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Risperidone inhibits voltage-gated sodium channels
European Journal of Pharmacology 728 (2014) 100–106
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Risperidone inhibits voltage-gated sodium channels Jan M. Brauner a,b, Sabine Hessler b, Teja W. Groemer a, Christian Alzheimer b, Tobias Huth b,n a b
Department of Psychiatry and Psychotherapy, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsstr. 17, 91054 Erlangen, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 11 October 2013 Received in revised form 26 December 2013 Accepted 26 January 2014 Available online 5 February 2014
In contrast to several other antipsychotic drugs, the effects of the atypical antipsychotic risperidone on voltage-gated sodium channels have not been characterized yet, despite its wide clinical use. Here we performed whole-cell voltage-clamp recordings to analyze the effects of risperidone on voltagedependent sodium currents of N1E-115 mouse neuroblastoma cells carried by either endogenous sodium channels or transfected NaV1.6 channels. Risperidone inhibited both endogenous and NaV1.6-mediated sodium currents at concentrations that are expected around active synaptic release sites owing to its strong accumulation in synaptic vesicles. When determined for pharmacologically isolated NaV1.6, risperidone inhibited peak inward currents with an IC50 of 49 mM. Channel block occurred in a statedependent fashion with risperidone displaying a fourfold higher affinity for the inactivated state than for the resting state. As a consequence of the low state dependence, risperidone produced only a small, but significant leftward shift of the steady-state inactivation curve and it required concentrations Z30 mM to significantly slow the time course of recovery from inactivation. Risperidone (10 mM) gave rise to a pronounced use-dependent block when sodium currents were elicited by trains of brief voltage pulses at higher frequencies. Our data suggest that, compared to other antipsychotic drugs as well as to local anesthetics and sodium channel-targeting anticonvulsants, risperidone displays an unusual blocking profile where a rather low degree of state dependence is associated with a prominent use-dependent block. & 2014 Elsevier B.V. All rights reserved.
Keywords: Risperidone Sodium channel NaV1.6 Use dependence Antipsychotic drug Schizophrenia Chemical compounds studied in this article: Risperidone (PubChem CID: 5073)
1. Introduction Risperidone (RIS), a benzisoxazole compound, is a clinically important atypical antipsychotic drug with a characteristic binding profile at dopamine D2 receptors and, in contrast to typical antipsychotic drugs (APD), to 5HT2A receptors (Schotte et al., 1996). Besides transmitter receptors, RIS, like other APD, also targets ion channels. For example, RIS might prolong the QT interval by inhibiting the cardiac potassium channel hERG (Crumb et al., 2006). More recently, inhibition of neuronal voltage-gated sodium channels (NaV) by APD has garnered considerable attention, as it might represent a novel mechanism of action. Being weak bases, APD get trapped in acidic synaptic vesicles and accumulate at intravesicular concentrations 50–120 times higher than their mean therapeutic plasma concentrations (Tischbirek et al., 2012). When APD are released by synaptic vesicle exocytosis, the synaptic cleft is exposed to high concentrations of APD and further
n
Corresponding author. Tel.: þ 49 9131 8522495; fax: þ 49 9131 8522497. E-mail address: [email protected] (T. Huth).
http://dx.doi.org/10.1016/j.ejphar.2014.01.062 0014-2999 & 2014 Elsevier B.V. All rights reserved.
exocytosis is inhibited by the block of presynaptic NaV in a usedependent fashion (Tischbirek et al., 2012; Yang and Wang, 2005). Whereas a direct interaction with NaV has been proven for some APD (Lenkey et al., 2010; Bolotina et al., 1992; Ito et al., 1997; Ogata et al., 1990; Wakamori et al., 1989) it is still unknown whether RIS, one of the most prescribed APD worldwide (Aparasu and Bhatara, 2006; Schwabe and Paffrath, 2012), also employs this mechanism. Here, we characterized the effects of RIS on neuronal NaV and found that, at clinically relevant concentrations, the drug inhibited sodium currents in a strongly use-dependent fashion. Unlike typical APD or “classical” NaV blockers like local anesthetics or anticonvulsants such as phenytoin, RIS exhibits little state dependence suggesting an unusual blocking profile.
2. Material and methods 2.1. Cell culture N1E-115 mouse neuroblastoma cells as previously described (Huth et al., 2008) were cultured at 37 1C in 5% CO2 in DMEM medium
J.M. Brauner et al. / European Journal of Pharmacology 728 (2014) 100–106
(Gibco, Karlsruhe, Germany) with 5 g/l glucose, supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany).
2.2. Plasmid and transfection mNaV1.6 in pCDNA3 was a gift from E. Leipold and S. Heinemann (Leipold et al., 2006). mNaV1.6r, a tetrodotoxin (TTX)-resistant mutant (Y371S) of mNaV1.6, was created with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Stratagene, USA) (Herzog et al., 2003; Wittmack et al., 2005). N1E-115 cells were trypsinized and plated in 3.5 cm dishes (Corning, Lowell, MA, USA). The following day, the cells were transfected with 0.9 mg of NaV1.6r and 0.4 mg of pEGFP-C1 (Mountain View, CA, USA) with the use of Nanofectin (PAA, Pasching, Austria), according to the manufacturer's protocol. Transfected cells were recorded 2 days after transfection.
