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Department of Neurophysiology, Leiden University Medical Center, Leiden, The Netherlands
Address correspondence to D. L. Ypey, PhD, Department of Neurophysiology, Leiden University Medical Center, PO Box 9604, 2300 RC Leiden, The Netherlands. Address e-mail to D.L.Ypey{at}lumc.nl.
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Abstract |
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20 µm diameter) dorsal root ganglion neurons from newborn mice, were inhibited by BAB with an IC50 value of 200 µM. Furthermore, 200 µM BAB accelerated T-type current activation, deactivation, and inactivation kinetics, comparable to earlier observations for N-type calcium channels. Finally, 200 µM BAB had no effect on the midpoint potential and slope factor of the activation curve, although it caused a 3 mV hyperpolarizing shift of the inactivation curve, without affecting the slope factor. We conclude that BAB inhibits T-type calcium channels with a mechanism associated with channel kinetics acceleration. |
Introduction |
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Although the T-type calcium currents were discovered in sensory neurons in 1984, and particularly in the small-size pain-sensing neurons (3), their exact role has never been made clear. In general, T-type currents are believed to be involved in neuronal pacemaker activity and in promoting calcium entry during short action potentials (4). However, in recent studies T-type calcium currents have been shown to play a role in pain signaling, and they have also been recognized as therapeutic targets (5–8). Therefore, it is of interest to investigate whether T-type calcium channels are inhibited by BAB.
The distinct biophysical properties of T-type calcium channels (low voltage activation and its transient nature resulting from fast inactivation) make it possible to separate effects of BAB on T-type channels from non-T-type calcium channels without the use of ion channel blockers. We were also interested in the question of whether an inhibition of T-type channels would be accompanied by an acceleration of channel kinetics, as has been observed in our earlier studies for various channel types, including native and cloned Kv1.1 channels (9) and native N-type calcium channels (2). The patch-clamp technique in the whole-cell voltage-clamp configuration allowed us to establish that BAB indeed inhibits T-type currents and, at the same time, accelerates T-type current kinetics in small-size (20 µm diameter) DRG neurons from neonatal mice. The functional implications of these findings are discussed.
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Methods |
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20 µm in diameter), mainly comprising nociceptive neurons (6), dissociated from DRG of neonatal mice were patch-clamped within 8 h of culture. Patch pipettes of borosilicate glass had resistances of 2.0 to 2.5 M
. Giga-seals were made in a microbath of 75 µL, continuously perfused (300 µL/min) with standard extracellular solution containing (in mM): NaCl 145, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, pH 7.4 (NaOH). The pipette solution contained (in mM): Cs-methanesulfonate 103, MgCl2 4, HEPES 9, EGTA 9, (Mg)ATP 4, (tris)GTP 1, (tris)phosphocreatine 14, pH 7.4 (CsOH). After establishment of the whole-cell configuration calcium channel currents were measured as barium currents during extracellular perfusion with (in mM): TEA-Cl 160, HEPES 10, EGTA 0.1, BaCl2 5, pH 7.4 (TEA-OH).
To minimize offset caused by the low Cl– pipette solution, the pipette holder (10) contained a Cl– rich solution at the Ag/AgCl electrode. Experiments were performed at room temperature (23°C) with a List EPC 7 amplifier (3 kHz filtering) and controlled by pClamp software (Axon Instruments, Foster City, CA). The membrane capacitance of the selected DRG neurons was 14 pF, the series resistance was largely (80%–90%) compensated, and the records were leak subtracted.
BAB (OPG Farma, Utrecht, The Netherlands) was added to the extracellular solution from a stock of BAB in ethanol (1–500 mM). Normalized data were corrected for rundown in the presence of vehicle (0.1% alcohol) at all potentials measured in control experiments (n = 8). At test pulses of –40 mV rundown was < 3% in 12 min.
The Hill equation, I/Io = (1 + ([BAB]/IC50)n)–1, was fitted to the concentration-inhibition data, where IC50 is the concentration at which the current is reduced by 50% and n is the Hill coefficient. The Boltzmann equation, I/Io = (1 + exp((V – V0.5)/k))–1, with V the prepulse potential, V0.5 the midpoint potential at which the current is half maximal, and k the slope factor, was fitted to the steady-state inactivation data. T-type current kinetics were fitted with a m2h Hodgkin-Huxley type model: I(t) = (m*)2 · (1 – exp(–t/
m))2 · (exp(–t/h) + h* · (1 – exp(–t/h))) · A, where m* is partial steady-state activation at –40 mV, obtained from the experiment in Figure 2, C and D, h* is a free parameter representing the partial steady-state inactivation, and A is an amplitude factor. The time constants for the m- and h-gate are m and h, respectively.
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Results are presented as mean ± sd for N cells (unless mentioned otherwise) and compared using paired or independent t-tests with the level of significance P = 0.05.
