Introduction

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Riluzole, a novel neuroprotective drug with anticonvulsant, sedative and anti-ischemic properties (Malgouris et al., 1989; Stutzmann et al., 1991; Romettino et al., 1991; Pratt et al., 1992; Bryson et al., 1996), has been used to prolong the survival of patients with amyotrophic lateral sclerosis (Bensimon et al., 1994; Bryson et al., 1996; Lacomblez et al., 1996).

Ischemic damage is known to be caused by the neurotoxic effects of excitatory amino acids released as a result of hyperexcitation of presynaptic nerves (Choi and Rothman, 1990; Rothman, 1984; Meldrum, 1985; Rothman and Olney, 1986). The excess activation of the glutamatergic receptors, especially the NMDA receptors, causes a massive influx of Ca⁺⁺ through the open channels leading to cell death. Riluzole blocked kainate and NMDA receptors (Debono et al., 1993); this would lessen the cell damage caused by ischemia. Several lines of evidence suggested that riluzole inhibited synaptic transmission by reducing the release of glutamate from the presynaptic nerve terminals (Martin et al., 1993; Boireau et al., 1995; Rothstein and Kuncl, 1995).

The neuroprotective effect of riluzole is also known to rely in part on inhibition of sodium channels (Hebert et al., 1994; Benoit and Escande, 1991), because the inhibition of sodium channels was correlated with postischemic neuronal protection (Prenen et al., 1988; Boening et al., 1989; Yamasaki et al., 1991). Riluzole had a higher affinity for the inactivated state than the resting state of sodium channels, which resulted in a selective block of damaged or depolarized nerve thereby preventing excess stimulation of the glutamatergic receptors (Song et al., 1996).

Because transmitter release from the nerve terminals is mediated by voltage-gated calcium channels, neuroprotective drugs such as riluzole could also block these channels, thereby preventing excess release of transmitter. The present study was undertaken to test this hypothesis. It was indeed demonstrated that riluzole blocked the HVA calcium channels, with much higher affinities for N-type and P/Q-type calcium channels than L-type channels.

	Methods

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Cell culture

Electrophysiology. Ionic currents were recorded under voltage-clamp conditions by the whole-cell patch clamp technique (Hamill et al., 1981). Pipette electrodes were made from 1.5-mm (outside diameter) borosilicate glass capillary tubes and had a resistance of 2 megohm when filled with standard internal solution. The transmembrane voltage was clamped at 80 mV. Unless otherwise indicated, a period of 10 min was allowed after rupture of the membrane to ensure adequate equilibration between the internal pipette solution and the cell interior. Cells showing a rundown rate of calcium currents more than 1%/min were excluded. Membrane currents passing through the electrode were recorded with an Axopatch amplifier (Axopatch-1B, Axon Instruments, Foster City, CA), and currents were stored in an SX 386 computer (DELL Computer Company, Austin, TX) using pCLAMP 6 software (Axon Instruments).

Drug application. The recording chamber was perfused continuously with the normal external solution by gravity at a rate of 1 ml/min. Unless otherwise described, all the drugs were applied to the bath by gravity. The total volume of the chamber was only 1 ml facilitating the rate of application and washout of the drug. All experiments were carried out at a room temperature of 20-23°C.

Data analysis. The dose-response relationship of riluzole inhibition of the transient and sustained calcium channel currents was fitted to a sigmoid curve as calculated by the four-parametric logistic function equation (Tandel Scientific, Corte Madera, CA):

(1)

where I_p represents the inhibition of calcium channel currents recorded at riluzole concentration C, and I_max is the maximal inhibition (%) of calcium channel currents. K_d and n denote the half-maximal effective concentration of riluzole and the Hill coefficient, respectively.

