Introduction

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Lubeluzole, the (+)-S-enantiomer of a benzothiazole derivative (Fig. 1), has a neuroprotective action in animal models of focal and global ischemia, in which it reduces sensorimotor deficits and the infarct volume (for review, see De Ryck, 1997). Experiments on neuronal cultures have shown that lubeluzole inhibits glutamate-induced nitric oxide-related neurotoxicity and that it blocks neurotoxicity induced by nitric oxide donors (Lesage et al., 1996; Maiese et al., 1997). Although a phase II clinical study seemed promising, a clear neuroprotective effect could not be demonstrated in phase III studies in patients with acute ischemic stroke (Diener, 1998).

Lubeluzole also affects ion channels. It blocks the fast sodium channel and antagonizes veratridine-induced neurotoxicity (Osikowska-Evers et al., 1995; Ashton et al., 1997). Lubeluzole blocks the transient low-voltage-activated Ca²⁺ channel current (iLVA or T-current) (Marrannes et al., 1998b) and the high-voltage-activated Ca²⁺ channel current (iHVA) in a concentration-, voltage-, and frequency-dependent manner (Hernández-Guijo et al., 1997; Marrannes et al., 1998b). The time needed for the block of Ca²⁺ channels to reach steady state suggests that lubeluzole penetrates the cell or cell membrane, or that another slow intracellular process is implied.

In the present study, we investigated the site of action of lubeluzole on Ca²⁺ channels. The question was addressed whether lubeluzole blocks Ca²⁺ channels from the extracellular or intracellular side and whether the protonated form of lubeluzole (HL⁺) or its uncharged basic form (L) produces the effects. To answer these questions, two methods were used and compared: 1) variation of the concentration of HL⁺ and L at both sides of the cell membrane, by testing the effect of lubeluzole (pK_a = 7.6) at different values of extracellular pH (pH_o) and intracellular pH (pH_i); and 2) application of lubeluzole and a permanently charged methyl iodide quaternary derivative of lubeluzole (R133121) (Fig. 1) via the extracellular solution or via the patch pipette.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structure of lubeluzole [(+)-(S)-4-[(2-benzothiazolyl)methylamino]--[(3,4-difluorophenoxy)methyl]-1-piperidineethanol] and its methyl iodide quaternary derivative R133121 (TRANS, S) 4-[(2-benzothiazolyl)methylamino]--[(3,4-difluorophenoxy)methyl]-1-methyl-1-piperidineethanol] iodide.

Variation of pH_o and pH_i by itself also influences Ca²⁺ channels. The activation and inactivation curves of iHVA shift positively after a reduction in pH_o (Krafte and Kass, 1988; Tombaugh and Somjen, 1996; Zhou and Jones, 1996) but negatively after a reduction in pH_i (Kaibara and Kameyama, 1988; Tombaugh and Somjen, 1997), as a consequence of a change in surface charges (Hille, 1992). In addition, protons can influence the channel conductance by other mechanisms (Iijima et al., 1986; Krafte and Kass, 1988; Prod'Hom et al., 1989; Zhou and Jones, 1996) and iHVA is blocked by a reduction in pH_o (Tombaugh and Somjen, 1996; Zhou and Jones, 1996) or pH_i (Kaibara and Kameyama, 1988; Tombaugh and Somjen, 1997) and iHVA increases after an alkaline change in pH_o and/or pH_i. Although iLVA is very sensitive to pH_o and decreases when pH_o falls, it was found to be insensitive to a change in pH_i (Tytgat et al., 1990; Tombaugh and Somjen, 1997). The study of the influence of pH_o and pH_i on the block of iLVA and iHVA by lubeluzole is also relevant to estimating the effect of a compound such as lubeluzole in ischemic situations, which are accompanied by changes in pH_o and pH_i (Lipton, 1999). Part of this study has been reported in abstract form (Marrannes et al., 1998a).

