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. 2002 May 1;540(Pt 3):761–770. doi: 10.1113/jphysiol.2001.013376

Roscovitine: a novel regulator of P/Q-type calcium channels and transmitter release in central neurons

Zhen Yan *, Ping Chi *,†, James A Bibb *, Timothy A Ryan †, Paul Greengard *
PMCID: PMC2290289  PMID: 11986366

Abstract

Roscovitine is widely used for inhibition of cdk5, a cyclin-dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca2+ channels and transmitter release in central neurons, was studied by whole-cell voltage-clamp recordings and time-lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3- to 6-fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L-channel activator Bay K 8644 was not occluded but additive. Roscovitine-induced potentiation of tail calcium currents was significantly blocked by the P/Q-channel blocker CgTx-MVIIC, indicating that the major target of roscovitine is the P/Q-type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild-type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5-independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1–43, consistent with the positive effect of roscovitine on the P/Q-type calcium channel, the major mediator of action potential-evoked transmitter release in the mammalian CNS.

In mammalian CNS neurons, a diversity of voltage-gated calcium channels play critical roles in regulating a wide range of cellular processes ranging from transmitter release to gene expression (Catterall, 1995). Pharmacological approaches have been extremely valuable in studying the properties and functions of these calcium channels (Wheeler et al. 1994; Randall & Tsien, 1995). The five major types of high voltage-activated calcium channels, termed L-, N-, P-, Q- and R-types, can be distinguished based on their different sensitivity to blockers such as dihydropyridine antagonists, toxins ω-CgTx GVIA, ω-AgTx IVA and ω-CgTx MVIIC (Tsien et al. 1988; Dunlap et al. 1995). So far, most of the calcium channel-specific pharmacological agents are channel inhibitors. The best-known calcium channel activator is Bay K 8644, a dihydropyridine agonist that selectively potentiates L-channels (Brown et al. 1984; Kokubun & Reuter, 1984; Fox et al. 1987; Jones & Jacobs, 1990). Since N- and P/Q-channels are localized in presynaptic terminals (Robitaille et al. 1990; Westenbroek et al. 1995), and participate in neurotransmitter release in mammalian CNS neurons (Luebke et al. 1993; Takahashi & Momiyama, 1993; Wheeler et al. 1994), identifying novel N- and P/Q-channel activators will provide new tools for understanding the role of these channels in the release machinery. These channel activators could also be used as potential therapeutic agents under pathological conditions when neurotransmitter release is impaired.

Roscovitine ((R)-2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine) is a widely used selective inhibitor of cdk5, a cyclin-dependent kinase expressed predominantly in the brain (Hellmich et al. 1992; Tsai et al. 1994). cdk5, in conjunction with its neuron-specific activator p35, plays a key role in brain development, neuronal migration, neurite outgrowth and neuronal cytoskeleton structure and organization (Tsai et al. 1994; Nikolic et al. 1996; Ohshima et al. 1996; Chae et al. 1997). Deregulation of cdk5 has been found to contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (Patrick et al. 1999). cdk5 protein and kinase activity increase as forebrain development progresses, and roscovitine has been used as a specific inhibitor for this constitutively active kinase in the mature nervous system (De Azevedo et al. 1997; Meijer et al. 1997). In this study, we have revealed a novel function of roscovitine by investigating its effect on calcium currents in central neurons.

Using whole-cell voltage-clamp recordings, we have found that roscovitine modifies the voltage-dependent gating of calcium channels in a unique and cdk5-independent way. In central neurons, extracellular application of roscovitine dramatically slowed the deactivation kinetics of P/Q-type calcium channels, therefore prolonging the open state of the channel and increasing calcium influx through these channels. Consistent with the positive effect of roscovitine on P/Q-type calcium channels, roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons measured with the vesicle trafficking dye FM1–43. These results suggest that roscovitine could be used as a novel pharmacological agent regulating P/Q-calcium channels and transmitter release in mammalian CNS neurons.

