Ca2+ Channel Antagonist U-92032 Inhibits Both T-Type Ca2+ Channels and Na+ Channels in Hippocampal CA1 Pyramidal Neurons

Robert B. Avery and Daniel Johnston

Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Avery, Robert B. and Daniel Johnston. Ca2+ channel antagonist U-92032 inhibits both T-type Ca2+ channels and Na+ channels in hippocampal CA1 pyramidal neurons. J. Neurophysiol. 77: 1023-1028, 1997. The effects of 7-[[4-[bis(4-fluoropheny l ) - m e t h y l ] - 1 - p i p e r a z i n y l ] m e t h y l ] - 2 - [ ( 2 - h y d r o x y e t h y l ) a m i n o ]4 -( 1 - m e t h y l e t h y l ) - 2 , 4 , 6 - c y c l o h e p t a t r i e n - 1 - o n e   ( U - 9 2 0 3 2 ) ,   anewly described Ca2+ channel blocker, on voltage-gated ionic currents were measured. Whole cell voltage-clamp records were obtained from acutely isolated CA1 hippocampal pyramidal neurons from 7- to 14-day-old rats. Dimethyl sulfoxide, at either 0.01% or 0.1%, partially inhibited T-type Ca2+ currents (~20% inhibition) but not high-voltage-activated (HVA) Ca2+ currents. Ethanol (0.2%) did not affect Ca2+ currents. U-92032 selectively inhibited T-type Ca2+ currents (median inhibiting concentration ~ 500 nM). HVA Ca2+ currents were less sensitive, with ~75% of the current resistant at 10 µM. Inhibition of Ca2+ currents was reversible. U-92032 inhibited Na+ currents at concentrations similar to those required for T-type currents (>33% block at 1 µM). Block of Na+ currents took several minutes to develop and was irreversible. Voltage-gated K+ currents were insensitive to U-92032 (1 or 10 µM). These results indicate that U-92032 inhibits both T-type Ca2+ channels and Na+ channels, constraining its utility in certain studies. Among Ca2+ channels, however, U-92032 should prove a useful tool for distinguishing physiological contributions of T-type channels.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Central neurons possess multiple types of voltage-gated Ca2+ channels (Carbone and Lux 1984; Fisher et al. 1990). The contribution of different types of channels to specific cellular functions is not clear. This is particularly true for T-type Ca2+ channels. In hippocampal neurons, proposed functions for T-type channels include promoting burst firing (Fraser and MacVicar 1991; Hablitz and Johnston 1981), influencing postsynaptic responses either electrically (Magee and Johnston 1995) or chemically (Magee et al. 1995), and influencing synaptic plasticity (Christie et al. 1996; Kamiya 1989; for review see Huguenard 1996).

Direct tests of these hypotheses have been hindered by a lack of specific blockers for T-type channels. Although several antagonists for T-type channels have been reported, their specificity for T-type channels over other neuronal Ca2+ channel types is not that impressive. Further, the quality of the blockade is quite variable across preparations. Probing the contribution of T-type channels to cellular physiology requires that antagonists be characterized in the cells of interest.

7 - [ [ 4 - [ B i s ( 4 - f l u o r o p h e n y l ) m e t h y l ] - 1 - p i p e r a z i n y l ]m e t h y l ] - 2 - [ ( 2 - h y d r o x y e t h y l ) a m i n o ] 4 - ( 1 - m e t h y l e t h y l ) 2,4,6-cycloheptatrien-1-one (U-92032) is a recently described T-type channel antagonist. U-92032 demonstrates about a 10-fold greater potency for T-type over L-type channels in atrial myocytes (Xu and Lee 1994), blocks T-type channels in a neuroblastoma cell line (NIE-115), and reduces ischemic damage assayed in hippocampal slices (Ito et al. 1994).

Whole cell voltage-clamp recordings were used to characterize the effects of U-92032 on voltage-gated conductances in hippocampal CA1 pyramidal neurons. We report that U-92032 is highly selective for T-type channels over other types of neuronal Ca2+ channels. However, concentrations that blocked T-type currents also inhibited Na+ currents.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Methods have been presented in more detail previously (Avery and Johnston 1996).