	Results

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Time- and Dose-Dependent Inhibition of HVA Ca⁺⁺ Channel Currents by Riluzole

With 10 mM barium as charge carrier, the T-type calcium channel current was activated by a test pulse to 40 mV from a holding potential of 80 mV. The current had fast inactivation kinetics and was found in only 15% of the cells tested. A test pulse to 20 mV from the same holding potential activated the HVA calcium channel currents which reached the maximal amplitude at a test pulse to 0 mV or 10 mV. Cells generating T-type calcium channel currents were discarded to avoid the contamination of HVA calcium channel currents.

Riluzole was applied to the bath while HVA calcium channel currents were evoked by 200-msec test pulses from 80 mV to +20 mV at 20-sec intervals. Riluzole at 300 µM inhibited the calcium channel currents, and the effect reached the maximum taking more than 3 min after bath perfusion (fig. 1). Both peak and steady-state amplitudes were suppressed. After washout, the riluzole inhibitory effect persisted for a while, and a complete recovery usually took more than 3 min. In 20% of the cells studied, a rebound to 118% of the control current amplitude was observed after washout (fig. 1). The slow onset and offset of the riluzole inhibitory effect and the rebound phenomenon suggest that intracellular components may be involved in the riluzole action.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Riluzole inhibition of the HVA calcium channel current. Currents are evoked by a step pulse to +20 mV from a holding potential of 80 mV at an interval of 20 sec. (A) Current traces a to e correspond to currents at times a to e in panel B. (B) Riluzole 300 µM inhibits the HVA channel current with a slow onset and offset. A rebound of the current amplitude occurs after washout of riluzole.

To study the dose-response relationship for the riluzole block of the HVA calcium channels, different concentrations of riluzole were perfused cumulatively. Riluzole inhibited both the peak current and sustained currents measured at the end of 200 msec pulse in a dose-dependent manner, and a complete block occurred at 300 µM riluzole. Using equation 1, the dose-response relationships for riluzole inhibition of peak and sustained calcium channel currents were fitted to sigmoid curves, which yielded apparent K_d values of 42.6 ± 7.4 µM (n = 10-16) and 39.5 ± 17.26 µM (n = 7-16), and the Hill coefficients of 1.1 ± 0.160 and 0.83 ± 0.212, respectively (fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   The dose-response relationship for riluzole inhibition of the peak transient () and sustained () components of HVA calcium currents with apparent Kd values of 42.6 ± 7.4 µM (n = 10-16) and 39.5 ± 17.26 µM (n = 7-16), respectively.

Effects of Riluzole on the Kinetics of HVA Calcium Channels

Activation. To study the effect of riluzole on the activation of calcium channel currents, current-voltage (I-V) relationships for the transient calcium channel currents were plotted by 5-mV incremental step pulses from 80 mV to +75 mV in the absence and presence of riluzole (fig. 3A). Because the I-V relationship was nonlinear in the positive potential range, the constant field theory (Goldman, 1943; Hodgkin and Katz, 1949) was used to calculate the maximal current amplitude at its full activation (Ohmori and Yoshii, 1977, Yoshii et al., 1988). The constant field equation for divalent cations is:

(2)

where I_Ba denotes the calcium channel current with barium as the charge carrier, F is the Faraday constant, E_m is the membrane potential, R is the gas constant, T is the absolute temperature, P_Ba is the permeability of barium and [Ba⁺⁺]_o is the external barium concentration. By normalizing the observed current amplitudes to the calculated maximal amplitude and by fitting to equation 2, a steady-state activation curve is plotted as a function of membrane potential (fig. 3B). The midpoint potentials for the activation curves were 1.6 ± 0.34 mV, 2.7 ± 0.23 mV and 1.2 ± 0.21 mV in the absence of riluzole (, n = 7), and in the presence of 30 µM (, n = 7) and 100 µM (, n = 3) riluzole, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of riluzole on the activation of HVA channels. (A) Current-voltage relationships for HVA channels in the absence () and presence of 30 µM () and 100 µM () riluzole. (B) The voltage dependence of activation curve for HVA channels is fitted by the constant field equation (see text). Riluzole at 30 and 100 µM reduces the mid-point potential from the control value of 1.6 ± 0.34 mV to 2.7 ± 0.23 mV (n = 7) and 1.2 ± 0.21 mV, respectively (P > .05). (C) The time to peak of HVA channel currents in the presence and absence of riluzole is measured as shown in the inset, and the data are plotted as a function of riluzole concentration. (D) The time to peak for HVA channel currents in the absence () or presence () of 30 µM riluzole is plotted against the step pulse potential. Riluzole reduction of the time to peak for HVA channel currents is not voltage dependent (n = 5).