METHODS

All procedures conformed with State University of New York at Buffalo Institutional Animal Care and Use Committee (IACUC) guidelines.

Acute dissociation procedure

Neurons from 4-week-old rats or mice were acutely dissociated using procedures similar to those previously described (Surmeier et al. 1995; Yan et al. 1997). In brief, rats and mice were anaesthetized by inhaling 2-bromo-2-chloro-1,1,1-trifluoroethane (1 ml (100 g)−1, Sigma) and decapitated; brains were quickly removed and then blocked for slicing. The blocked tissue was cut in 400 μm slices with a vibratome (Technical Products International, Inc., St Louis, MO, USA) while bathed in a low Ca2+ (100 μm), Hepes-buffered salt solution (mm: 140 sodium isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose and 15 Hepes; pH 7.4, 300–305 mosmol l−1). Slices were then incubated for 1–6 h at room temperature (20–22 °C) in a NaHCO3-buffered saline bubbled with 95 % O2 and 5 % CO2 (mm: 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3 1.25 NaH2PO4 and 10 glucose; pH 7.4 with NaOH, 300–305 mosmol l−1). All reagents were obtained from Sigma. Slices were then removed from the low Ca2+ buffer, regions of dorsal neostriatum, somatosensory cortex or hippocampus dissected, and placed in an oxygenated Cell-Stir chamber (Wheaton, Inc., Millville, NJ, USA) containing pronase (Sigma protease Type XIV, 1–3 mg ml−1) in Hepes-buffered Hanks' balanced salt solution (HBSS, Sigma) at 35 °C. After 30 min of enzyme digestion, tissue was rinsed three times in the low Ca2+, Hepes-buffered saline and mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35 mm Lux Petri dish mounted on the stage of a Zeiss inverted microscope containing Hepes-buffered HBSS saline.

Whole-cell recording

Whole-cell recordings of Ba2+ currents through Ca2+ channels employed standard techniques (Hamill et al. 1981; Surmeier et al. 1995; Yan & Surmeier, 1996). Electrodes were pulled from Corning 7052 glass and fire-polished prior to use. The internal solution consisted of (mm): 180 N-methyl-d-glucamine (NMG), 40 Hepes, 4 MgCl2, 5 BAPTA, 12 phosphocreatine, 2 Na2ATP, 0.2 Na3GTP and 0.1 leupeptin; pH 7.2–7.3 with H2SO4, 265–270 mosmol l−1. The pH of NMG solutions was measured with a Corning Model 476570 probe. The external solution consisted of (mm): 135 NaCl, 20 CsCl, 1 MgCl2, 10 Hepes, 0.001 TTX, 5 BaCl2 and 10 glucose; pH 7.3 with NaOH, 300–305 mosmol l−1. Roscovitine and olomoucine were obtained from Dr L. Meijer (Station Biologique de Roscoff, CNRS UPR, Roscoff cedex, Bretagne, France) or Alexis Co. (San Diego, CA, USA). Nifedipine, (±)-Bay K 8644 and ω-conotoxin MVIIC were obtained from RBI, Inc. (Natick, MA, USA). Drugs were dissolved in water or DMSO to make a concentrated (e.g. ×1000) stock solution, followed by dilution into the recording saline. When DMSO was used, vehicle controls were used in the electrophysiology and FM1–43 experiments.

Recordings were obtained with an Axon Instruments 200B patch-clamp amplifier, controlled and monitored with an IBM PC running pCLAMP (v. 6.0) with a DigiData 1200 series interface (Axon instruments, Foster City, CA, USA). Electrode resistances were typically 2–4 MΩ in the bath. After seal rupture, series resistance (4–10 MΩ) was compensated (70–90 %) and periodically monitored. Only currents with small leaks (< 50 pA) were used, and current records were not leak-subtracted. Drugs were applied with a gravity-fed ‘sewer pipe’ system. To avoid cross contamination, different drugs were applied via different tubes. The application capillary (ca 150 μm i.d.) was positioned a few hundred microns from the cell under study. Solution changes were effected by altering the position of the array with a DC drive system controlled by a microprocessor-based controller (Newport-Klinger, Inc., Irvine, CA, USA). Solution changes typically were complete within less than 1 s; the time constant of Cd2+ block of Ca2+ current was 300 ms.