2.3. Electrophysiology Transfected N1E-115 cells were identified by green fluorescence. TTX (1 mM, Biotrend AG, Wangen, Switzerland) was used to isolate NaV1.6r-mediated currents from TTX-sensitive endogenous sodium currents (Fig. 1A and B). Current signals were recorded in whole-cell voltage-clamp mode at room temperature (217 1 1C) using a Multiclamp 700B amplifier in conjunction with a Digidata1322A and pClamp10 software (Molecular Devices, Sunnyvale, CA). Recordings were sampled at 100 kHz (20 kHz for use dependence experiments) and filtered with a 6 kHz Bessel filter. P/5 leak correction was applied to activation protocols and P/4 leak correction to recovery protocols. Patch electrodes were made from borosilicate glass (Harvard Apparatus, Edenbrigde, Kent, UK) using a DMZ-Universal Puller (Zeitz-Instruments, Munich, Germany). Pipette resistance in the bath solution was 1.5–2.1 MΩ. Series resistance in whole-cell mode was o6 MΩ before compensation ( Z75%). Pipette solution was composed of (in mM) 125 CsCl, 5 NaCl, 2 Mg2ATP, 10 Hepes free acid and 5 EGTA adjusted to pH 7.2 with CsOH. Bath solution contained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 D-Glucose, 10 Hepes free acid and 10 TEA adjusted to pH 7.4 with NaOH. Risperidone (RIS, Sigma-Aldrich, Deisenhofen, Germany) was dissolved in DMSO (Sigma-Aldrich, Deisenhofen, Germany) and added to the bath solution. The total concentration of DMSO in the bath solution was 0.03%, 0.1%, 0.3%, and 1% for 3 μΜ, 10 μΜ, 30 μΜ and 100 μΜ RIS, respectively. To exclude possible DMSO effects, DMSO in the respective concentration was also added to the control solutions. RIS solution or control solution was applied using a rapid, gravity-driven Y-tube system. The experimental protocol was as follows: recording started 4 min after whole-cell access. A sequence of voltage protocols was recorded in bath solution. Then RIS solution was applied using a rapid, gravitydriven Y-tube system. 5 min after perfusion, the inhibition had reached its maximal amplitude (data not shown) and the same sequence of voltage protocols was recorded again. To exclude possible effects not attributable to RIS, control experiments (equal DMSO concentration) were performed following the same procedure. Each cell was subjected to just one concentration of RIS.
2.4. Data analysis From the activation protocols, the whole-cell sodium conductance G was calculated for every command potential V according
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to the following equation: G¼
I : V Erev
The equilibrium potential Erev for sodium was 85.3 mV under our experimental conditions. G was normalized and fitted with a Boltzmann function of the form V V mid : Gnorm ¼ 1= 1 þ exp k For dose–response relationships, we used the peak inward current of I/V-curves. The normalized current was fitted with a logistic relation: ½RIS p I norm ¼ 1= 1 þ : IC50 Steady-state inactivation was analyzed by normalizing the sodium current to the maximum peak current of the protocol (100% availability). The dependence on the pre-pulse potential V was fitted by a Boltzmann function V V mid I norm ¼ 1= 1 þexp : k For the calculation of the dissociation constant Ki of RIS from inactivated channels, we used the equation of Bean et al. (1983) for 1:1 binding stoichiometry: ½RIS ½RIS ΔV ¼ k ln 1 þ 1þ ; kr ki where ΔV is the drug-induced shift in the steady-state inactivation curve, and kr and ki the dissociation constants for the resting and inactivated channel states, respectively. Recovery from inactivation was analyzed as follows: for every inter-pulse interval Δt, the peak current of the second (test) pulse was divided by the current of the first (conditioning) pulse. For control experiments, the data points were fitted by a monoexponential function of the form Δt I norm ¼ 1 exp :
τ
However, in the presence of Z30 mM RIS, the data points were more adequately fitted with the sum of two exponentials using the following equation: Δt Δt I norm ¼ 1 Af ast exp Aslow exp ;
τf ast
τslow
where Afast and Aslow are the percentages of fast and slow recovering currents, respectively, and τfast and τslow are the respective time constants. All curve fitting was performed in Origin 9pro (OriginLab Corporation, Northampton, MA, USA) using the Levenberg–Marquardt algorithm. 2.5. Data and statistical analysis Data are given as mean 7 S.E.M. In order to exclude possible effects of DMSO, we chose not to use paired statistics (before vs. during application of RIS), but to test instead the effect of RIS vs. the effect of DMSO alone (unpaired statistics). Shift of halfmaximal activation and inactivation was analyzed as follows. The voltage shift Vmid of control vs. DMSO treatment was calculated for the control group. The shift of control vs. RIS treatment was
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Fig. 1. Risperidone inhibits endogenous and heterologously expressed NaV1.6r sodium channels in N1E-115 cells. (A) Overlay of endogenous N1E-115 current responses of a representative cell evoked by depolarizing voltage steps of increasing amplitude before and during application of 10 mM risperidone and after application of 1 mm TTX. (Inset) Activation protocol. (B) Overlay of NaV1.6r current responses of a representative cell evoked by depolarizing voltage steps of increasing amplitude before, during and after application of 10 mM risperidone. (Inset) Activation protocol. (C) I–V-relationships for endogenous sodium currents (red data points and curves) and isolated NaV1.6r currents (black data points and curves) were determined in the absence (dotted curves) and presence of 10 mM risperidone (solid curves) using the activation protocol of (A). (D) Steady-state activation curves in the absence and presence of 10 μΜ RIS were calculated from the like-labeled I–V curves of (B) as stated in Section 2. (E) Dose–response relationship for inhibition of NaV1.6r currents by RIS. Normalized peak sodium currents of the activation protocol were plotted against RIS concentration. Data points were fitted with a logistic function. IC50 ¼ 45.8 7 4.3 μΜ; P¼ 0.87 70.08. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
calculated for the RIS group. Both groups were compared with a two sample t-test using Origin 9pro. Recovery from inactivation was tested similarly. The percent current difference of control and DMSO vs. control and RIS was point-wise compared with a two sample t-test using Origin 9pro. In each category (endogenous current at 10 mM RIS; NaV1.6r at 3/10/30/100 mM RIS) the DMSO control group consisted of three cells. Use dependence of block was tested with a paired two sample t-test (Origin 9pro), because the used DMSO concentration was low (0.1%). Significance was determined at P o0.05.