Cell preparation

Transverse hippocampal slices 500 µm thick were cut from 7- to 14-day-old rats in ice-cold, oxygenated dissecting saline (see Solutions and drugs). Slices were incubated in a papain solution (10 U/ml, Worthington) at 37°C for 30 min with oxygen flowing over the solution surface. Slices were transferred to a room-temperature holding chamber. As needed, two slices were removed from the holding chamber and the CAI region was dissected out in 1 mg/ml bovine serum albumin. Cells were isolated by gently triturating the tissue through a series of fire-polished pasteur pipettes, plated on a clean coverslip, and allowed to settle for 5 min, at which time the dissecting saline was gradually replaced with the recording solution.

Recordings

Patch pipettes were pulled from borosilicate glass (Drummond) on a two-stage vertical puller (Adams-List) and had a resistance in the bath of 2-5 MOmega . Cells were visualized with the use of an inverted microscope (Zeiss) equipped with Hoffman modulation optics. Target cells had pyramidal-shaped somata and were patch clamped with an Axopatch 1-C amplifier (Axon Instruments). The average cell capacitance was 18.1 ± 7.9 (SD) pF and the average series resistance was 6.1 ± 1.4 (SD) MOmega . Series resistance was typically compensated to 75%. Depolarizing test pulses were usually delivered once every 15 s and records were leak subtracted off-line. The cell was positioned in the outlet stream of a multi-inlet microperfusion pipette, which allowed exchange of the solution bathing the cell within a few seconds. For all drug applications, the control saline included ethanol (EtOH). Cells were used within 5 h after dissection.

Solutions and drugs

The dissecting saline included (in mM) 110 sodium piperazine-N,N'-bis(2-ethanesulfonic acid), 20 NaCl, 3 KCl, 2 MgCl2, 10 dextrose, and 1 kynurenic acid, pH 7.4, 300 mosM.

Composition of the external recording salines used to isolate the different ionic currents is presented in Table 1.

 
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TABLE 1. Composition of external recording salines

Composition of the internal solutions is presented in Table 2. The tip was filled with the indicated solution, and then the pipette was backfilled with the same solution plus an ATP regenerating system composed of 4 mM tris(hydroxymethyl)aminomethane (Tris)-ATP, 0.3 mM Tris-guanosine 5'-triphosphate, 14 mM Tris-phosphocreatine, 50 U/ml creatine phosphokinase, and 0.1 mM leupeptin.

 
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TABLE 2. Composition of internal recording salines

U-92032 was provided by Pharmacia and Upjohn (Kalamazoo, MI). Tetraethylammonium chloride and CsCl were obtained from Aldrich Chemicals (Milwaukee, WI). All other drugs were purchased from Sigma (St. Louis, MO).

Statistical significance was determined with the use of a two-tailed, paired-sample t-test.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

DMSO reduced T-type currents

T-type currents were isolated by stepping to -50 mV from a holding potential of -80 mV. In 2 mM Ca2+, this protocol evoked a current with two kinetically distinct components; one inactivating and one noninactivating. We have previously shown that the inactivating component results from the opening of T-type channels, but other types of channels contribute to the noninactivating component (Avery and Johnston 1996). To quantify effects on T-type channels, we measured only the inactivating current by taking the difference in the current amplitude from the peak to the end of a 600-ms step (illustrated as the measure bar on the control trace of Fig. 1A). In the same cell, high-voltage-activated (HVA) Ca2+ channels were isolated by holding at -50 mV to inactivate T-type channels (Mogul and Fox 1991). Subsequent depolarizations to 0 mV elicited a composite current representing activation of all the other known types of Ca2+ channels in hippocampal pyramidal neurons (Avery and Johnston 1996). HVA currents were measured at the the peak of the current. Initially, we dissolved U-92032 in dimethyl sulfoxide (DMSO). We observed a small but consistent decrease in the amplitude of the T-type current when switching from control saline to saline with DMSO only (Fig. 1A). This effect is quantified in Fig. 1B, which shows a slight reduction in T-type but not HVA currents. The inhibition was not reversible and was the same magnitude for either 0.01 or 0.1% DMSO. Therefore we used EtOH as the solvent in all experiments presented in this paper. At the highest concentrations used (0.2%), EtOH did not affect Ca2+ currents (Fig. 1B).