Data analyses were performed with AxoGraph (Axon Instruments) and Kaleidagraph (Albeck Software, Reading, PA, USA) software. Box plots were used for graphic presentation of the data because of the small sample sizes (Tukey, 1977). The box plot represents the distribution as a box with the median as a central line and the hinges as the edges of the box (the hinges divide the upper and lower halves of the distributions in half). The inner fences (shown as a line originating from the edges of the box) run to the limits of the distribution excluding outliers (defined as points that are more than 1.5 times the interquartile range beyond the interquartiles). Some plots show the mean ± s.e.m. values for a sample of neurons tested.

Photometric measurement of neurotransmitter release

Hippocampal cultures were derived from postnatal 3- to 4-day-old Sprague-Dawley rats (Ryan et al. 1993). Cells were used 2–4 weeks after plating, and the coverslip was mounted in a low volume laminar superfusion microscope chamber and superfused in a saline solution consisting of 119 mm NaCl, 2.5 mm KCl, 1 mm CaCl2, 2 mm MgCl2, 25 mm Hepes, 30 mm glucose, 10 μm CNQX and 50 μm AP-5. The dye loading solution consisted of 15 μm FM1-43 (Molecular Probes, Eugene, OR, USA) in the standard saline. Action potentials were stimulated by passing 1 ms current pulses yielding fields of 10 V cm−1 through the chamber. Scanning fluorescence and DIC images were acquired at a rate of 1 image per 2.2 s using a modified Bio-Rad MRC 500 laser scanning unit coupled to a Zeiss IM-35 inverted microscope, and were stored digitally. Fluorescence was excited using the 488 nm line of an argon laser, and emissions were detected through a 500 nm pass filter. Digital time-lapse sequences were analysed as previously described (Ryan et al. 1993). Regions of interest, typically 1.5 × 1.5 μm2, were selected to overlap the largest portion of fluorescence spots. A brightness centre of mass was computed and used to recentre a square region of interest whose pixel intensities were then averaged together to obtain a measure of the local fluorescence intensity in individual synaptic boutons over time.

RESULTS

Roscovitine rapidly and reversibly enhanced the tail calcium current in a dose-dependent manner

To test the effect of roscovitine on Ca2+ channels in central neurons, several different cell types, including neostriatal cholinergic interneurons and medium spiny neurons, cortical and hippocampal pyramidal neurons, were chosen for recording. Bath application of roscovitine rapidly and reversibly enhanced the tail calcium current following repolarization from depolarized voltages in all the cells tested (n = 34). Since the fractional contributions of different Ca2+ channels are similar in these cells (Bargas et al. 1994; Lorenzon & Foehring, 1995; Yan & Surmeier, 1996) and roscovitine has similar effects in them, averaged data were pooled across cell types. The recording from a neostriatal cholinergic interneuron is shown in Fig. 1A–C. This effect was dose dependent, with the IC50 around 20 μm (n = 5). The average enhancement in the amplitude of a slow tail current component (measured at 5 ms after the initiation of repolarization) produced by roscovitine (50 μm) was 352 ± 139 % (mean ± s.d., n = 20). The onset of roscovitine modulation of calcium currents had fast kinetics with a time constant of 1.3 s (n = 3). Application of the calcium channel blocker Cd2+ (250 μm) completely eliminated the roscovitine-induced potentiation of tail currents (Fig. 1D), suggesting that roscovitine was acting on voltage-dependent calcium channels. In contrast to the potent effect of roscovitine on calcium currents, olomoucine (100 μm), a compound that has a structure similar to that of roscovitine and also acts as a cyclin kinase inhibitor, had no effect on calcium currents in central neurons (data not shown).

Figure 1. Roscovitine reversibly enhanced calcium tail currents.