	Materials and Methods

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Cell Preparation. DRG neurons were isolated from male Wistar rats (250 g) as described previously (Marrannes et al., 1998b), with some modifications. Briefly, DRGs were digested at 37°C in 1 ml of 0.5% collagenase medium for 30 min, to which 1 ml of 0.25% trypsin medium was then added, and digestion was allowed to continue for a further 30 min. The collagenase medium contained 0.5% collagenase (Boehringer Mannheim, Mannheim, Germany), and 120 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 3 mM D-glucose, 0.1 mM CaCl₂, 19.7 mM NaHCO₃, and 31.4 mM HEPES at pH 8. The trypsin medium contained 0.25% trypsin (Life Technologies, Merelbeke, Belgium), and 136.7 mM NaCl, 2.7 mM KCl, 16.3 mM Na₂HPO₄, and 1.5 mM KH₂PO₄. After washing and trituration of the dorsal root ganglia, purification of the cells in a Percoll gradient and removal of nonneuronal cells, the DRG cells were seeded in the center of Petri dishes that had previously been coated with poly(L-lysine) at 100 µg/ml (1 h) and then with laminin at 10 µg/ml (4 h). The cells were incubated in DMEM-fetal calf serum at 37°C in a humidified atmosphere (95% air, 5% CO₂) for 4 h. DMEM-fetal calf serum is DMEM to which 10% fetal calf serum and 6 g/l D-glucose were added, together with 0.3 mM L-cysteine, 0.4 mM L-alanine, 0.4 mM L-asparagine, 0.4 mM L-aspartic acid, 0.4 mM L-proline, and 0.4 mM L-glutamic acid as amino acid supplements. Then 4 ml of the HEPES-buffered solution used to perfuse the experimental chamber (see below) was added to the medium of each Petri dish, and the cells were stored at 4°C to retard the decline of iLVA. The cells were used for experiments on the day of isolation and the following day. Except when a different cell size is specified in the text, we used medium-sized DRG cells (35-40 µm) having a large LVA Ca²⁺ channel current (Scroggs and Fox, 1992).

Electrophysiological Recording. Whole-cell voltage clamp (Hamill et al., 1981) was performed as described previously (Marrannes et al., 1998b). The electrode resistance ranged from 1.5 to 2.5 M when measured in the bath. After the patch had been broken, the cells were allowed to equilibrate with the contents of the electrode for 5 min before stimulation, except in the experiments in which lubeluzole, R133121, or the corresponding amount of DMSO was applied via the microelectrode.

Solutions. Lubeluzole and R133121 (both from Janssen Pharmaceutica, Beerse, Belgium) were prepared in 10 mM stock solutions in DMSO. The concentration of DMSO in the control solutions was always the same as in the corresponding solutions with one of these compounds.

Variation of Extracellular and Intracellular pH to Change the Extracellular and Intracellular Concentration of the Protonated and Basic Form of Lubeluzole. Lubeluzole is a weak base (pK_a = 7.6). The fraction of the extracellular lubeluzole concentration in the HL⁺ and in the L form can be calculated as follows:

(1)

(2)

where [T]_o is the total extracellular lubeluzole concentration ([T]_o = [HL⁺] _o + [L] _o), K_a is the dissociation constant of HL⁺, and [H⁺]_o is the extracellular proton concentration. Part of L that penetrates the cell membrane is protonated in the cell to HL⁺ as a function of its K_a and pH_i. If HL⁺ does not cross the cell membrane and L diffuses only passively through the cell membrane and if lubeluzole does not disappear rapidly via the electrode or metabolism, one can assume that in steady state the intracellular concentration of L ([L]_i) will approach [L]_o or the following:

(3)

Because

(4)

combining eqs. 2, 3, and 4 yields the following:

(5)

This is valid on the condition that eq. 3 is true. The calculated values for [HL⁺]_o/[T]_o, [L]_o/[T]_o, and [HL⁺]_i/[T]_o at different values of pH_o and pH_i are shown in Table 1.

Statistical Analysis. The data are expressed as mean ± S.D., except in Figs. 6 and 8, where S.E. is used. The difference between groups was evaluated by means of the two-sided Student's t test for independent samples. The difference between the values of the inactivation parameters before and after application of lubeluzole was evaluated with a two-sided paired t test (Fig. 6). The dependence of a measured variable on pH_i was evaluated by means of a two-sided Jonckheere-Terpstra test for ordered alternatives. Values of P < .05 were considered to indicate statistical significance.