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FIG. 1. Effects of the solvents dimethyl sulfoxide (DMSO) and ethanol (EtOH) on Ca2+ currents. A, left: Ca2+ currents at -50 mV in control and in the presence of 0.01% DMSO. T-type currents were assayed by measuring the difference between the peak current and the amplitude of current at the end of a 600-ms step (measure bar on the control trace). DMSO partially reduced T-type currents. A, right: high-voltage-activated (HVA) currents during the same application. The HVA current was insensitive to DMSO. B: summary data for DMSO and EtOH. The amplitudes of the T-type and HVA currents are graphed as a % of their control values (shorter bars mean greater block). DMSO inhibited T-type currents similarly at 0.01 and 0.1%, but HVA currents were not affected. Ca2+ currents were insensitive to EtOH (0.2%). Values expressed as % of control (mean ± SE)---0.01% DMSO: T type, 82 ± 4% (n = 7), HVA, 103 ± 2% (n = 7); 0.1% DMSO: T type, 77 ± 5% (n = 5), HVA, 101 ± 3% (n = 5); 0.2% EtOH: T type, 101 ± 4% (n = 5), HVA, 99 ± 2% (n = 5). DMSO effects on the T-type current were statistically significant, with P < 0.05.

U-92032 selectively inhibited T-type Ca2+ channels

We determined whether U-92032 was selective for T-type channels over other types of Ca2+ channels in hippocampal neurons. Figure 2 shows the effects of U-92032 on Ca2+ currents. T-type currents were sensitive to U-92032, with a median inhibiting concentration of ~500 nM. In contrast, the HVA current was much less sensitive. At 10 µM, ~75% of the HVA current was resistant to the drug. For T-type currents, the on rate of block varied from a blocking time constant of ~3 min at 500 nM to ~2 min at 10 µM. Block was completely reversible and the time course of recovery was independent of concentration, with an average unblocking time constant of ~5 min. Measurements of U-92032 inhibition were complicated by rundown, particularly for the HVA current. On average, the HVA current ran down to half of its control value in ~30 min. The amplitude of the control current was extrapolated to the time of the drug measurement (illustrated by - - - in Fig. 2B). The cell in Fig. 2 is the only cell in this study exposed to more than one application of the drug. The HVA currents in Fig. 2 showed the greatest block by 10 µM U-92032 of the six cells tested. In a preliminary set of experiments, Ca2+ currents in CA3 pyramidal neurons showed a similar sensitivity to U-92032 (n = 13 cells, varying from 1 to 50 µM).


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FIG. 2. U-92032 selectively inhibited T-type Ca2+ channels. A: effect of U-92032 on Ca2+ currents. Top traces: Ca2+ currents at -50 mV in the presence of baseline (control 1), 1 µM of drug, a 2nd baseline (control 2), and 10 µM of drug. Bottom traces: HVA currents during the same applications. B: time course of U-92032 effects for the cell shown in A. Top plot: amplitude of the T-type current over time. Time 0: onset of whole cell recording (break-in). Bottom plot: peak amplitude of the HVA current over time. For measurements, the amplitude of the control current was estimated by interpolating the baseline before and after the drug application (for example, - - - in 10 µM exposure). Arrows: timepoints for the control traces in A. C: dose response of Ca2+ currents. The amplitudes of the T-type current (bullet ) and the HVA current (black-square) are plotted as % of the control amplitude. Half-maximal inhibition of T-type currents occurred near 500 nM. HVA currents were much less sensitive, with ~75% of the current resistant at 10 µM. Error bars for the HVA points are obscured by the symbols. Values expressed as % of control (mean ± SE)---0.1 µM: T type, 76 ± 5% (n = 4), HVA, 97 ± 2% (n = 3); 0.5 µM: T type, 47 ± 4% (n = 8), HVA, 97 ± 1% (n = 6); 1 µM: T type, 31 ± 3% (n = 5), HVA, 89 ± 2% (n = 3); 5 µM: T type, 20 ± 3% (n = 3); 10 µM: T type, 6 ± 4% (n = 7), HVA, 76 ± 2% (n = 6).

U-92032 inhibited Na+ currents but not K+ currents

We next tested the effects of U-92032 on Na+ currents. Na+ currents were isolated with steps from -60 to 0 mV. As demonstrated in Fig. 3, U-92032 significantly reduced Na+ currents at concentrations of 1 and 10 µM. The block developed slowly (blocking time constant ~ 10 min at 10 µM) and was not reversible. Because of the slow kinetics and irreversibility of the inhibition, we arbitrarily quantified the block by measuring the peak amplitude of the Na+ current in control and after 10 min of exposure to the drug. Because the block had not reached steady state at this time, this measure underestimated the equilibrium block. We also attempted to estimate current rundown by measuring a separate group of cells 10 min into a mock application (Fig. 3C, control). It is clear that concentrations of U-92032 that inhibit T-type Ca2+ channels will strongly block Na+ channels as well.