Figure 1

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A, current traces in the absence or presence of different concentrations (5, 20 and 50 μm) of roscovitine recorded from a neostriatal cholinergic interneuron. Membrane potential was held at −80 mV. Calcium currents were activated by depolarizing to 0 mV and then deactivated by repolarizing to −55 mV. B, expanded view of the tail current in A. C, plot of tail current in A (measured 5 ms into the tail) as a function of time and drug application. Roscovitine potentiated tail current in a dose-dependent manner. D, plot of tail current as a function of time and drug application. The effect of roscovitine (50 μm) was eliminated by the calcium channel blocker Cd2+ (250 μm).

Roscovitine modulated calcium currents by slowing the deactivation kinetics

Since roscovitine markedly enhanced the tail current, neurons were repolarized to different voltages from depolarized potentials to test the effect of roscovitine on deactivation kinetics of calcium channels. A representative example is shown in Fig. 2A and B. Roscovitine (50 μm) increased the amplitude of a slow tail current component by ∼4- to 5-fold when neurons were repolarized to levels near the resting potential (Fig. 2C; n = 4; P < 0.001, paired t test). Fitting the tail currents with single exponential curves, we found that roscovitine (50 μm) increased the deactivation time constant 3- to 6-fold when repolarizing to different voltages (Fig. 2D; n = 4; P < 0.001, paired t test). Because the time constants for tail currents at extreme negative voltages could be inaccurate in control conditions, this effect of roscovitine could even be underestimated. These results suggest that roscovitine modifies calcium currents by slowing the process of channel closing, therefore prolonging the open state of calcium channels. In contrast to the potent effect of roscovitine on deactivation kinetics, the voltage dependence of channel activation (Fig. 2E) and inactivation (Fig. 2F) was not significantly altered by roscovitine (n = 4; P > 0.05, paired t test).

Figure 2. Roscovitine slowed the deactivation kinetics of calcium channels.

Figure 2

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A and B, current traces in the absence (control) or presence of roscovitine (50 μm) recorded from a neostriatal cholinergic interneuron. Calcium tail currents were evoked by repolarizing to different voltages (−10 to −80 mV) from 0 mV. C, plot of tail current amplitudes (measured 3 ms into the tail) against membrane potentials after repolarization in a sample of four neurons. Circles indicate mean values; error bars indicate s.e.m.D, plot of deactivation time constants against membrane potentials after repolarization in a sample of four neurons. Deactivation time constants were obtained by fitting tail currents with single exponential curves. E, activation plot obtained by applying the Goldman-Hodgkin-Katz constant field transformation to the I-V plot constructed with normalized peak calcium currents for a sample of four neurons activated by step voltage commands (see stimulation protocol in inset). Curves are the Boltzmann fit of the transformed permeability. Parameters (mean ± s.e.m.): Vh = −18.5 ± 1.5 mV (control); Vh = −.2 ± 1.3 mV (roscovitine); Vc = 7.37 ± 0.8 mV (control); Vc = 7.45 ± 0.9 mV (roscovitine). F, inactivation plot constructed with normalized peak calcium currents for a sample of four neurons elicited by 0 mV commands after depolarization prepulses (see stimulation protocol in inset). Curves are the best-fit Boltzmann relationships to the data. Parameters: V1/2 = −38.3 ± 3.2 mV (control); V1/2 = −42 ± 3.5 mV (roscovitine).

The potentiation of tail currents caused by roscovitine and Bay K 8644 was additive