	Results

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Extracellular Application of Lubeluzole. Figure 2 illustrates the effects of 3 µM lubeluzole at pH_o 7.4, with a pipette solution of pH_i 7.2. First the activation of iHVA was investigated in the control solution (Fig. 2A). Thereafter, a constant pulse sequence with test pulses to 50 and 20 mV was given every 30 s to activate iLVA and iHVA, respectively, and 3 µM lubeluzole was applied via the extracellular solution (Fig. 2B). Lubeluzole induced a rapid decrease of iLVA and iHVA, followed by a gradual further decrease for at least 5 min. Thereafter, the activation of iHVA was tested again (Fig. 2C). At 30 mV lubeluzole accelerated the apparent inactivation of iHVA and this was even more pronounced at 20 mV. The observation that lubeluzole accelerates the apparent inactivation of iHVA more at test potentials at which iHVA is more activated is consistent with an open channel block of iHVA by lubeluzole. Lubeluzole did not decrease the time constant of inactivation of iLVA, in contrast to that of iHVA.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Influence of extracellular application of lubeluzole at pHo 7.4 and pHi 7.2. A, activation of iHVA in the control solution. From a 100-mV HP, a 200-ms test pulse to 50 mV was given to elicit and inactivate iLVA nearly completely. Thereafter, 155-ms test pulses varying from 50 to +10 mV were given to activate iHVA. The sweep interval was 20 s. B, transition from the control solution to 3 µM lubeluzole. Every 30 s the shown pulse sequence was given. The last sweep in the control solution is shown together with the first 10 sweeps in the presence of 3 µM lubeluzole. Lubeluzole was applied immediately after the last sweep in the control solution. C, activation of iHVA after 5-min application of 3 µM lubeluzole. D, I-V relationships of iHVA derived from the sweeps in A and C. Filled symbols: peak inward current. Unfilled symbols: current at the end of the 155-ms test pulse.

Influence of pH_o on the Block of iLVA and iHVA by Lubeluzole. To see mainly the contribution of extracellular HL⁺ to the block of iLVA and iHVA and to a lesser extent the contribution of L and intracellular HL⁺, the influence of lubeluzole was tested at pH_o 6 (Table 1). A decrease in pH_o reduced iLVA and iHVA (Fig. 3A). At 30 s after extracellular application of 3 µM lubeluzole at constant pH_o 6 there was a clear inhibition of iLVA and a much smaller relative block of iHVA, which changed only little thereafter (n = 5) (Fig. 3B). When lubeluzole was superfused only 15 s before the next pulse sequence (given every 30 s), there was already some block of iLVA and iHVA but this block was clearly more extensive 30 s later, after which it was approximately stable (n = 5) (Fig. 3C). The equilibration time of the effect of lubeluzole at pH_o 6 was shorter than that at pH_o 7.4 (Fig. 2B), probably because it reflects the rapid equilibration of the extracellular effect of HL⁺ on iLVA and iHVA, given the lower [L] and reduced penetration of L and intracellular accumulation of HL⁺ at pH_o 6. It is remarkable that 3 µM lubeluzole accelerated either very little or not at all the apparent inactivation of iHVA at pH_o 6, which indicates that extracellular HL⁺ is not sufficient to produce this acceleration. A large delayed acid-induced inward current (Waldmann et al., 1999), starting 2.1 ± 2.3 min (mean ± S.D., n = 29) after the decrease in pH_o, terminated the experiment prematurely in most cells at pH_o 6. 

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Influence of pHo on the effects of lubeluzole on iLVA and iHVA. A, sweeps recorded at pHo 7.4 and 30 s after switching to pHo 6 in the absence of lubeluzole. B, block of iLVA and iHVA by 3 µM lubeluzole at constant pHo 6. Lubeluzole was applied immediately after the sweep in the control solution. Sweeps are shown every 30 s until 3.5 min after application of lubeluzole. Same cell as in A. Note the difference in scale. C, in another cell 3 µM lubeluzole was given 15 s after the control sweep and 15 s before the next sweep. Sweeps are shown in the control solution and 15, 45, and 75 s after application of lubeluzole. D, sweeps recorded at pHo 7.4 and 30 s after switching to pHo 9 in the absence of lubeluzole. Same cell as in E, F, and H. E, at constant pHo 9 the block of iLVA and iHVA by 3 µM lubeluzole was more progressive than at pHo 6 (compare E with B). F, sweeps are shown after 5-min equilibration in 3 µM lubeluzole at pHo 9 and 30 s after pHo was lowered to 7.4 while the external lubeluzole concentration was kept constant at 3 µM. G, time course of the experiment shown in B. ILVA is quantified as the difference between the peak inward current and the current at the end of the test pulse to elicit iLVA (at 40 mV); iHVApeak and iHVAend are the peak inward current and the current at the end of the test pulse to elicit iHVA, respectively. H, time course of the experiment shown in D to F.

