current by phenytoin illustrates the progressive increase in block with increasing concentrations. Horizontal bars indicate time of drug application. C, current traces show effects of 10 (left) and 300 (right) µM phenytoin, respectively, on inward 1H current in another HEK cell. Note that 10 µM phenytoin produced more block (about 25%) and 300 µM phenytoin blocked less block (only about 40%) than for the cell in A. D, the temporal record of the effects of phenytoin on peak current for the cell in C shows that for this cell there is little difference in inhibition by phenytoin over the range of 30 to 300 µM with maximal block occurring at less than 50%. E, concentration dependencies for block by phenytoin is displayed for the two categories of cells: those in which phenytoin produces a partial, but higher affinity, block (IC50 = 8.3 µM; maximum = 44.8%; n = 10 cells) and those in which phenytoin produces a complete, but lower affinity, block (IC50 = 192 µM; n = 13 cells). F, concentration dependencies of block of 1H current by phenytoin are compared with block of DRG current and 1G current by phenytoin (from Fig. 1E).

Effects of Succinimides on 1G and 1H Current. MPS is another anticonvulsant reported to have only partial blocking effects on DRG T current (Todorovic and Lingle, 1998), although it has been reported to produce complete block of thalamic neuron T current (Coulter et al., 1990). The effects of 300 µM and 3 mM MPS on 1G current are shown in Fig. 3A, whereas the effects of four different MPS concentrations on peak 1G current are summarized in Fig. 3B. MPS, like phenytoin, appears able to produce a nearly complete block of 1G current with sufficiently high concentrations. The IC₅₀ for block of 1G current by MPS was 1.7 ± 0.3 mM (n = 1.7 ± 0.3) with maximal block near 100%.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   MPS produces a low affinity, complete block of 1G and 1H current, which differs from blockade of DRG T current. A, left and right panels display 1G currents blocked by 0.3 and 3 mM MPS, respectively. B, temporal record of the peak current amplitude over the course of an experiment illustrates the concentration dependence of MPS inhibition of 1G current. 6 mM MPS produces an almost complete block of 1G current. C, left and right panels show examples of the inhibition of DRG T current by 0.3 and 3 mM MPS, respectively. Note that 3 mM MPS produces little additional inhibition over that achieved with 0.3 mM MPS. D, a temporal record of the inhibition of peak DRG T current by 1 and 3 mM MPS shows that MPS maximally blocks only about 30% of the peak current in this case. E, blockade of 1H current by 6 mM MPS is illustrated. F, the concentration dependencies of MPS block of DRG T current (filled circles), 1G current (open circles), and 1H current (diamonds) are displayed. Vertical lines represent S.D. Solid lines are best fits of eq. 1. For MPS inhibition of DRG T current (n = 6 cells), the IC50 was 0.14 ± 0.1 mM (h = 1.2) with a maximal block of 33%. For inhibition of 1G current (n = 5 cells), the IC50 was 1.7 ± 0.3 mM (h = 1.6) with maximal block constrained to 100%. For 1H current (n = 19 cells), the IC50 was 2.3 ± 0.4 mM (h = 1.5) with maximal block constrained to 100%.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   The anticonvulsant, ES, blocks 1G and 1H current similarly. A, traces show block of 1G current produced by either 10 and 100 mM ES. B, peak inward current activated every 20 s from the experiment shown in A is plotted as a function of elapsed time. C, traces show block of 1H current produced by 100 mM ES. D, peak inward current activated every 20 s from the experiment shown in C is plotted as a function of elapsed time. E, the concentration dependence of 1G and 1H current reduction by ES is plotted. Each point is the average of the block produced by ES applications to at least five different cells, with vertical lines indicating S.D. Solid lines are best fit of eq. 1, yielding for 1G current, an IC50 of 14 ± 3.7 mM (h = 0.9) with the fit constrained to a maximal block of 100% and, for 1H current, an IC50 = 22.4 ± 3.0 mM (h = 1.5) with maximal block constrained to 100%.

View this table: [in this window] [in a new window]   TABLE 1 A comparison of sensitivities of native T currents in DRG cells and 1G, 1H, and 1E currents in HEK cells

Lack of Effect of Convulsants on 1G Current. Because thalamic T currents have been proposed to play an important role in the initiation of epileptic activity (Chung et al., 1993; Huguenard and Prince, 1994; Tsakiridou et al., 1995), we tested two convulsant compounds on 1G current. Pentylenetetrazol at 1 mM had no effect on 1G current in five cells (4.4 ± 6% inhibition). This was similar to the reported insensitivity to this agent of T current in DRG cells (Todorovic and Lingle, 1998) and thalamic cells (Coulter et al., 1990). TBPS is a potent GABA_A receptor antagonist (Casida et al., 1985). In four HEK cells, 50 µM TBPS had no effect on 1G current.