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FIG. 3. U-92032 inhibited Na+ but not K+ currents. A: traces showing the effect of U-92032 on Na+ currents. Trace labeled 10 µM was taken 10 min into the drug application. B: time course of U-92032 effects for the cell shown in A. The peak amplitude of the Na+ current is plotted over time. U-92032 irreversibly reduced the current amplitude over several min. Magnitude of the block was quantified 10 min into the drug exposure (arrows). C: summary data of the dose response of Na+ currents. Height of bars: amplitude of the peak of the Na+ current expressed as % of the baseline value. Control bar: cells subjected to mock applications. U-92032 reduced Na+ currents in a dose-dependent manner. D: traces showing no effect of U-92032 on K+ currents. Cells were held at -80 mV and stepped to +40 mV. Arrows on left traces: points at which current amplitudes were measured. E: summary data of U-92032 effects on K+ currents. Bars: amplitude of the peak and sustained component of the current at +40 mV, expressed as % of their control values. K+ currents were insensitive at concentrations up to 10 µM. Values expressed as % of baseline (mean ± SE)---Na+ currents: control, 96 ± 7% (n = 7), 1 µM, 66 ± 9% (n = 7); 10 µM, 19 ± 5% (n = 4); K+ currents: 1 µM, peak 101 ± 2%, sustained 101 ± 5%, (n = 5), 10 µM, peak 103 ± 3%, sustained 101 ± 6%, (n = 4). U-92032 effects on Na+ currents were statistically significant, with P < 0.02.

In contrast to Ca2+ and Na+ channels, U-92032 did not affect voltage-gated K+ channels (Fig. 3D). As an inclusive screen for voltage-gated K+ currents, cells were held at -80 mV and stepped to +40 mV. To quantify the effect of the drug, we measured both the amplitude of the peak and the current remaining at 300 ms. Both were unaffected by U-92032 (Fig. 3E).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We consistently observed a reduction in T-type currents with concentrations of DMSO as low as 0.01%. This warrants caution when designing and interpreting experiments in which DMSO is used as a solvent.

Among Ca2+ channels, U-92032 proved quite selective for T-type channels, with a median inhibiting concentration of ~500 nM. With a potency >20-fold higher for T-type channels than other Ca2+ channels, U-92032 compares favorably with other T-type channel blockers. For example, Ni2+, the most commonly used T-type channel antagonist, showed a potency ~15-fold greater for T-type than HVA currents in similar experiments in hippocampal neurons (Avery and Johnston 1996). Our stimulation paradigm would have minimized activity-dependent block. In atrial myocytes, U-92032 blocks L-type channels more effectively with higher-frequency stimulation (Xu and Lee 1994). If this is also the case for neuronal L-type channels, high-frequency activity could compromise the selectivity of U-92032. Nevertheless, U-92032 should provide a useful experimental tool for probing the contributions of T-type Ca2+ channels to specific cellular functions.

U-92032 appears to be at least as potent at blocking Na+ channels as T-type Ca2+ channels. In contrast to Ca2+ currents, the inhibition of Na+ currents developed more slowly and was not reversible. Because we arbitrarily quantified block of Na+ currents after 10 min, we underestimated the steady-state block. It is possible that at equilibrium U-92032 has a greater potency for Na+ channels than Ca2+ channels. It is intriguing that several other T-type channel blockers also inhibit Na+ currents, including phenytoin (MacDonald and McLean 1982) and zonisamide (Suzuki et al. 1992). This may provide insight to the structure of T-type Ca2+ channels, which have so far eluded cloning. It is also interesting to note that the dual inhibitory actions of phenytoin have been postulated to underlie its effective anticonvulsant actions for epilepsies involving the hippocampus (Yaari et al. 1987).

    ACKNOWLEDGEMENTS

  We thank K. Gibson and R. Shebuski of Pharmacia and Upjohn for providing U-92032.

  This work was supported by National Institutes of Health Grants MH-10473 to R. Avery, and NS-11535, MH-44754, and MH-48432 to D. Johnston.

    FOOTNOTES

  Address for reprint requests: R. B. Avery, Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

  Received 30 August 1996; accepted in final form 23 October 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society