3. Results We studied the effects of risperidone (RIS) on endogenous (Fig. 1A) and transfected sodium currents (Fig. 1B) in a neuroblastoma cell line (N1E-115) using whole-cell voltage-clamp recordings. In the first set of experiments, sodium currents were gradually activated from a holding potential of 90 mV by depolarizing voltage steps of
increasing amplitude. As shown in Fig. 1A and B, RIS (10 mM) produced a reversible inhibition of sodium currents. To obtain I/V-relationships, we plotted the amplitude of peak inward currents as a function of the command voltage (Fig. 1C). For endogenous as well as for transfected NaV1.6r-mediated current, RIS (10 mM) reduced the peak current by about 25% (endogenous current: 27.375.7%, n¼10; NaV1.6r: 24.675.9%, n¼11). When we transformed I/V-curves to steady-state activation curves (see Section 2), it became obvious that RIS at concentrations up to 100 mM did not significantly alter the voltage dependence of activation (Fig. 1D, endogenous current, 10 μΜ RIS: Vmid from 17.571.0 mV to 19.1 70.8 mV, n ¼10; NaV1.6r, 10 μΜ RIS: from 23.17 1.2 mV to 25.8 71.5 mV, n ¼11; NaV1.6r, 30 μΜ: from 21.670.8 mV to 24.97 0.8 mV, n¼ 14; NaV1.6r, 100 μΜ : Vmid from 20.4 71.3 mV to 21.971.3 mV, n ¼15). We next used the activation protocol to determine the dose– response relationship for the inhibition of NaV1.6-mediated currents by RIS (Fig. 1E). The dose–response curve was well fitted by a logistic function, yielding half-maximal inhibition at
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Fig. 2. Risperidone suppresses NaV1.6r currents in a use-dependent fashion. (A, B) Overlay of NaV1.6r current responses evoked by brief repetitive depolarizing voltage steps (see the inset) at 20 Hz before (A) and during 10 mM RIS (B). Recordings were leak-corrected off-line. (C, D) Peak currents were normalized to amplitude of the first pulse and plotted against time for trains delivered at 1 Hz (C) and 20 Hz (D) in the absence (black data points) and presence of 10 mM RIS (blue data points). nnnPo 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
45.8 7 4.3 μΜ (5 r n r 19) and a power factor P ¼ 0.87 7 0.08, suggesting a 1:1 stoichiometry of drug binding. To explore the extent of use dependence in the blocking action of RIS, we determined NaV1.6r responses during repetitive activation by brief depolarizing voltage pulses, which were delivered at frequencies of either 1 or 20 Hz (Fig, 2A and B). Under control conditions, the current amplitudes remained constant at 1 Hz (n ¼10) and exhibited only a slight decrease at 20 Hz (reduction to 88.1 70.9% of I0, n ¼10; Fig. 2C and D, black data points). The small frequency-dependent reduction in current amplitude under control conditions was dramatically enhanced in the presence of 10 mM RIS. Whereas the drug was only moderately effective at 1 Hz (decrease of NaV1.6r response to 92.0 72.4%, n ¼10, P o0.001), more than half of the current was suppressed at 20 Hz (decrease to 44.5 72.0%, n ¼10, P o0.001, Fig. 2). Note that the data points depicted in Fig. 2C and D were normalized to the current amplitude attained in the absence or presence of RIS before a train of voltage pulses was delivered. Thus, the data points show the use-dependent (dynamic) block of NaV1.6 that occurred in addition to an already existing tonic block. To investigate the state dependence of the channel block by RIS, we employed a steady-state inactivation protocol, where conditioning pulses of 1 s to various potentials were followed by a test pulse to 0 mV. Steady-state inactivation curves were obtained by plotting the normalized peak current amplitude as a function of the conditioning potential (Fig. 3A). The curves were well fitted by a Boltzmann function. RIS (10 mM) produced a slight shift of the half-inactivation potential Vmid to more negative values for both the endogenous current (from 69.8 71.5 mV to 72.9 7 1.5 mV, n ¼10, Po 0.05 vs. DMSO control group) and NaV1.6r (from 71.1 72.5 mV to 74.1 72.7 mV, n ¼11, P o0.05). The halfinactivation potentials were shifted from 72.4 71.2 mV to 78.6 7 2.0 mV (n ¼13, P o0.001), and from 69.4 71.3 mV to 80.7 7 1.5 mV (n ¼ 13, Po 0.001) for 30 μΜ RIS and 100 μΜ RIS, respectively.
The shift in the voltage dependence of steady-state inactivation indicates a preferential binding of RIS to the inactivated channel. We determined the apparent dissociation constant Ki of RIS for the inactivated state, based on a four channel state model (Bean et al., 1983). Using this model, Ki is calculated from the shift of the inactivation curves (see Section 2). Here, our data yielded Ki values of 12.3, 11.9 and 9.2 μΜ for 10, 30 and 100 μΜ RIS, respectively. Compounds that produce a shift in steady-state inactivation are also expected to slow recovery from inactivation (Bean et al., 1983; Jo and Bean, 2011). We examined the channel repriming using a double-pulse protocol (Fig. 3B). Two 5 ms depolarizing voltage steps to 0 mV with variable inter-pulse interval Δt at 90 mV were applied and the peak sodium current of the second pulse was expressed as a fraction of that of the first pulse. When examined at 10 mM, RIS did not significantly affect the mono-exponential time course of recovery for endogenous sodium currents (n¼10) or for NaV1.6r (Fig. 3C, n¼ 11). However, higher concentrations (30– 100 mM) of RIS produced a considerable slowing of NaV1.6r so that even after a 1 s interval, the amplitude of the second current reached only 90–95% of that of the first current (Fig. 3D). In quantitative terms, NaV1.6 recovered from inactivation along a mono-exponential curve with a time constant of 6.870.7 ms (n¼11) under control conditions. In the presence of 30 μΜ and 100 μΜ RIS, the time course of recovery required a bi-exponential fit. Whereas the majority of the current recovered almost as fast as under control conditions (30 μΜ RISː τfast ¼7.970.9 ms, n¼ 13; 100 μΜ RIS: 8.971.1 ms, n¼13), a percentage of channels displayed much slower repriming. For 30 μM RIS, 7.670.3% (percentages calculated from the amplitude factors of the bi-exponential fit) of current recovered with τslow ¼10447113 ms. For 100 μM RIS, the percentage of slowly recovering channels increased to 19.972.1% and τslow was 13487109 ms (Fig. 3D). The appearance of a slow component of recovery in the presence of higher drug concentrations suggests that, as the concentration was raised, more channels were in
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Fig. 3. Risperidone shifts steady-state inactivation and impairs recovery from inactivation. (A) Voltage protocol with conditioning pre-pulses (inset) was used to construct steady-state inactivation curves of endogenous sodium currents (red data points and curves) and NaV1.6r currents (black data points and curves) before (dotted lines) and during 10 μM RIS (solid lines). (B) Representative current traces from a double-pulse protocol with increasing time interval Δt that was used to determine recovery from inactivation. Sodium currents were evoked by brief voltage steps to 0 mV from a holding potential of 90 mV. Note that time axis was logarithmically transformed. Numbers above current responses indicate duration of Δt in ms. (C) Double-pulse protocol of (B) was used to study the effect of 10 mM RIS on repriming of endogenous and NaV1.6r channels. Same labeling as in (A). Data points were fitted by a mono-exponential function. A point-wise comparison between control and RIS for every inter-pulse interval was made as stated in Section 2. (D) High concentration of RIS (100 mM) significantly impaired repriming of NaV1.6r channels as indicated by the appearance of a second, much slower time constant of recovery. nPo 0.05, nnPo 0.01, and nnnP o 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
drug-bound state, from where repriming required additional time (Bean et al., 1983).