Previous studies have found that Bay K 8644, a dihydropyridine derivative, can slow the tail current mediated by L-type calcium channels (Brown et al. 1984; Fox et al. 1987; Jones & Jacobs, 1990; Bargas et al. 1994). It seemed possible that roscovitine had the same effect on calcium channels as Bay K 8644. One sign of this would be an occlusion in their modulation of calcium currents. To test this, roscovitine and Bay K 8644 were applied alone and together (n = 6). Data from one of these experiments is shown in Fig. 3A where tail current is plotted as a function of time and drug application. Application of Bay K 8644 (2.5 μm) and roscovitine (25 μm) alone enhanced tail currents by 566.7 and 400 %, respectively. Co-application of the two compounds induced an additive enhancement of tail currents (933 %, Fig. 3). Comparing the roscovitine-potentiated tail current (Fig. 1B) with the Bay K 8644-potentiated tail current (Fig. 3C), it is quite clear that roscovitine mainly enhanced the fast component of tail current (1–15 ms following repolarization), while the Bay K 8644 effect persisted in the slow component of tail current (15–40 ms following repolarization). Similar results were observed in all six cells tested. The lack of occlusion of roscovitine and Bay K 8644-induced potentiation of tail currents suggests that these two drugs target different subtypes of calcium channels or act through different mechanisms.

Figure 3. Roscovitine-induced potentiation of tail current was not occluded by Bay K 8644.

Figure 3

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A, plot of tail current (measured 4 ms into the tail) evoked by repolarizing to −55 mV from 0 mV as a function of time and drug application. The recording was obtained from a neostriatal cholinergic interneuron. Bay K 8644 (2.5 μm) alone and roscovitine (25 μm) alone enhanced tail currents. Co-application of Bay K 8644 and roscovitine produced additional enhancement. B, current traces from the data used to construct A. C, expanded view of the tail current in B.

Roscovitine targeted P/Q-type calcium channels

In most central neurons, there are five major types of high voltage-activated calcium channels: L-, N-, P-, Q- and R-types (Brown et al. 1993; Bargas et al. 1994; Tsien et al. 1995; Yan & Surmeier, 1996). To determine which of these channels were affected by roscovitine, specific channel antagonists were used. As shown in Fig. 4A and B, application of the P/Q-channel antagonist ω-CgTx MVIIC (2 μm, Hillyard et al. 1992) drastically reduced the effect of roscovitine (25 μm). Prior to block of P/Q-channels, roscovitine markedly enhanced tail currents. Subsequent to the application of MVIIC, roscovitine had little effect on tail currents. Washing off MVIIC led to the recovery of roscovitine modulation. On the other hand, block of L-type channels with nifedipine (5 μm) only slightly reduced the effect of roscovitine on tail currents (Fig. 4C and D). The insets in Fig. 4B and D are box plots summarizing the percentage block of roscovitine effect by P/Q-type and L-type channel blockers, respectively (n = 5). On average, around 80 % of the roscovitine modulation targeted P/Q-type channels, and only 20 % involved L-type channels. Since nifedipine may have a weak blocking action on non-L-currents (Brown et al. 1994), the 20 % inhibition of L-current is an upper limit.

Figure 4. Roscovitine mainly enhanced P/Q-type calcium currents.

Figure 4

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A, plot of tail current (measured 4 ms into the tail) evoked by repolarizing to −55 mV from 0 mV as a function of time and drug application. The recording was obtained from a neostriatal cholinergic interneuron. Application of the P/Q-channel antagonist ω-CgTx MVIIC (2 μm) greatly blocked the effect of roscovitine (25 μm). Washing off MVIIC led to the recovery of roscovitine modulation. B, current traces from the data used to construct A. The inset is a box plot summarizing the percentage block of roscovitine effect by MVIIC (n = 5). C, plot of tail current as a function of time and drug application. Application of the L-channel antagonist nifedine (5 μm) only slightly reduced the effect of roscovitine (25 μm). D, current traces from the data used to construct C. The inset is a box plot summarizing the percentage block of roscovitine effect by nifedipine (n = 5).