Inactivation. To study the kinetics of channel inactivation, calcium channel currents were generated at a test pulse to +20 mV preceded by a 5-sec conditioning prepulse to various potentials and were normalized to the current associated with 100 mV prepulse. Plot of the normalized current amplitude against the prepulse potential yielded a steady-state inactivation curve for the calcium channel, which could be fitted by two exponential functions, which indicates the existence of at least two groups of HVA calcium channels with distinct inactivation kinetics (fig. 4A). Riluzole at 30 µM shifted the half-inactivation potentials for one group of calcium channels from 48.2 ± 1.31 mV to 55.4 ± 1.70 mV (P < .05), and for the other group from 8.1 ± 1.98 mV to 9.0 ± 2.53 mV (P > .05) (n = 7). The slope factor was changed by riluzole from 9.6 ± 0.81 mV to 11.6 ± 1.29 mV for the former (P < .05), and from 7.6 ± 1.09 mV to 7.91 ± 1.49 mV for the latter (P > .05) (n = 7). Thus, riluzole selectively shifted the steady-state inactivation curve for one group of HVA calcium channels in the hyperpolarizing direction.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   (A) The steady-state inactivation curve for HVA channels in the absence () and presence () of 30 µM riluzole showing two components. Riluzole shifts the half-inactivation potential from 48.2 ± 1.31 mV to 55.4 ± 1.7 mV for one component (P < .05), and from 8.1 ± 1.98 mV to 9.0 ± 2.53 mV for the other component (P > .05) (n = 7). (B) Riluzole accelerates the inactivation kinetics. The time course of inactivation of HVA channel currents generated by step pulses from 80 mV to +20 mV for a duration of 300 msec can be fitted to a double-exponential curve. The time constants of the HVA channel inactivation are reduced by rulizole to 47.5 ± 5.49% and 41.4 ± 5.71% of control for the fast and slow inactivation, respectively.

Deactivation. The tail current evoked upon step hyperpolarization of the membrane after a depolarizing test pulse reflects the fraction of calcium channels remaining open at the end of the test pulse. The decay component of this tail current represents the deactivation or closure of the activation gate of the channel. With an increase in the duration of depolarizing pulse, the tail current amplitude reached a maximum and gradually decreased (fig. 5, A and B). To ensure the full activation of the channel and to minimize the effect of inactivation, the subsequent tail current experiments were performed with a 10-msec test pulse. The rate of decay of the tail current varied with the level of repolarization after a depolarizing pulse to a fixed potential level (fig. 5Ca), and could be fitted by a double-exponential curve (fig. 5Cb). Figure 5D plots the fast time constant against various repolarizing potentials after the same depolarizing pulse in the presence and absence of 30 µM riluzole. Riluzole decreased the fast time constant (fig. 5D), but not the slow time constant (data not shown) for the decay of tail current. At repolarizing potentials more positive then 40 mV, riluzole clearly reduced the fast time constant (n = 7, P < .05), whereas at the more negative repolarizing potentials, this effect was less pronounced because of the fast deactivation kinetics. As shown in figure 5E, the deactivation kinetics did not change significantly by varying the depolarizing potential preceding the same repolarizing potential (, n = 5). However, riluzole reduction of the fast time constant for the decay of tail current was absent if preceded by large positive depolarizing test pulses (, n = 5). Thus, riluzole accelerates the deactivation kinetics of HVA calcium channels by reducing the fast time constant for the decay phase in a voltage-dependent manner.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of riluzole on the deactivation of HVA channels. (A) The tail current amplitude is time dependent. The currents are evoked by step pulses from 80 mV to +20 mV with 1- to 14-ms durations. (B) Normalized tail current amplitude as a function of pulse duration. (C) HVA channel currents are generated by the same test pulse which is followed by repolarization to various potentials. Decay of the calcium tail current is slowed with repolarization to less negative potentials (Ca) and can be fitted to a double-exponential curve (Cb). (D) Riluzole at 30 µM reduces the fast time constant for the decay of HVA channel tail current, and the effect is more pronounced at more positive repolarizing potentials. (E) HVA channel currents are evoked by the protocol shown in the inset. Plot of the fast time constant for the decay of HVA tail current against the test pulse potential. Riluzole reduces the fast time constant in a voltage-dependent manner.