4. Discussion We have studied the effects of RIS on endogenous sodium currents of cultured N1E-115 cells, which are mainly mediated by sodium channel subtypes NaV1.1, 1.2, and 1.3 (Benzinger et al., 1999; Jo and Bean, 2011), and on a TTX-insensitive variant of NaV1.6, which was transfected into the same cell line. All these channel subtypes are widely expressed in central and peripheral neurons (Candenas et al., 2006). Notably, NaV1.6 is thought to act as an axonal “detonator” owing to its comparably low activation potential and its strategic location at the axon initial segment, the trigger site for action potentials, where it is present at high density (Royeck et al., 2008). The use of the TTX-resistant NaV1.6r channel variant is an established method to record selectively NaV1.6 mediated currents in cells with endogenous TTX-sensitive channels (Herzog et al., 2003; Wittmack et al., 2005). Resistance to TTX is introduced by a point mutation in the TTX binding region (Y371S). We do not expect the effect of RIS on the mutant channel to be different from the effect on wildtype NaV1.6, because the established local anesthetic binding site does not overlap with the TTX binding site (Catterall, 2012). RIS inhibited voltage-gated sodium channels in a use- and state-dependent fashion. The dissociation constant for resting NaV1.6r was Kr ¼45.8 μΜ. RIS (10 μM) shifted the steady-state inactivation curve to more negative potentials. In principle, small shifts in voltage dependence might arise from a voltage error due to residual series resistance. RIS inhibits the peak sodium current
and could therefore reduce the voltage drop across residual series resistance. This would cause a shift in the apparent voltage dependence of sodium current. However, such a shift was not observed for activation curves in the presence of r100 μM RIS (66% peak current inhibition). Furthermore, reducing the voltage drop across residual series resistance would be expected to shift the steady-state inactivation curve to more positive potentials (Marty and Neher, 1995; Traynelis, 1998). The mean dissociation constant (Ki ¼ 11.17 1.0 μΜ) was calculated from the dosedependent shift of the steady-state inactivation curves. The resulting state dependence (K r =K i ¼ 4:1) indicated a four times higher affinity to the inactivated states. At higher concentrations (30–100 μΜ) RIS caused a percentage of channels to recover from inactivation with slower kinetics, while the majority of current recovered with kinetics similar to control conditions. This observation is compatible with the notion that a percentage of channels binds RIS while in open or inactivated states and drug-bound channels reprime more slowly. Similar effects on recovery are typically obtained with local anesthetics (Bean et al., 1983). Our study demonstrates that RIS is able to inhibit neuronal NaV activity. Although psychotic patients treated with RIS typically show plasma concentrations in the range of 0.1 μΜ, the pronounced accumulation of the drug in synaptic vesicles (see Introduction) has been calculated to entail intravesicular concentrations of about 7 μM (Tischbirek et al., 2012). During strong neuronal activity, when RIS is synaptically co-released with neurotransmitters, pre- and postsynaptic NaV located in the vicinity of synapses will be hence exposed to low micromolar concentrations, which we show here to be able to exert a considerable impact on sodium channel gating. Notably, the usedependent inhibition of NaV by RIS does not only arise from the
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way in which its delivery is contingent upon synaptic activity. Our experiments indicate that use dependence is also an important intrinsic feature of the interaction of the drug with the channel. A characteristic property of clinically used NaV blockers is their preferential binding to sodium channels in their inactivated state, whereas the affinity to resting channels is much lower. This state dependence is expressed as the ratio between the apparent dissociation constants of the drug for the resting and the inactivated state (Kr/Ki). In a recent study, we found that the state dependence of the typical antipsychotic drug haloperidol for NaV1.6 was 57 (Tischbirek et al., 2012). Another typical antipsychotic, chlorpromazine, inhibited the endogenous sodium currents of N1E-115 cells in a similar experimental setup with a state dependence of 13 (Ogata et al., 1990). In a survey that probed a number of widely used antipsychotics, antidepressants, anticonvulsants and local anesthetics that all act on NaV, values for their state dependence were in the same order of magnitude (Lenkey et al., 2010). Here, we found that RIS behaves differently. With a state dependence of only 4.1, RIS displays an unusually low binding preference for the inactivated state. As a consequence, RIS produced only a minor shift of steady-state channel availability towards more negative potentials. Also, repriming of sodium channels after depolarization-induced inactivation was not appreciably affected by RIS. A significant slowing of recovery from inactivation was only observed when we applied RIS at concentrations (Z 30 mM) well exceeding the calculated level at synaptic release sites. So far, all neuropsychiatric drugs that were found to inhibit NaV displayed a pronounced state dependence (Lenkey et al., 2010; Tischbirek et al., 2012; Ogata et al., 1990). By contrast, the extent of use dependence appeared more variable and, of note, this feature was completely absent in several drugs. For example, the anticonvulsant carbamazepine shows a 44 times higher affinity for inactivated than for resting sodium channels, but its block does not occur in a use-dependent fashion (Lenkey et al., 2010; Ragsdale et al., 1991; see also Lang et al. (1993)). Thus, compared to other NaV-targeting neuropsychiatric drugs, the blocking profile of RIS appears unusual in that it exerts a powerful use-dependent (dynamic) block that is accompanied by little state-dependent (tonic) inhibition. It remains to be determined whether this profile is a particular characteristic of RIS or a more general feature of atypical antipsychotic drugs, because data on the effect of atypical APD on NaV are still very sparse. It is widely believed that a pronounced state dependence is an essential requirement for a NaV blocker to be considered a safe drug: the greater its state dependence, the more reliable the prediction that the drug leaves normal firing largely intact and suppresses mainly aberrant discharges. Do our data then question the safety of RIS? In view of the particular mechanism of drug