Influence of Intracellular pH on the Block of iHVA by Lubeluzole. To distinguish the contribution of intracellular HL⁺ from that of L, the effect of lubeluzole was tested at different values of pH_i, but at a constant pH_o of 7.4. Figure 4 shows the effect of extracellular application of 3 µM lubeluzole (pH_o 7.4) with a pipette solution of pH_i 9. At pH_i 9 the intracellular equilibrium concentration of HL⁺ is expected to be 65 times lower than at pH_i 7.2, whereas [HL⁺]_o, [L]_o, and [L]_i are unchanged (Table 1). If the acceleration of the apparent inactivation of iHVA by lubeluzole is due to an effect of HL⁺ from the intracellular side, then this acceleration is predicted to be much less at pH_i 9 than at pH_i 7.2, which indeed was observed (compare Figs. 2 and 4). At pH_i 6 [HL⁺]_i is expected to be 15.8 times higher than at pH_i 7.2 (Table 1). Accordingly, at pH_i 6 lubeluzole induced a much faster voltage-dependent acceleration of the apparent inactivation of iHVA than at pH_i 7.2 (compare Figs. 2 and 5). The block of iHVA and the acceleration of the apparent inactivation of iHVA by lubeluzole are thus strongly dependent on pH_i and much more pronounced at lower pH_i, at which [HL⁺]_i would be expected to be higher.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Influence of 3 µM lubeluzole on the activation of iHVA at pHi 9 (and pHo 7.4). A, activation of iHVA in the control solution. B, transition from the control solution to 3 µM lubeluzole. Every 30 s the shown pulse sequence was given. The last sweep in the control solution is shown together with the first 10 sweeps in the presence of 3 µM lubeluzole. C, activation of iHVA after 5-min application of lubeluzole. D, I-V relationships of iHVA derived from the sweeps in A and C. Filled symbols: peak inward current. Unfilled symbols: current at the end of the 155-ms test pulse.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Influence of 3 µM lubeluzole at pHi 6 (and pHo 7.4). A, activation of iHVA in the control solution. B, transition from the control solution to 3 µM lubeluzole. Every 30 s the shown pulse sequence was given. The last sweep in the control solution is shown together with the first 10 sweeps in the presence of 3 µM lubeluzole. C, activation of iHVA after 5-min application of lubeluzole. D, I-V relationships of iHVA derived from the sweeps in A and C. Filled symbols: peak inward current. Unfilled symbols: current at the end of the 155-ms test pulse.