Pharmacological Dissection of HVA Calcium Channels

Effects of dihydropyridines. Nimodipine was used as a dihydropyridine antagonist to block the L-type calcium channel current because of its less voltage-dependent action. Nimodipine at 10 µM suppressed HVA channel currents reversibly (fig. 6A). Bay K 8644 (5 µM), a dihydropyridine agonist known to prolong the opening of dihydropyridine-sensitive calcium channels (Fox et al., 1987; Nowycky et al., 1985), potentiated HVA calcium channel currents (fig. 6, A and C). Current traces a to d in figure 6B correspond to the currents at times a to d in figure 6A. Subtracting the current in nimodipine (trace b) from the control current (trace a) yields the L-type current blocked by 10 µM nimodipine, whereas subtracting current trace c from current trace d yields the L-type current in the present of 5 µM Bay K 8644 (fig. 6B2). Thus, 5 µM Bay K 8644 potentiated the L-type current 3.1 ± 0.53 times (n = 5). Note that the decay of tail current was prolonged by Bay K 8644 (fig. 6B2). To test if 10 µM nimodipine can completely block the L-type current, 5 µM Bay K 8644 was applied in the absence and presence of 10 µM nimodipine (fig. 6C). Bay K 8644 potentiation of the L-type current was observed in the control and was completely abolished in the presence of 10 µM nimodipine. Thus, nimodipine, at a concentration of 10 µM, was sufficient to block all the L-type current.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of dihydropyridines on HVA channel currents. (A) Nimodipine at 10 µM inhibits the HVA channel current and Bay K 8644 at 5 µM potentiates it. (B) Current traces a to d were recorded at times a to d of panel A. (C) Nimodipine at 10 µM inhibits 5 µM Bay K 8644 potentiation of HVA channel currents. Thus, 10 µM nimodipine inhibits the dihydropyridine-sensitive L-type current completely.