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Results |
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The LVA barium current at membrane voltages of –40 mV is carried mainly through T-type calcium channels and is characterized by a relatively fast and nearly complete inactivation. Other indications confirming that the current we observed was T-type include the crossing of the current traces at successive voltage steps (Fig. 2C) and a relatively slow deactivation compared with that of the HVA currents elicited by stronger depolarizing steps to 0 mV (Fig. 3C) (11). The amplitude of the peak barium current, elicited by voltage pulses to –40 mV preceded by a 80 ms prepulse to –120 mV from a holding potential of –80 mV, amounted to 415 ± 357 pA (N = 46) under control conditions. That evoked by 0 mV pulses was 4.7 ± 1.3 nA (N = 37). The T-type barium currents were reversibly reduced by BAB in a concentration-dependent manner. At the concentration of 200 µM, barium currents at –40 mV were diminished by 52% ± 8% (n = 6) (Fig. 1C). After washout the currents completely recovered to 96% ± 10% of the control amplitude. In Figure 1D, the concentration-response relation is shown for the peak of the T-type barium current. This relation was described using the Hill equation, resulting in an IC50 of 178 ± 21 µM and a Hill coefficient of 1.5 ± 0.3 (N = 40). This IC50 is similar to that found for the N-type barium current evoked at 0 mV (220 µM) (2). The reversal potential (EREV) of the T-type currents could not be measured separately, but the EREV of the total barium current was not affected by BAB, with values of +56 ± 4 mV (N = 8) and +55 ± 6 mV (N = 8) for control and 200 µM BAB, respectively.
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A hyperpolarizing shift in the inactivation curve has been shown to be a possible current-reducing mechanism of action of BAB for sodium channels (12,13). The typical inactivating time course of the T-type current makes this current an excellent model for examining the effects of BAB on inactivation of calcium channels. For that reason, we measured the steady-state inactivation of the T-type barium current by using a test pulse to –40 mV after applying a 350 ms prepulse to varying potentials from the holding potential –80 mV. The interval between the test pulses was 15 s. This was performed both in the absence and presence of 200 µM BAB. Plotting the relative current as a function of prepulse voltage resulted in the inactivation curves shown in Figure 2A. A Boltzmann equation fitted to the data of the individual cells yielded midpoint potentials of the inactivation curves under control conditions with a mean value of –52 ± 4 mV (N = 5). Application of 200 µM BAB reduced the currents to <50% and induced only a small but significant shift of the midpoint to –55 ± 4 mV (P = 0.001), which could be reversed by washout towards –53 ± 3 mV (P = 0.002). The normalized voltage-dependent inactivation curves are shown in Figure 2B. BAB induced a shift of the midpoint of –2.8 ± 0.8 mV, which was not accompanied by a significant change in slope factor, with values of 3.6 ± 0.3, 3.4 ± 0.4, and 3.5 ± 0.2 mV for control, BAB, and washout, respectively. It is clear from Figure 2, A and B that the small BAB-induced hyperpolarizing shift of the steady-state inactivation curve of T-type calcium channels is not responsible for the current reduction observed at the test pulse of –40 mV.
The T-type activation curve can only be obtained in a limited range of voltages because of the overlap with the activation curves of the HVA calcium channels at the more depolarized potentials. In Figure 2C currents are shown elicited by depolarizing steps to various potentials ranging from –75 mV to –40 mV from a holding potential of –80 mV. This voltage range was limited to these values to only activate the T-type currents. The resulting peak values were converted to conductance assuming a linear relation between current and driving force with an EREV of +50 mV, unaffected by BAB. Subsequently, the conductance-voltage relations for the individual cells were fitted with a Boltzmann equation, which also described the activation curve for the higher range of potentials by extrapolation. The resulting values were averaged to obtain the mean activation curve, which showed no significant shift of the midpoint potential with –41 ± 5 mV for control and –41 ± 3 mV in the presence of 200 µM BAB (N = 9). Nor was there a difference in slope factor with –4.5 ± 0.9 mV and –4.3 ± 0.8 mV for control and BAB, respectively.
BAB caused and overall inhibition of T-type currents, thus, although the steady-state activation/inactivation properties of the currents were not, or were hardly, affected. Only the midpoint potential of the inactivation curve was slightly shifted in the hyperpolarizing direction.
The current during a maintained depolarizing step to –40 mV from a holding potential of –80 mV for control and with BAB is shown in Figure 3A. Scaling of the currents to the control peak value showed an accelerating effect of 200 µM BAB on the currents (Fig. 3B). This acceleration could well be quantified by describing the T-type current traces with an m2h Hodgkin-Huxley model (14). The time constant of the activation process (m) reduced significantly in the presence of BAB from 11.3 ± 2.5 ms to 8.7 ± 3.0 ms (P < 0.001, N = 9). The inactivation process (h) accelerated as well, with time constants for control and BAB of 91 ± 52 ms and 40 ± 14 ms, respectively (P = 0.007, N = 9).