release that requires strong synaptic activation, it seems unlikely that the ambient concentration of RIS in the overall extracellular space will reach levels that appreciably interfere with normal firing. Remember that RIS inhibited resting channels with an IC50 of 46 mM, whereas its concentration in synaptic vesicles was assumed to attain 7 mM at therapeutic plasma levels of about 0.1 mM. Furthermore, owing to its selective accumulation in intracellular vesicles, pharmacologically effectual concentrations of RIS will only be transiently reached at and around highly active synapses. Thus, rather than having all axonal and somatodendritic sodium channels exposed to a fairly equal drug level, considerable channel block should be restricted to a confined space around synaptic “hotspots”. Whereas our experiments in a heterologous expression system firmly establish an intriguing and unusual role of RIS in NaV gating, this approach inevitably leaves a number of questions open regarding the impact that these effects will have in intact neuronal
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networks. Let us consider, for example, an inhibitory axo-axonic synapse that impinges upon the initial segment of a pyramidal cell axon. Assuming that RIS will be co-released with GABA, one might expect that RIS supports the inhibitory action of GABA by blocking the NaV1.6 “detonator” of the initial segment, but the drug might also cause a feed-back inhibition of GABA release by blocking presynaptic NaV, thereby curtailing synaptic inhibition. What effect will prevail? Another question relates to glutamatergic synapses, where RIS might interfere with high frequency activity required for synaptic plasticity, and thus impair essential mechanisms of cognitive functions, learning and memory. However, in view of the glutamate hypothesis of schizophrenia, which posits that interneuron dysfunction leads to disinhibition of glutamatergic neurons and excessive glutamate release (Moghaddam and Javitt, 2012), the use-dependent block of overactive glutamatergic synapses might emerge as a therapeutic action of risperidone. Only with issues like these resolved, we will be in a position to address two more fundamental questions: first, do the effects of RIS on NaV contribute to its therapeutic benefits or do they rather mediate adverse reactions, and, second, are the distinct blocking properties of RIS at NaV, that we have explored here and compared to those of haloperidol, linked to the different psychopharmacological profiles of the two APD in psychotic patients?
Acknowledgments We thank Iwona Izydorczyk for technical assistance. T.G. was supported by a grant from the Interdisciplinary Centre for Clinical Research Erlangen (IZKF). C.A. and T.H. were supported by intramural grants of the University of Erlangen-Nürnberg.
References Aparasu, R.R., Bhatara, V., 2006. Antipsychotic use and expenditure in the United States. Psychiatr. Serv. 57, 1693. Bean, B.P., Cohen, C.J., Tsien, R.W., 1983. Lidocaine block of cardiac sodium channels. J. Gen. Physiol. 81, 613–642. Benzinger, G.R., Tonkovich, G.S., Hanck, D.A., 1999. Augmentation of recovery from inactivation by site-3 Na channel toxins. A single-channel and whole-cell study of persistent currents. J. Gen. Physiol. 113, 333–346. Bolotina, V., Courtney, K.R., Khodorov, B., 1992. Gate-dependent blockade of sodium channels by phenothiazine derivatives: structure-activity relationships. Mol. Pharmacol. 42, 423–431. Candenas, L., Seda, M., Noheda, P., Buschmann, H., Cintado, C.G., Martin, J.D., Pinto, F.M., 2006. Molecular diversity of voltage-gated sodium channel alpha and beta subunit mRNAs in human tissues. Eur. J. Pharmacol. 541, 9–16. Catterall, W.A., 2012. Voltage-gated sodium channels at 60: structure, function and pathophysiology. J. Physiol. 590, 2577–2589. Crumb , W.J., Ekins, S., Sarazan, R.D., Wikel, J.H., Wrighton, S.A., Carlson, C., Beasley , C.M., 2006. Effects of antipsychotic drugs on I(to), I (Na), I (sus), I (K1), and hERG: QT prolongation, structure activity relationship, and network analysis. Pharm. Res. 23, 1133–1143. Herzog, R.I., Cummins, T.R., Ghassemi, F., Dib-Hajj, S.D., Waxman, S.G., 2003. Distinct repriming and closed-state inactivation kinetics of NaV1.6 and NaV1.7 sodium channels in mouse spinal sensory neurons. J. Physiol. 551, 741–750. Huth, T., Schmidtmayer, J., Alzheimer, C., Hansen, U.P., 2008. Four-mode gating model of fast inactivation of sodium channel NaV1.2a. Pflug. Arch. 457, 103–119. Ito, K., Nishimura, Y., Uji, Y., Yamamoto, T., 1997. Haloperidol effects on Na current in acutely isolated rat retinal ganglion cells. Japan. J. Ophthalmol. 41, 221–225. Jo, S., Bean, B.P., 2011. Inhibition of neuronal voltage-gated sodium channels by brilliant blue G. Mol. Pharmacol. 80, 247–257. Lang, D.G., Wang, C.M., Cooper, B.R., 1993. Lamotrigine, phenytoin and carbamazepine interactions on the sodium current present in N4TG1 mouse neuroblastoma cells. J. Pharmacol. Exp. Ther. 266, 829–835. Leipold, E., Hansel, A., Borges, A., Heinemann, S.H., 2006. Subtype specificity of scorpion beta-toxin Tz1 interaction with voltage-gated sodium channels is determined by the pore loop of domain 3. Mol. Pharmacol. 70, 340–347. Lenkey, N., Karoly, R., Lukacs, P., Vizi, E.S., Sunesen, M., Fodor, L., Mike, A., 2010. Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study. PLOS One 5, e15568. Marty, A., Neher, E., 1995. Tight-seal whole-cell recording. In: Sakmann, B., Neher, E. (Eds.), Single-Channel Recording. Plenum, New York
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Moghaddam, B., Javitt, D., 2012. From revolution to evolution: the glutamate hypothesis of schizophrenia and its implication for treatment. Neuropsychopharmacology 37, 4–15. Ogata, N., Yoshii, M., Narahashi, T., 1990. Differential block of sodium and calcium channels by chlorpromazine in mouse neuroblastoma cells. J. Physiol. 420, 165–183. Ragsdale, D.S., Scheuer, T., Catterall, W.A., 1991. Frequency and voltage-dependent inhibition of type IIA Na þ channels, expressed in a mammalian cell line, by local anesthetic, antiarrhythmic, and anticonvulsant drugs. Mol. Pharmacol. 40, 756–765. Royeck, M., Horstmann, M.T., Remy, S., Reitze, M., Yaari, Y., Beck, H., 2008. Role of axonal NaV1.6 sodium channels in action potential initiation of CA1 pyramidal neurons. J. Neurophysiol. 100, 2361–2380. Schotte, A., Janssen, P.F., Gommeren, W., Luyten, W.H., Van, G.P., Lesage, A.S., De, L.K., Leysen, J.E., 1996. Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding. Psychopharmacology 124, 57–73. Schwabe, U., Paffrath, D., 2012. Arzneiverordnungs Report. Springer, Heidelberg.