Influence of pH_i on the Inactivation of iLVA and Block of iLVA by Lubeluzole. At constant pH_o 7.4, the block of iLVA by lubeluzole was smaller at pH_i 9 than at pH_i 7.2 and even more extensive at pH_i 6 (Figs. 2B, 4B, 5B, and 8). This might be due to an intracellular contribution of HL⁺ to the inhibition of iLVA. Alternatively, this could be partly a consequence of possible pH_i-dependent shifts of the inactivation curve of iLVA. The inactivation of iLVA was therefore studied at different values of pH_i, in the absence and presence of 1 µM lubeluzole (Fig. 6). In the absence of lubeluzole, the inactivation curve was clearly shifted to the left by intracellular acidosis and slightly shifted to the right by intracellular alkalosis (Fig. 6A).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Influence of pHi and lubeluzole on the inactivation of iLVA. The 10-s conditioning prepulse was varied from 60 to 120 mV, with the 155-ms test pulse maintained at 50 mV. For each test solution, the curve of the amplitudes of iLVA (I) was fitted as a function of the potential (V) of the conditioning prepulse in accordance with the Boltzmann equation I = Imax/(1 + exp((V  Vh)/Sl)), which yielded the parameters Imax, Vh, and Sl, where Imax is the amplitude of iLVA in the absence of inactivation, Vh is the conditioning potential at which the inactivation was half-maximal, and Sl is the slope factor. The iLVA values measured and the fitted curves were normalized by division by the individual Imax. The results are presented as mean ± S.E. A, influence of pHi on the inactivation of iLVA, measured 10 min after the patch was broken in the absence of lubeluzole. Vh (mV), Sl (mV), and the current density of Imax (pA/pF) were 91.7 ± 1.1, 5.0 ± 0.1, and 77 ± 13, respectively, at pHi 6 (n = 6); 87.6 ± 0.9, 4.8 ± 0.1, and 117 ± 9 at pHi 6.6 (n = 16); 82.3 ± 1.1, 5.1 ± 0.2, and 87 ± 8 at pHi 7.2 (n = 13); and 77.1 ± 1.2, 4.0 ± 0.1, and 118 ± 20 at pHi 9 (n = 6). These findings show that the Vh of iLVA was more negative at a lower pHi (P < .0001). B, the ratio of Imax after 5 min of extracellular application of 1 µM lubeluzole to Imax in the control solution is shown as a function of pHi. C, influence of 1 µM lubeluzole on the inactivation gating of iLVA at pHi 6, 6.6, 7.2, and 9. The curves are normalized by division by Imax. At 5 min after extracellular application of 1 µM lubeluzole, Vh (mV), Sl (mV), and the current density of Imax (pA/pF) were 101.3 ± 1.2, 6.0 ± 0.2, and 32 ± 7, respectively, at pHi 6 (n = 6); 97.6 ± 1.0, 6.5 ± 0.2, and 53 ± 7 at pHi 6.6 (n = 16); 88.6 ± 1.0, 6.2 ± 0.2, and 51 ± 6 at pHi 7.2 (n = 13); and 82.8 ± 1.6, 5.2 ± 0.2, and 78 ± 13 at pHi 9 ( = 6). At all tested pHi values lubeluzole produced a significant negative shift in Vh, an increase in Sl and a reduction in Imax (two-sided paired t test). The lubeluzole-induced change in Vh and ratio Imax were pHi dependent and larger at lower pHi (P < .0001).

Influence of the Uncharged Form of Lubeluzole on iLVA and iHVA. To isolate the contribution of L to the block of iLVA and iHVA, the influence of lubeluzole was tested at pH_o 9 and pH_i 9, at which [HL⁺]_o and [HL⁺]_i are reduced (Table 1; Fig. 7). At 5 min after application of 3 µM lubeluzole at pH_o 9 and pH_i 9, rLVA, rHVApeak, and rHVAend were decreased to 0.502 ± 0.093, 0.758 ± 0.038, and 0.598 ± 0.057, respectively (mean ± S.D., n = 4). This suggests that also L can contribute to some extent to the block of iLVA and iHVA. However, at normal pH_o 7.4 the contribution of L to the block should be smaller than at pH_o 9, at which [L] is 2.5 times higher.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Contribution of the uncharged form of lubeluzole to the block: influence of 3 µM lubeluzole at pHo 9 and pHi 9. A, voltages applied and sweeps in the control solution and every 30 s up to 5 min after application of 3 µM lubeluzole. The numbers in parentheses refer to the time point at which the sweeps were recorded, as shown in B and C. B, time course of the difference between the peak inward current and the current at the end of the 200-ms test pulse to 50 mV (iLVA), the peak current at the 155-ms test pulse to 20 mV (iHVApeak) and the current at the end of the test pulse to 20 mV (iHVAend). C, current ratios rLVA, rHVApeak, and rHVAend. To obtain these ratios, the time courses of iLVA, iHVApeak, and iHVAend in the control period were fitted exponentially and extrapolated to the end of the experiment. Division of each measured current amplitude by the value of the corresponding calculated curve obtained by fitting, at the same time point, yielded the ratios. This was done to quantify the effect of lubeluzole in the presence of continuous run-down of the Ba2+ currents. Same cell as in A and B.

Influence of Variation of Both pH_o and pH_i on the Block of Ca²⁺ Channels by Lubeluzole. We also tested whether lubeluzole blocks iLVA and iHVA to a different degree when both pH_o and pH_i are reduced, as is the case in ischemia (Lipton, 1999). At pH_o 6.8/pH_i 6.6 lubeluzole blocked iLVA and iHVA more than at pH_o 7.4/pH_i 7.2 with the same nominal transmembrane pH gradient (P < .01; unpaired two-sided t test) (Fig. 8).