Various types of calcium channels. -Aga is known to block the P-type calcium channel (Mintz et al., 1992a, b; Mintz and Bean, 1993). It also blocks the Q-type channel in certain preparations (Randall et al., 1993; Sather et al., 1993; Wheeler et al., 1994; Randall and Tsien, 1995; Rusin and Moises, 1995). -CTx blocks the N-type calcium channel selectively (McCleskey et al., 1987). The residual current insensitive to dihydropyridine antagonists, -Aga and -CTx, is designated as the R-type current, which can be blocked by the nonspecific calcium channel blocker CdCl₂ (Ellinor et al., 1993; Zhang et al., 1993; Randall and Tsien, 1995). To separate various types of calcium channels present in the same cell, 10 µM nimodipine, 1 µM -CTx, 300 nM -Aga and 150 µM CdCl₂ were applied to the bath in sequence (fig. 7A). Current traces a to e in figure 7B represent the currents indicated at times a to e in figure 7A. Similar to figure 6, L-, N-, P- and R-type calcium channel currents were isolated by subtracting the current after application of respective blocker from that before application (fig. 7B). Thus, the precentages of L-, N-, P/Q- and R-type calcium channel currents out of the total current are 10.5 ± 1.10, 62.8 ± 2.35, 22.8 ± 1.81 and 9.1 ± 1.62%, respectively (n = 4-20). To test whether -CTx and -Aga block part of the L-type current, 10 µM nimodipine was applied in the presence of 1 µM -CTx and 300 nM -Aga (fig. 7C). Nimodipine was able to block a small portion of the HVA calcium channel current still remaining in the presence of -CTx and -Aga. Thus -CTx and -Aga do not seem to block the L-type current.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   (A) Sequential applications of various calcium channel blockers including 10 µM nimodipine, 1 µM -CTx, 300 nM -Aga and 150 µM CdCl2. (B) Currents subtracted before and after the administration of respective HVA channel blockers yield L-, N-, P/Q- and R-type currents. (C) Nimodipine 10 µM, applied after 1 µM -GTx and 300 nM -Aga, still blocks the HVA channel current. L-, N-, P/Q- and R-type currents represent 10.5 ± 1.10%, 62.8 ± 2.35%, 22.8 ± 1.81%, and 9.1 ± 1.62% of the total current, respectively (n = 4-20).

Riluzole Inhibition of Various Types of HVA Calcium Channel Currents

Because riluzole affects only one of the two groups contained in the steady-state inactivation curve (fig. 4A), riluzole may have selective actions on some of the HVA calcium channels. To prove this hypothesis, riluzole inhibition of HVA calcium channel currents was studied in the presence of various calcium channel blockers. As shown in figure 8, 30 µM riluzole blocked HVA calcium channel currents by 17.1 ± 0.83%, 17.1 ± 1.49%, 3.9 ± 0.58% and 9.3 ± 1.45% in the control (n = 30), in the presence of 10 µM nimodipine (n = 11), 1 µM -CTx (n = 11) and 300 nM -Aga (n = 10), respectively. Thus, -CTx-GIVA and -Aga-IVA, but not nimodipine, partially block riluzole inhibition of HVA calcium channel currents.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Riluzole inhibition of various HVA channel currents. Riluzole at 30 µM inhibits 17.1 ± 0.83, 17.1 ± 1.49, 3.9 ± 0.58 and 9.3 ± 1.45% in the control (n = 30), in the presence of 10 µM nimodipine (n = 11), 1 µM -CTx (n = 11), and 300 nM -Aga (n = 10), respectively.

To determine the efficacy of riluzole on various HVA calcium channels, riluzole inhibitory effect was studied further in the absence and presence of different calcium channel blockers in the same cell. Nimodipine did not affect riluzole inhibition of HVA calcium channel currents (fig. 9A), which indicates that the L-type channel is not the target of riluzole. However, riluzole reduced the fast time constant for the decay of tail current in Bay K 8644-modified calcium channel from 0.33 ± 0.039 ms to 0.25 ± 0.034 ms (n = 4, fig. 9, B and C). Thus, riluzole does not affect the native L-type current, but accelerates the deactivation of the Bay K 8644-modified L-type calcium channel.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Riluzole inhibitory effect on the HVA channel current is tested in the presence and absence of dihydropyridines in the same cell. (A) Riluzole 30 µM inhibition of HVA channel currents is not affected by 10 µM nimodipine. (B) Riluzole inhibitory effect on HVA channel currents in the presence of 5 µM Bay K 8644. (C) Riluzole still accelerates the decay of the Bay K 8644-modified HVA channel tail current. Thus, 30 µM riluzole does not affect unmodified L-type current, but accelerates the decay of Bay K 8644-modified L-type tail current.