Tischbirek, C.H., Wenzel, E.M., Zheng, F., Huth, T., Amato, D., Trapp, S., Denker, A., Welzel, O., Lueke, K., Svetlitchny, A., Rauh, M., Deusser, J., Schwab, A., Rizzoli, S.O., Henkel, A.W., Muller, C.P., Alzheimer, C., Kornhuber, J., Groemer, T.W., 2012. Usedependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs. Neuron 74, 830–844. Traynelis, S.F., 1998. Software-based correction of single compartment series resistance errors. J. Neurosci. Methods 86, 25–34. Wakamori, M., Kaneda, M., Oyama, Y., Akaike, N., 1989. Effects of chlordiazepoxide, chlorpromazine, diazepam, diphenylhydantoin, flunitrazepam and haloperidol on the voltage-dependent sodium current of isolated mammalian brain neurons. Brain Res. 494, 374–378. Wittmack, E.K., Rush, A.M., Hudmon, A., Waxman, S.G., Dib-Hajj, S.D., 2005. Voltage-gated sodium channel NaV1.6 is modulated by p38 mitogen-activated protein kinase. J. Neurosci. 25, 6621–6630. Yang, T.T., Wang, S.J., 2005. Effects of haloperidol and clozapine on glutamate release from nerve terminals isolated from rat prefrontal cortex. Synapse 56, 12–20.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Risperidone inhibits voltage-gated sodium channels Jan M. Brauner a,b, Sabine Hessler b, Teja W. Groemer a, Christian Alzheimer b, Tobias Huth b,n a b
Department of Psychiatry and Psychotherapy, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsstr. 17, 91054 Erlangen, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 11 October 2013 Received in revised form 26 December 2013 Accepted 26 January 2014 Available online 5 February 2014
In contrast to several other antipsychotic drugs, the effects of the atypical antipsychotic risperidone on voltage-gated sodium channels have not been characterized yet, despite its wide clinical use. Here we performed whole-cell voltage-clamp recordings to analyze the effects of risperidone on voltagedependent sodium currents of N1E-115 mouse neuroblastoma cells carried by either endogenous sodium channels or transfected NaV1.6 channels. Risperidone inhibited both endogenous and NaV1.6-mediated sodium currents at concentrations that are expected around active synaptic release sites owing to its strong accumulation in synaptic vesicles. When determined for pharmacologically isolated NaV1.6, risperidone inhibited peak inward currents with an IC50 of 49 mM. Channel block occurred in a statedependent fashion with risperidone displaying a fourfold higher affinity for the inactivated state than for the resting state. As a consequence of the low state dependence, risperidone produced only a small, but significant leftward shift of the steady-state inactivation curve and it required concentrations Z30 mM to significantly slow the time course of recovery from inactivation. Risperidone (10 mM) gave rise to a pronounced use-dependent block when sodium currents were elicited by trains of brief voltage pulses at higher frequencies. Our data suggest that, compared to other antipsychotic drugs as well as to local anesthetics and sodium channel-targeting anticonvulsants, risperidone displays an unusual blocking profile where a rather low degree of state dependence is associated with a prominent use-dependent block. & 2014 Elsevier B.V. All rights reserved.
Keywords: Risperidone Sodium channel NaV1.6 Use dependence Antipsychotic drug Schizophrenia Chemical compounds studied in this article: Risperidone (PubChem CID: 5073)
1. Introduction Risperidone (RIS), a benzisoxazole compound, is a clinically important atypical antipsychotic drug with a characteristic binding profile at dopamine D2 receptors and, in contrast to typical antipsychotic drugs (APD), to 5HT2A receptors (Schotte et al., 1996). Besides transmitter receptors, RIS, like other APD, also targets ion channels. For example, RIS might prolong the QT interval by inhibiting the cardiac potassium channel hERG (Crumb et al., 2006). More recently, inhibition of neuronal voltage-gated sodium channels (NaV) by APD has garnered considerable attention, as it might represent a novel mechanism of action. Being weak bases, APD get trapped in acidic synaptic vesicles and accumulate at intravesicular concentrations 50–120 times higher than their mean therapeutic plasma concentrations (Tischbirek et al., 2012). When APD are released by synaptic vesicle exocytosis, the synaptic cleft is exposed to high concentrations of APD and further
n
Corresponding author. Tel.: þ 49 9131 8522495; fax: þ 49 9131 8522497. E-mail address: [email protected] (T. Huth).
http://dx.doi.org/10.1016/j.ejphar.2014.01.062 0014-2999 & 2014 Elsevier B.V. All rights reserved.
exocytosis is inhibited by the block of presynaptic NaV in a usedependent fashion (Tischbirek et al., 2012; Yang and Wang, 2005). Whereas a direct interaction with NaV has been proven for some APD (Lenkey et al., 2010; Bolotina et al., 1992; Ito et al., 1997; Ogata et al., 1990; Wakamori et al., 1989) it is still unknown whether RIS, one of the most prescribed APD worldwide (Aparasu and Bhatara, 2006; Schwabe and Paffrath, 2012), also employs this mechanism. Here, we characterized the effects of RIS on neuronal NaV and found that, at clinically relevant concentrations, the drug inhibited sodium currents in a strongly use-dependent fashion. Unlike typical APD or “classical” NaV blockers like local anesthetics or anticonvulsants such as phenytoin, RIS exhibits little state dependence suggesting an unusual blocking profile.