View larger version (37K): [in this window] [in a new window]   Fig. 8.   Influence of pHo and pHi on the block of iLVA and iHVA by lubeluzole. The histograms show rLVA, rHVApeak, and rHVAend after 5-min application of 3 µM lubeluzole at different combinations of pHo and pHi. On the abscissa, pHo 6.8/pHi 6.6 stands for measurements at pHo 6.8 and simultaneously pHi 6.6. The first two combinations were at a constant pHo of 6.8. The next four combinations were at a constant pHo of 7.4. The last two combinations were at a constant pHo of 9. Results are expressed as mean ± S.E. The number of cells tested is given in parentheses.

Application of Lubeluzole via the Patch Electrode. As another method of testing whether lubeluzole affects Ca²⁺ channels via the intracellular side of the cell membrane, 10 µM lubeluzole or the corresponding solvent (DMSO) was added to the electrode solution (pH_i 7.2 and pH_o 7.4). In these experiments the cells were stimulated with a 200-ms pulse to 50 mV, followed by a 155-ms pulse to 20 mV, every 30 s after entry into the whole-cell mode. The LVA and HVA Ba²⁺ current of DRG cells to which 10 µM lubeluzole was applied via the microelectrode alone (Fig. 9A) (n = 11) was difficult to distinguish from that of cells measured with the normal pipette solution (n = 8) or a solution containing 0.1% DMSO in addition (n = 4). Intracellular application of lubeluzole via the microelectrode tended to produce only a small acceleration of the apparent inactivation of iHVA, if any (Fig. 9A), in comparison with cells in which no lubeluzole was added to the microelectrode (Fig. 2A). In contrast, extracellular application of 10 µM lubeluzole to the same cells produced a pronounced block of iLVA and iHVA (Fig. 9, B-D).

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Influence of application of 10 µM lubeluzole via the patch electrode and via the extracellular solution. A, activation of iHVA 10 min after entry into the whole-cell mode, with 10 µM lubeluzole in the electrode and no lubeluzole in the extracellular solution. From a 100-mV HP, a 200-ms test pulse to 50 mV was given to elicit and inactivate iLVA nearly completely. Thereafter, 155-ms test pulses varying from 50 to +10 mV were given to activate iHVA. The sweep interval was 20 s. Rs = 3.1 M; Cm = 58.8 pF. B, same cell as in A. In contrast to intracellular application of 10 µM lubeluzole alone, extracellular application of 10 µM lubeluzole produced a pronounced block of iLVA and iHVA. Every 30 s the shown pulse sequence was given. The last sweep in the control solution is shown together with the first 10 sweeps in the presence of 10 µM lubeluzole. Lubeluzole was applied immediately after the last sweep in the control solution. C, activation of iHVA after 5-min extracellular application of 10 µM lubeluzole to the same cell. D, I-V relationships of iHVA derived from the sweeps in A and C. Filled symbols: peak inward current. Unfilled symbols: current at the end of the 155-ms test pulse to 20 mV.

Extracellular and Intracellular Application of a Quaternary Ammonium Derivative of Lubeluzole. To avoid the complication arising from transmembrane diffusion of the uncharged form, a methyliodide quaternary ammonium derivative of lubeluzole (R133121) (Fig. 1) was synthesized and used, on the assumption that such a permanently charged compound would not be able to cross the cell membrane. If extracellular application of R133121 produced the same effects as lubeluzole, this would suggest that lubeluzole blocks Ca²⁺ channels from the extracellular side.