Blockade of 1G Current by Parenteral Anesthetics and Related Compounds. Various anesthetics have also been reported to inhibit T-type currents in various cells, in some cases at concentrations that overlap their clinical usage. All parental anesthetics used in this study did produce complete block of 1G currents and, when tested, complete block of 1H currents. Blocking potency was, in general, quite comparable with previous results on T current in DRG neurons.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Parenteral general anesthetics block 1G currents in HEK cells. A, traces display inward currents activated by a 200-ms voltage step from 90 mV to 35 mV before, during, and after application of 0.3 and 1 mM octanol. Note the faster rate of inactivation in the presence of 300 µM octanol. B, peak inward current over the course of the experiment for the cell shown in A is displayed to illustrate the concentration dependence and reversible nature of block by octanol. Currents were activated every 20 s. Horizontal bars indicate time of application of the indicated concentrations of octanol. C, the decay phase of normalized currents before, during, and after application of 300 µM octanol are shown to illustrate the effect of octanol on current inactivation rate. Inactivation time constants were 10.7, 5.5, and 10.4 ms before, during, and after octanol application, respectively, from single exponential fits to the current decay phase. D, average concentration-response curves for blockade of peak 1G current by propofol, etomidate, ketamine, and octanol. All points are averages of at least five different cells. Vertical bars indicate S.D. of multiple determinations, and solid lines are best fits of the Hill equation (eq. 1, Materials and Methods). Fits were constrained to 100% block with IC50 values of 20.0 ± 2.0 µM (h = 1.3), 158.9 ± 25.5 µM (h = 1.6), 152.3 ± 29.7 µM (h = 1.4), and 1.14 ± 0.17 mM (h = 1.4), for propofol, etomidate, octanol, and ketamine, respectively.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Octanol and propofol block 1H and 1G current similarly. A, traces display 1H currents activated by 200-ms voltage steps from 90 mV to 35 mV before, during, and after application of 0.3 and 1 mM octanol. Note the faster rate of inactivation in the presence of 300 µM octanol. B, peak inward current over the course of the experiment for the cell shown in A is displayed to illustrate the concentration dependence and reversible nature of block by octanol. C, traces show the effects of 6 and 60 µM propofol on 1H current activated as in A. D, average concentration-response curves are shown for blockade of peak 1H current by propofol and octanol. All points are averages of at least five different cells. Solid lines are best fits of the Hill equation (eq. 1, Materials and Methods). Fits were constrained to 100% block with an IC50 for propofol of 27 ± 3 µM (h = 1.2) and an IC50 for octanol of 218.6 ± 44.5 µM (h = 1.4). Lines with smaller symbols are the fitted concentration-response curves for block of 1G current by propofol (filled circles) and octanol (open circles) from Fig. 5.

Effects of Isoflurane on 1G Current. Volatile anesthetics, like isoflurane and halothane, are reported to block T currents in DRG cells in clinically relevant concentrations (Todorovic and Lingle 1998). Of the volatile anesthetics that have been tested on T currents, isoflurane appears to have the most pronounced blocking effects at concentrations likely to occur during clinical use (Todorovic and Lingle, 1998). Examples of the effects of isoflurane on 1G current are shown in Fig. 7A. Blockade was rapid and readily reversible (Fig. 7B). At sufficiently high concentrations, isoflurane blocked 1G current essentially completely. In all experiments, actual concentrations of isoflurane in the bath were measured using gas chromatographic analysis. Like octanol, but not propofol, isoflurane increased the apparent rate of inactivation of 1G current (Fig. 7C). The concentration-response curve for blockade of 1G current by isoflurane is shown in Fig. 7D, yielding an IC₅₀ of 277 ± 24 µM and h of 2.2 ± 0.4. This compares to an IC₅₀ for blockade of DRG T current by isoflurane of 303 µM (Todorovic and Lingle, 1998).