2. Material and methods 2.1. Cell culture N1E-115 mouse neuroblastoma cells as previously described (Huth et al., 2008) were cultured at 37 1C in 5% CO2 in DMEM medium
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(Gibco, Karlsruhe, Germany) with 5 g/l glucose, supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany).
2.2. Plasmid and transfection mNaV1.6 in pCDNA3 was a gift from E. Leipold and S. Heinemann (Leipold et al., 2006). mNaV1.6r, a tetrodotoxin (TTX)-resistant mutant (Y371S) of mNaV1.6, was created with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Stratagene, USA) (Herzog et al., 2003; Wittmack et al., 2005). N1E-115 cells were trypsinized and plated in 3.5 cm dishes (Corning, Lowell, MA, USA). The following day, the cells were transfected with 0.9 mg of NaV1.6r and 0.4 mg of pEGFP-C1 (Mountain View, CA, USA) with the use of Nanofectin (PAA, Pasching, Austria), according to the manufacturer's protocol. Transfected cells were recorded 2 days after transfection.
2.3. Electrophysiology Transfected N1E-115 cells were identified by green fluorescence. TTX (1 mM, Biotrend AG, Wangen, Switzerland) was used to isolate NaV1.6r-mediated currents from TTX-sensitive endogenous sodium currents (Fig. 1A and B). Current signals were recorded in whole-cell voltage-clamp mode at room temperature (217 1 1C) using a Multiclamp 700B amplifier in conjunction with a Digidata1322A and pClamp10 software (Molecular Devices, Sunnyvale, CA). Recordings were sampled at 100 kHz (20 kHz for use dependence experiments) and filtered with a 6 kHz Bessel filter. P/5 leak correction was applied to activation protocols and P/4 leak correction to recovery protocols. Patch electrodes were made from borosilicate glass (Harvard Apparatus, Edenbrigde, Kent, UK) using a DMZ-Universal Puller (Zeitz-Instruments, Munich, Germany). Pipette resistance in the bath solution was 1.5–2.1 MΩ. Series resistance in whole-cell mode was o6 MΩ before compensation ( Z75%). Pipette solution was composed of (in mM) 125 CsCl, 5 NaCl, 2 Mg2ATP, 10 Hepes free acid and 5 EGTA adjusted to pH 7.2 with CsOH. Bath solution contained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 D-Glucose, 10 Hepes free acid and 10 TEA adjusted to pH 7.4 with NaOH. Risperidone (RIS, Sigma-Aldrich, Deisenhofen, Germany) was dissolved in DMSO (Sigma-Aldrich, Deisenhofen, Germany) and added to the bath solution. The total concentration of DMSO in the bath solution was 0.03%, 0.1%, 0.3%, and 1% for 3 μΜ, 10 μΜ, 30 μΜ and 100 μΜ RIS, respectively. To exclude possible DMSO effects, DMSO in the respective concentration was also added to the control solutions. RIS solution or control solution was applied using a rapid, gravity-driven Y-tube system. The experimental protocol was as follows: recording started 4 min after whole-cell access. A sequence of voltage protocols was recorded in bath solution. Then RIS solution was applied using a rapid, gravitydriven Y-tube system. 5 min after perfusion, the inhibition had reached its maximal amplitude (data not shown) and the same sequence of voltage protocols was recorded again. To exclude possible effects not attributable to RIS, control experiments (equal DMSO concentration) were performed following the same procedure. Each cell was subjected to just one concentration of RIS.