Roscovitine modification of calcium currents was cdk5 independent

Since roscovitine is a potent and selective inhibitor of cdk5 (Meijer et al. 1997), it is of interest to know whether roscovitine modifies calcium currents by inhibiting cdk5 phosphorylation of calcium channels. To test the role of cdk5, we examined the effect of roscovitine on calcium currents in mutant mice lacking p35, a neuronal-specific activator of cdk5 (Chae et al. 1997). Previous studies have shown that cdk5 kinase activity is largely eliminated in adult brain lysates of p35−/- mice (Chae et al. 1997). However, in p35-deficient cortical neurons, roscovitine enhanced the deactivation tail current in a manner similar to that found in wild-type neurons (Fig. 5). Moreover, intracellular perfusion of roscovitine (50 μm, 20 min) failed to modulate calcium currents in whole-cell recordings (n = 4, data not shown). These results suggest that roscovitine acts on extracellular sites of calcium channels in a cdk5-independent way.

Figure 5. Roscovitine regulated calcium currents via a cdk5-independent mechanism.

Figure 5

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A, plot of tail current (measured 4 ms into the tail) as a function of time and drug application in a cortical neuron from a p35−/- mouse. B, a box plot summarizing the effect of roscovitine (50 μm) on tail currents in wild-type (n = 5) and p35−/- (n = 7) neurons.

Roscovitine potentiated glutamate release at presynaptic terminals of hippocampus

Neurotransmitter release is steeply dependent upon Ca2+ influx through calcium channels into presynaptic terminals (Augustine et al. 1985; Wheeler et al. 1994; Dunlap et al. 1995; Mintz et al. 1995). To examine the functional consequences of roscovitine coupling to calcium channels, we assessed the impact of roscovitine on neurotransmitter release by using the fluorescent membrane dye FM1–43. This dye can be taken up by newly endocytosed synaptic vesicles during electrical stimulation, and can be subsequently released from the vesicles during action potential-evoked exocytosis. Therefore it is possible to measure transmitter release quantitatively by monitoring the increase and subsequent decrease in dye fluorescence (Betz et al. 1992; Reuter, 1995; Ryan & Smith, 1995).

Cultured hippocampal nerve terminals were loaded with FM1–43 by giving a train of 600 action potentials (AP) at 20 Hz. FM1–43 was left for an additional 1 min to allow complete endocytosis. Cultures were then rinsed in an FM1–43-free solution for 10 min before a subsequent unloading experiment with a train of 1500 action potentials at 10 Hz. Destaining (decrease of fluorescence) at individual synaptic boutons was monitored when the cells were washed in different perfusion solutions. Destaining kinetics of FM1–43 are well described by a single exponential decay (Reuter, 1995; Ryan & Smith, 1995). The time constant of FM1–43 destaining represents the kinetics of synaptic vesicle turnover. FM1–43 destaining data were normalized to the total loss of fluorescence during the train of AP, determined by subtracting the average of the final three time points from that of the first five time points before stimulation for each individual bouton. Time constants for FM1–43 were obtained by fitting the destaining curves to single exponential decays. A representative experiment is shown in Fig. 6A and B (40 single boutons were analysed). When the dye was unloaded in the control external solution, the fluorescence intensity decreased exponentially with a decay time constant of 32.0 s. However, when the dye was unloaded in the external solution with 50 μm roscovitine, the fluorescence intensity decreased with a faster decay time constant of 26.5 s. When the dye was unloaded in the external solution containing roscovitine (50 μm) plus ω-CgTx MVIIC (1 μm), the fluorescence intensity decay time constant recovered to 39.2 s, which was close to the decay rate with ω-CgTx MVIIC alone (τ = 40.3 s, data not shown). Fluorescence decay time constants obtained during destaining in control, roscovitine, MVIIC or roscovitine plus MVIIC for four such experiments (20–50 boutons were analysed in each experiment) are shown in Fig. 6C. Application of roscovitine significantly reduced the destaining time constant (control: τ = 30.4 ± 0.9 s; roscovitine: τ = 22.9 ± 2.1 s; mean ± s.e.m.; P < 0.05, paired t test), and this effect was eliminated in the presence of MVIIC (MVIIC: τ = 38.6 ± 3.3 s; roscovitine plus MVIIC: τ = 36.9 ± 2.3 s; P > 0.05, paired t test). The data suggest that roscovitine increased the rate of transmitter release in response to action potential stimulation at hippocampal synapses by activating P/Q-type calcium channels.