View larger version (30K): [in this window] [in a new window]   Fig. 10.   Influence of intracellular and extracellular application of 100 µM R133121. A, time course of iLVA, iHVApeak, and iHVAend from the establishment of the whole-cell configuration. DMSO (1%) was added to the standard microelectrode solution (pHi 7.2/pHo 7.4). The stimulation sequence shown in E was given every 30 s. Rs = 2.2 M; Cm = 46.3 pF. B, same stimulation protocol in another cell with an electrode solution to which 100 µM R133121 (+1% DMSO) was added (pHi 7.2/pHo 7.4). Rs = 3.0 M; Cm = 42.7 pF. C, activation of iHVA 10 min after establishment of the whole-cell configuration with an electrode containing 1% DMSO. After a 200-ms prepulse to 50 mV 155-ms test pulses varying from 50 to +10 mV were given every 20 s. Same cell as in A. D, activation of iHVA 10 min after establishment of the whole-cell configuration with an electrode containing 100 µM R133121 (+1% DMSO). Same cell as in B. The apparent inactivation of iHVA was accelerated with 100 µM R133121 in the electrode. E, influence of extracellular application of 30 µM R133121 for 5 min, and thereafter 100 µM R133121 for 2.5 min. The electrode solution contained neither DMSO nor R133121. F, influence of extracellular application of 100 µM R133121 17 min after establishment of the whole-cell mode with an electrode containing 100 µM R133121 (+1% DMSO). Same cell as in B and D. The cell was stimulated every 30 s. The graph shows the last sweep in the extracellular control solution (to which 1% DMSO had been added) and the first six sweeps after the switch to an extracellular solution containing 100 µM R133121 (+1% DMSO). In contrast to intracellular application, extracellular application of even the very high concentration of 100 µM R133121 was not able to accelerate the apparent inactivation of iHVA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The experiments extend our earlier observations that lubeluzole blocks iLVA and iHVA (Marrannes et al., 1998b). The results presented in this article show the following: 1) the effects of lubeluzole on Ca²⁺ channels depend on pH_o and pH_i; 2) application of lubeluzole via the patch electrode blocks iLVA and iHVA only minimally in comparison with extracellular application; and 3) extracellular and intracellular application of the less potent quaternary ammonium derivative R133121 block iLVA and iHVA.

Influence of pH on Ca²⁺ Channels. The observed influence of pH_o on iLVA and iHVA, and the pH_i-dependent shift of the activation curve of iHVA agree with earlier findings with other cell types (Kaibara and Kameyama, 1988; Prod'Hom et al., 1989; Tytgat et al., 1990; Tombaugh and Somjen, 1996; Zhou and Jones, 1996). Previous studies reported that iLVA of heart cells (Tytgat et al., 1990) and hippocampal neurons (Tombaugh and Somjen, 1997) was not influenced by pH_i. However, we found a clear dependence of the half-inactivation potential of iLVA of DRG cells on pH_i, probably due to a change in intracellular surface potential of the cell membrane and T-channel (Hille, 1992).

Site of Action of Lubeluzole on iLVA. Lubeluzole clearly blocked iLVA at pH_o 6. Because at pH_o 6 97.5% of the extracellular lubeluzole is in the protonated form and the predicted [HL⁺]_i is low, this suggests that HL⁺ can block iLVA from the extracellular side. That no intracellular penetration of lubeluzole is needed to achieve block of iLVA is supported by the observation that extracellular application of the quaternary ammonium derivative of lubeluzole (R133121) also blocks iLVA. After switching pH_o from 9 to 7.4 in the continuous presence of lubeluzole, the relative change in iLVA was greater than after a pH_o change of the same magnitude in the absence of lubeluzole. This indicates that a rapid replacement of extracellular L by HL⁺ increased the block of iLVA, and consequently that extracellular HL⁺ blocks iLVA more than L.

Site of Action of Lubeluzole on iHVA. The partial block of the peak amplitude of iHVA by 3 µM lubeluzole at pH_o 6 and after extracellular application of 30 µM R133121 suggest that extracellular HL⁺ contributes somewhat to the block of iHVA. That iHVA is also blocked partly by 3 µM lubeluzole at pH_o 9/pH_i 9 suggests that L may contribute to the block, as well.

Extracellular versus Intracellular Application of Lubeluzole. Application of lubeluzole via the patch electrode in the whole-cell recording configuration had only minimal effects on iLVA and iHVA in comparison with extracellular application. As shown by the other experiments in this article, this does not necessarily mean that L and HL⁺ block Ca²⁺ channels only from the extracellular side; the effect after intracellular application may have been small because of efflux of L, resulting in a low concentration of lubeluzole in and near the cell membrane. The membrane permeability of L is probably high owing to its high lipophilicity (octanol/water partition ratio = 10^4.8) (Harris, 1960) and 28% of lubeluzole is in the uncharged form at pH_i 7.2. Because of the easy efflux of L via the large surface area of the cell membrane, the steady state concentrations of L in the cell membrane and [HL⁺]_i just below the membrane remained probably low and were determined more by the extracellular lubeluzole concentration than by that of the solution in the electrode, which communicated with the membrane via the much smaller area of the electrode tip and via a longer diffusional pathway than the transmembrane diffusion distance.