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Isoflurane blocks 1G currents at concentrations likely to occur in clinical usage. A, traces show currents activated by voltage steps to 35 mV in the presence and absence of isoflurane. Concentrations were determined by gas chromatography (Evers et al., 1986). Similar to octanol, isoflurane increased the current inactivation rate. B, a temporal record of the effects of three concentrations of isoflurane on peak 1G current for the cell shown in A is plotted. Open circles represent peak current evoked every 20 s. C, the effect of 410 µM isoflurane on the normalized time course of current inactivation is shown. i was 17.7, 8.2, and 15.2 ms for control, 410 µM isoflurane, and following wash. D, percent blockade of peak 1G current is plotted as a function of isoflurane concentration for isoflurane applications to six cells. All isoflurane values were determined by gas chromatography. The solid line is the best fit of eq. 1 yielding an IC50 of 262 ± 16.6 µM and h of 2.3 ± 0.3 with maximal block constrained to 100%.

Blockade of 1G and 1H Current by Barbiturates. Barbiturates are interesting compounds, because they are both general anesthetics and antiepileptics. Pentobarbital, methohexital, thiopental, and phenobarbital block DRG T currents in adult rats, although at concentrations higher than those achieved during clinical use (Todorovic and Lingle, 1998).

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Blockade of 1G current by barbiturates. A, traces show 1G currents activated by voltage steps to 35 mV in the presence and absence of thiopental applied at the concentrations indicated on the figure. B, traces show 1G currents activated as in A before, during, and after the application of the indicated concentrations of phenobarbital. In contrast to the effects of octanol and isoflurane shown in Figs. 5 and 6, the barbiturates did not affect the time course of current inactivation. In A, in control saline, i = 14.4 ms; with 1 mM thiopental, i = 15.5 ms. In B, in control saline, i = 12.0 ms, whereas with 5 mM phenobarbital, i = 12.2 ms. C, the time course of block for multiple concentrations of phenobarbital and thiopental is plotted from the same cell as in A and B. Bars indicate time of application. D, concentration-response curves for three barbiturates are shown, with each point being the average of at least five different cells. Vertical lines are the S.D. The solid lines are best fits of eq. 1. IC50 values for block of 1G current were 263 ± 31 µM (h = 1.3) for thiopental, 311 ± 31 µM (h = 1.3) for pentobarbital, and 1.5 ± 0.2 mM (h = 1.4) for phenobarbital. E, traces show 1H currents activated as in A in the presence and absence of the indicated concentrations of pentobarbital. F, the concentration dependence for block of both 1H and 1G current by pentobarbital is compared. The IC50 for block of 1H current was 345 ± 94 µM (h = 1.1).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The low threshold for activation of T type Ca²⁺ currents allows them to play a critical role in the regulation of cellular excitability, both in neurons and other cell types (Huguenard, 1996). Despite general similarity among various T-type currents in different cells, some differences in kinetic behaviors and pharmacological sensitivities have been observed (Huguenard, 1996). With the availability of cloned T-type current variants (Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999a), it is now possible to examine physiological and pharmacological properties of T currents of defined molecular components. Here, we have examined the sensitivity of cloned T-type current variants to anticonvulsants and anesthetics. The results suggest that, although anesthetic sensitivity does not vary among DRG, 1H, and 1G currents, blocking effects of some anticonvulsants may depend on specific subunits contributing to the T channel.

Blockade of T Current by Anticonvulsants. Interest in the possible role of T current inhibition in the action of anticonvulsants arose from reports that ES and MPS may inhibit T current in thalamic relay neurons (Coulter et al., 1989, 1990). These succinimide compounds belong to a class of anticonvulsants used to treat petit mal-generalized absence seizures (Macdonald and McLean, 1986). However, although some results support a potential contributory role of T current in experimental models of epilepsy (Chung et al., 1993; Tsakiridou at el., 1995), more recent work indicates that 1 mM ES does not inhibit T current on thalamic cells (Leresche et al., 1998). The lack of effect of ES on thalamic T currents is comparable with the relative insensitivity (IC₅₀ values greater than 10 mM) of native T currents (Herrington and Lingle, 1992; Todorovic and Lingle, 1998) and 1G and 1H current that we observe. The results would suggest that the anticonvulsant action of ES is unlikely to arise from effects on T-type currents.

Blockade of T currents Arising from 1G and 1H Subunits by General Anesthetics. Anesthetics and anesthetic-like compounds blocked all three T-type currents similarly and are of limited utility in distinguishing among T-type currents. Table 1 shows the sensitivity of cloned 1E currents to these same compounds (Nakashima et al., 1998), indicating that anesthetics are relatively ineffective in distinguishing between T-type current and at least one type of HVA Ca²⁺ current.