Figure 6. Roscovitine potentiated transmitter release at hippocampal synapses.

Figure 6

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Synaptic boutons were loaded with FM1-43 prior to measuring the kinetics of release of the dye in response to action potential (AP) stimuli. A, the ensemble average of the normalized fluorescence intensity time courses obtained from 40 individual boutons during destaining in control, roscovitine (50 μm) or roscovitine (50 μm) plus MVIIC (1 μm). B, the fluorescence time courses in the different destaining conditions for the same boutons shown in A are plotted on a semilogarithmic scale to permit better kinetic comparison. C, a bar graph showing the mean fluorescence decay time constants for the different destaining conditions in four experiments. The exocytosis time constants were obtained by fitting FM1–43 destaining curves with single exponential equations. *P < 0.05, paired t test.

DISCUSSION

Specific pharmacological agents targeting different subtypes of calcium channels have been valuable tools for studying Ca2+-associated cellular processes in central neurons (Tsien et al. 1988; Dunlap et al. 1995). Roscovitine has gained considerable interest as a selective cdk5 inhibitor (De Azevedo et al. 1997; Meijer et al. 1997). In this study, we revealed a novel function of this important drug: modifying calcium currents. Roscovitine enhanced calcium entry by slowing the deactivation kinetics and prolonging the open state of P/Q-calcium channels. This potent effect had rapid onset kinetics and only occurred with extracellular application of the drug. It suggests that roscovitine may directly bind to the channel at extracellular sites, and thus interfere with gating, rather than act through inhibition of cdk5 phosphorylation of calcium channels or other proteins. The concentrations (5–50 μm) of roscovitine used here are comparable to those used to inhibit kinases in other studies (Bibb et al. 1999; Li et al. 2001).

Another well-known calcium channel agonist that can enhance the tail current is Bay K 8644. The potentiation of tail currents by roscovitine and by Bay K 8644 was not occlusive, but additive, suggesting that they either target different channels and/or act through different mechanisms. Pharmacological and electrophysiological analyses have revealed distinct actions of these two compounds on calcium channels. First, roscovitine and Bay K 8644 target different subtypes of calcium channels. Bay K 8644 selectively enhances the L-current-dominated slow component of deactivation tail current (Brown et al. 1984; Fox et al. 1987; Jones & Jacobs, 1990; Bargas et al. 1994), while roscovitine primarily enhances the P/Q-current-mediated rapid component of deactivation tail current. This suggests that Bay K 8644 targets L-channels and roscovitine targets P/Q-channels. Secondly, roscovitine and Bay K 8644 modify calcium currents through different mechanisms. Bay K 8644, in addition to enhancing the tail current, also increases the peak current and produces a leftward shift in the current-voltage relationship (Sanguinetti et al. 1986; Bargas et al. 1994; Yan & Surmeier, 1996). The underlying mechanism for Bay K 8644 is to increase the mean open time by promoting the long-lasting open state of the channel (Kokubun & Reuter, 1984). Roscovitine, on the other hand, has only a small effect on the peak current and does not affect the activation or inactivation kinetics. The underlying mechanism for roscovitine awaits further analysis at the single channel level. From macroscopic currents, it is easy to see that roscovitine slows the transition from the open to the closed state of calcium channels. We speculate that roscovitine prolongs the open state of calcium channels by slowing the deactivation process following repolarization during action potentials. Alternatively, since a tail current can be a burst of openings, roscovitine may induce more openings per tail.