Figure 10A shows the riluzole inhibitory effect on the HVA calcium channel current in the presence and absence of 1 µM -CTx in a representative cell. Current traces a to d in figure 10B represent the currents at times a to d in figure 10A. Current trace a minus current trace b (a  b), 13.42% of the total HVA current, represents the portion of the total HVA calcium channel current blocked by 30 µM riluzole. Because -CTx blocks the N-type current, current trace c minus current d (c  d), 4.6% of the total HVA calcium channel current, equals the portion of the current other than the N-type current blocked by 30 µM riluzole. Thus, (a  b) minus (c  d), 8.8%, equals the N-type current that was blocked by riluzole. The total N-type current equals (a  c) 62.8%. Therefore, the percentage of the total N-type current blocked by 30 µM riluzole is 14.2% (8.8  62.8) in this cell. Through similar calculation, 30 µM riluzole blocks 1.7 ± 2.88%, 20 ± 1.84%, 32.5 ± 2.35% of the total L- (n = 10), N- (n = 12) and P/Q (n = 10)-type currents, respectively (fig. 10C). Thus, 30 µM riluzole blocks P/Q- and N-type calcium currents selectively.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   (A) Riluzole at 30 µM inhibits the current to a lesser extent in the presence of 1 µM -CTx than in the absence. (B) Current traces a to d were recorded to at times a to d in A. (C) Riluzole at 30 µM inhibits L-, N- and P/Q-type currents 1.7 ± 2.88%, 20 ± 1.84% and 32.5 ± 2.35%, respectively.

	Discussion

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The HVA channels in neonatal rat DRG neurons contained various types with relative fractions of N-  P/Q- > L- ~R-type. Riluzole inhibited these channel types with relative efficacies of P/Q- > N-  L-type.

HVA calcium channels in rat DRG neurons. There are at least four pharmacologically distinguishable HVA calcium channels in rat DRG neurons, i.e., L-, N-, P/Q- and R-type. Without separating the Q-type current from the P-type current, our results show that 62.8% of the HVA channel currents are a -CTx-sensitive N-type current, 22.8% are a -Aga-sensitive P/Q-type current, and 10.5% are a dihydropyridine-sensitive L-type current. The remaining 9.1% of the HVA channel currents are insensitive to the these blockers but sensitive to Cd⁺⁺; they are termed as R-type currents.

Differential block of HVA channel currents by riluzole. Riluzole differentially inhibits HVA channel currents. First, riluzole affects only one of the two components of steady-state inactivation curve for the HVA channel current (fig. 4). Second, -CTx and -Aga, but not nimodipine, partially abolish riluzole suppression of HVA channel currents, which indicates the involvement of N- and P/Q-type currents and the exclusion of L-type current in riluzole's inhibitory effect (fig. 8). Third, the inhibitory action of riluzole on the various types of HVA channel currents is in the order of P/Q- > N-  L-type currents (fig. 10C). Because P- and Q-type currents are different in the inactivation kinetics and the sensitivity to -Aga (Randall et al., 1993; Randall and Tsien, 1995; Rusin and Moises, 1995), riluzole may not block P- and Q-type currents equally.

Mechanism of riluzole block of HVA calcium channel currents. The kinetic values of deactivation and inactivation of HVA channel currents are altered by riluzole, but the activation kinetics remains unchanged. Riluzole selectively blocks the sodium channel in the inactivated state without affecting the activation kinetics (Hebert et al., 1994; Benoit and Escande, 1991; Song et al., 1996). However, there are differences between sodium and calcium block by riluzole. Riluzole blocks sodium channels rapidly and the effect is reversed rapidly after washout, which suggests a direct action on the channel (Benoit and Escande, 1991; Song et al., 1996). In contrast, the slow time course of the onset and offset of riluzole inhibitory effect on HVA channel currents suggests an indirect mechanism involving riluzole interactions with some intracellular components. This idea is further supported by the rebound phenomenon after washout of riluzole, reminiscent of the rebound of baclofen effect on the calcium channel (Matsushima et al., 1993).