On repolarization to –80 mV, after a 15 ms pulse to –40 mV, the tail current was measured, representing the deactivation of T-type channels (Fig. 3, C & D). In control solution the tail currents decayed with a time constant of 1.70 ± 0.23 ms (N = 5). Application of 200 µM BAB significantly reduced this to a time constant of 1.17 ± 0.23 ms (P < 0.001). This effect was completely reversible with a time constant of 1.84 ± 0.36 ms (P < 0.001) after washout. Thus, besides inhibiting T-type currents, BAB also accelerates activation, inactivation and deactivation kinetics of this current.
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Discussion |
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200 µM and accelerated the kinetics of activation, deactivation, and inactivation of this current. These effects of BAB on T-type current are very similar to those on HVA N-type current (2). In the Hodgkin-Huxley model describing ion channel behavior, altered kinetics are typically reflected in a change of the midpoint potential and slope factor of the steady-state activation and inactivation curves. However, the BAB-induced accelerating effects on T-type current kinetics were not, or were only weakly, accompanied by changes in these variables. This would imply that the voltage-dependent rates of gate opening and closing are roughly proportionally increased. In this interpretation, the current inhibition by BAB cannot be fully explained by the observed kinetic changes because the maximal currents are reduced. Therefore, we consider factors other than purely kinetic Hodgkin-Huxley mechanisms.
The faster deactivation with BAB argues against a classical open-channel block because that type of block is rather accompanied by slowed deactivation (15). The described effects on T-type currents are also similar to those found for Kv1.1 potassium channels (9). BAB accelerated both activation and deactivation kinetics without shifting the midpoint potential of activation for Kv1.1 current. In addition there was also an accelerated current decay or inactivation of these currents. These results were explained by an allosteric mechanism with BAB biasing the channels towards nonconducting channel states. Vedantham and Cannon (16) hypothesized a preferential binding to intermediate closed states, causing increased inactivation in those states and a hyperpolarizing shift of the inactivation curve. Although they studied lidocaine's effects, others have found confirming results for benzocaine, which is structurally very similar to BAB (17). A similar mechanism might act in T-type calcium channels. However, more experiments are needed to reach definitive conclusions about the underlying mechanisms.
The T-type calcium current seems to be a common neuronal process for mediating excitability. However, its biophysical properties determine that it can have complex and paradoxical roles. The activation of the current in the low voltage range has a depolarizing effect, leading to a faster recruitment of sodium channels and therefore to the firing of an action potential. Raman and Bean (18) showed, in Purkinje neurons, that blocking the T-type current using mibefadril resulted in a 30% slowing of the firing rate, which indicated that the presence of T-type current enhances excitability. McCallum et al. (19), however, showed an increased excitability in sensory neurons as a result of a T-type current blockade, which means that the T-type presence would be responsible for less excitability. These authors suggested that the relatively slow deactivation of the T-type current results in prolonged calcium entry at the end of the action potential. Taking this calcium influx away would lead to higher excitability. How these counteracting mechanisms act in pain signaling is not yet established.
It should also be considered that the T-type current may be carried by three different 
-subunits, each with a specific expression pattern in the body. These three pore-forming subunit isotypes contribute differently to neuronal excitability through their different biophysical properties (20). It is this variety of actions that makes it difficult to predict the role of inhibit-ing T-type calcium currents by BAB in its pain-suppressing actions. Nevertheless, modifying the T-type currents in vivo has shown that T-type currents are involved in pain signaling. Drugs that selectively enhance T-type currents result in exaggerated thermal and mechanical nociception, whereas T-type current reducing drugs do the opposite (6). Moreover, suppressing CaV3.2 (1H) T-type currents—which are expressed in DRG neurons—using the mRNA antisense technique results in antinociceptive, antihyperalgesic, and antiallodynic effects (8). These studies suggest an enhancing nociceptive role for T-type currents. However, mice lacking the CaV3.1 (1G) gene (21), i.e., those without those T-type currents in the thalamic neurons, showed hyperalgesia, suggesting an antinociceptive role for central T-type currents. This indicates that T-type currents have different roles depending on where they are located along the pain pathway.
The ultralong duration of the pain suppression seems to be attributable to a slow steady release of BAB from the suspension in the confined epidural space (22). Hence, the calcium channels present in this space will be subjected continuously to submaximal solubility concentrations of BAB. Although it is unlikely that inhibiting the T-type calcium current in sensory neurons alone can explain the described pain suppressing effects of epidural BAB, directly or via the interplay with other BAB-affected channels (KV, and NaV), BAB's effects on T-type calcium channels are likely to play a role if T-type channels are expressed by dorsal root fibers. However, in the light of the above discussion of the role of T-type channels in pain signal generation in the peripheral nerve endings, the present results do implicate inhibition of T-type channels in BAB-containing topical skin applications.
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Footnotes |
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Reprints will not be available from the authors.
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