Because of the significant role of Ca2+ channels in the regulation of neurotransmitter release in mammalian CNS neurons (Luebke et al. 1993; Takahashi & Momiyama, 1993; Wheeler et al. 1994; Mintz et al. 1995), the effect of roscovitine on Ca2+ channels allows this drug to have a potent impact on synaptic transmission. In hippocampal neurons, synaptic transmission was only partially (∼50 %) blocked by the N-channel inhibitor ω -CgTx GVIA and the rest was blocked by the Q-channel inhibitor ω-CgTx MVIIC (Wheeler et al. 1994). Previous measurement of exocytosis from single presynaptic terminals with the styryl dye FM1–43 showed that several types of Ca2+ channels co-exist in individual hippocampal boutons (Reuter, 1995). Blocking N-channels with ω-CgTx GVIA (2–10 μm) completely inhibited exocytosis in 45 % of boutons, while only partially (0–38 %) inhibited exocytosis in the other 55 % of boutons. In contrast, blocking P/Q-channels with a high (1 μm) concentration of ω-AgTx IVA homogeneously inhibited exocytosis in all synapses by 40 % (Reuter, 1996). This indicates that, in addition to N-type channels, P/Q-type channels play a major role in exocytosis in a broad spectrum of synapses. In agreement with these results, we found that when the deactivation of P/Q-channels was slowed by roscovitine, action potential-triggered glutamate release at hippocampal synapses was potentiated. By prolonging the open state of P/Q-channels, roscovitine may have caused an increase in calcium influx during action potentials, leading to the enhancement of glutamate release. Blocking P/Q-type Ca2+ channels with ω-CgTx MVIIC eliminated the roscovitine effect on transmitter release, suggesting that roscovitine regulates release by specifically acting on P/Q-channels. Of course, our experiments do not rule out the possibility that roscovitine could affect release by other mechanisms.

In a recent paper showing that roscovitine could block long-term potentiation (Li et al. 2001), it was mentioned that roscovitine did not have a detectable effect on EPSPs recorded in slices (data not shown), suggesting that roscovitine does not affect transmitter release. In our experimental preparations, we found that roscovitine can induce a strong modulation of tail calcium currents in voltage-clamped dissociated neurons and a modest change of FM1–43 destaining kinetics in cultured hippocampal neuronal terminals, suggesting that roscovitine can affect transmitter release. The seemingly inconsistent results could be due to several reasons. First, the concentration (5 μm) of roscovitine used in the Li et al. (2001) paper is relatively low, which could only induce a relatively small effect on tail calcium currents. This small change may not be able to induce a strong increase in transmitter release that is detectable with electrophysiological approaches. Secondly, the commercially available roscovitine is not as pure as the roscovitine we obtained from Dr L. Meijer (Station Biologique de Roscoff, CNRS UPR, Roscoff cedex, Bretagne, France). We found that the commercial roscovitine (Alexis Co.) inhibited peak calcium currents, which could prevent the increase of calcium influx. Thirdly, different approaches (electrophysiology vs. FM1–43 imaging) were used to measure transmitter release in the two studies, which could give potentially different results due to the limitation of each method. Over the last decade, FM1–43 has been proven to be a very useful probe for membrane trafficking. Because it stains membranes in an activity-dependent manner, FM1–43 is ideal for studies of neurotransmitter release mechanisms such as synaptic vesicle recycling, exocytosis, and endocytosis (for review, see Cochilla et al. 1999). Comparing with electrophysiological techniques, the major advantages of FM1–43 are twofold. On one hand, FM1–43 specifically measures presynaptic vesicle turnover without being affected by postsynaptic factors such as receptor saturation or desensitization. On the other hand, FM1–43 directly measures single boutons instead of the average of many boutons (in which case the number of synapses is hard to control). Given these advantages, we have adopted the FM1–43 approach to test the effect of roscovitine on transmitter release.

In summary, the results described here have revealed a novel activator of P/Q-type calcium channels in central neurons. Roscovitine not only acts as a specific inhibitor of cdk5, but also modifies calcium channels and hence transmitter release. Investigation of the interaction of roscovitine with calcium channels may provide new insights not only into a unique function of this important drug for potential clinical use, but also into structures involved in calcium channel gating.

Acknowledgments

We thank Dr Li-Huei Tsai at Harvard Medical School for providing p35 knockout mice. This work was supported by a NARSAD Young Investigator Award (to Z. Y.) and US Public Health Service Grants MH 40899 and DA 10044 (to P. G.).

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