2.4. Data analysis From the activation protocols, the whole-cell sodium conductance G was calculated for every command potential V according
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to the following equation: G¼
I : V Erev
The equilibrium potential Erev for sodium was 85.3 mV under our experimental conditions. G was normalized and fitted with a Boltzmann function of the form V V mid : Gnorm ¼ 1= 1 þ exp k For dose–response relationships, we used the peak inward current of I/V-curves. The normalized current was fitted with a logistic relation: ½RIS p I norm ¼ 1= 1 þ : IC50 Steady-state inactivation was analyzed by normalizing the sodium current to the maximum peak current of the protocol (100% availability). The dependence on the pre-pulse potential V was fitted by a Boltzmann function V V mid I norm ¼ 1= 1 þexp : k For the calculation of the dissociation constant Ki of RIS from inactivated channels, we used the equation of Bean et al. (1983) for 1:1 binding stoichiometry: ½RIS ½RIS ΔV ¼ k ln 1 þ 1þ ; kr ki where ΔV is the drug-induced shift in the steady-state inactivation curve, and kr and ki the dissociation constants for the resting and inactivated channel states, respectively. Recovery from inactivation was analyzed as follows: for every inter-pulse interval Δt, the peak current of the second (test) pulse was divided by the current of the first (conditioning) pulse. For control experiments, the data points were fitted by a monoexponential function of the form Δt I norm ¼ 1 exp :
τ
However, in the presence of Z30 mM RIS, the data points were more adequately fitted with the sum of two exponentials using the following equation: Δt Δt I norm ¼ 1 Af ast exp Aslow exp ;
τf ast
τslow
where Afast and Aslow are the percentages of fast and slow recovering currents, respectively, and τfast and τslow are the respective time constants. All curve fitting was performed in Origin 9pro (OriginLab Corporation, Northampton, MA, USA) using the Levenberg–Marquardt algorithm. 2.5. Data and statistical analysis Data are given as mean 7 S.E.M. In order to exclude possible effects of DMSO, we chose not to use paired statistics (before vs. during application of RIS), but to test instead the effect of RIS vs. the effect of DMSO alone (unpaired statistics). Shift of halfmaximal activation and inactivation was analyzed as follows. The voltage shift Vmid of control vs. DMSO treatment was calculated for the control group. The shift of control vs. RIS treatment was
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Fig. 1. Risperidone inhibits endogenous and heterologously expressed NaV1.6r sodium channels in N1E-115 cells. (A) Overlay of endogenous N1E-115 current responses of a representative cell evoked by depolarizing voltage steps of increasing amplitude before and during application of 10 mM risperidone and after application of 1 mm TTX. (Inset) Activation protocol. (B) Overlay of NaV1.6r current responses of a representative cell evoked by depolarizing voltage steps of increasing amplitude before, during and after application of 10 mM risperidone. (Inset) Activation protocol. (C) I–V-relationships for endogenous sodium currents (red data points and curves) and isolated NaV1.6r currents (black data points and curves) were determined in the absence (dotted curves) and presence of 10 mM risperidone (solid curves) using the activation protocol of (A). (D) Steady-state activation curves in the absence and presence of 10 μΜ RIS were calculated from the like-labeled I–V curves of (B) as stated in Section 2. (E) Dose–response relationship for inhibition of NaV1.6r currents by RIS. Normalized peak sodium currents of the activation protocol were plotted against RIS concentration. Data points were fitted with a logistic function. IC50 ¼ 45.8 7 4.3 μΜ; P¼ 0.87 70.08. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
calculated for the RIS group. Both groups were compared with a two sample t-test using Origin 9pro. Recovery from inactivation was tested similarly. The percent current difference of control and DMSO vs. control and RIS was point-wise compared with a two sample t-test using Origin 9pro. In each category (endogenous current at 10 mM RIS; NaV1.6r at 3/10/30/100 mM RIS) the DMSO control group consisted of three cells. Use dependence of block was tested with a paired two sample t-test (Origin 9pro), because the used DMSO concentration was low (0.1%). Significance was determined at P o0.05.
3. Results We studied the effects of risperidone (RIS) on endogenous (Fig. 1A) and transfected sodium currents (Fig. 1B) in a neuroblastoma cell line (N1E-115) using whole-cell voltage-clamp recordings. In the first set of experiments, sodium currents were gradually activated from a holding potential of 90 mV by depolarizing voltage steps of
increasing amplitude. As shown in Fig. 1A and B, RIS (10 mM) produced a reversible inhibition of sodium currents. To obtain I/V-relationships, we plotted the amplitude of peak inward currents as a function of the command voltage (Fig. 1C). For endogenous as well as for transfected NaV1.6r-mediated current, RIS (10 mM) reduced the peak current by about 25% (endogenous current: 27.375.7%, n¼10; NaV1.6r: 24.675.9%, n¼11). When we transformed I/V-curves to steady-state activation curves (see Section 2), it became obvious that RIS at concentrations up to 100 mM did not significantly alter the voltage dependence of activation (Fig. 1D, endogenous current, 10 μΜ RIS: Vmid from 17.571.0 mV to 19.1 70.8 mV, n ¼10; NaV1.6r, 10 μΜ RIS: from 23.17 1.2 mV to 25.8 71.5 mV, n ¼11; NaV1.6r, 30 μΜ: from 21.670.8 mV to 24.97 0.8 mV, n¼ 14; NaV1.6r, 100 μΜ : Vmid from 20.4 71.3 mV to 21.971.3 mV, n ¼15). We next used the activation protocol to determine the dose– response relationship for the inhibition of NaV1.6-mediated currents by RIS (Fig. 1E). The dose–response curve was well fitted by a logistic function, yielding half-maximal inhibition at
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Fig. 2. Risperidone suppresses NaV1.6r currents in a use-dependent fashion. (A, B) Overlay of NaV1.6r current responses evoked by brief repetitive depolarizing voltage steps (see the inset) at 20 Hz before (A) and during 10 mM RIS (B). Recordings were leak-corrected off-line. (C, D) Peak currents were normalized to amplitude of the first pulse and plotted against time for trains delivered at 1 Hz (C) and 20 Hz (D) in the absence (black data points) and presence of 10 mM RIS (blue data points). nnnPo 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
45.8 7 4.3 μΜ (5 r n r 19) and a power factor P ¼ 0.87 7 0.08, suggesting a 1:1 stoichiometry of drug binding. To explore the extent of use dependence in the blocking action of RIS, we determined NaV1.6r responses during repetitive activation by brief depolarizing voltage pulses, which were delivered at frequencies of either 1 or 20 Hz (Fig, 2A and B). Under control conditions, the current amplitudes remained constant at 1 Hz (n ¼10) and exhibited only a slight decrease at 20 Hz (reduction to 88.1 70.9% of I0, n ¼10; Fig. 2C and D, black data points). The small frequency-dependent reduction in current amplitude under control conditions was dramatically enhanced in the presence of 10 mM RIS. Whereas the drug was only moderately effective at 1 Hz (decrease of NaV1.6r response to 92.0 72.4%, n ¼10, P o0.001), more than half of the current was suppressed at 20 Hz (decrease to 44.5 72.0%, n ¼10, P o0.001, Fig. 2). Note that the data points depicted in Fig. 2C and D were normalized to the current amplitude attained in the absence or presence of RIS before a train of voltage pulses was delivered. Thus, the data points show the use-dependent (dynamic) block of NaV1.6 that occurred in addition to an already existing tonic block. To investigate the state dependence of the channel block by RIS, we employed a steady-state inactivation protocol, where conditioning pulses of 1 s to various potentials were followed by a test pulse to 0 mV. Steady-state inactivation curves were obtained by plotting the normalized peak current amplitude as a function of the conditioning potential (Fig. 3A). The curves were well fitted by a Boltzmann function. RIS (10 mM) produced a slight shift of the half-inactivation potential Vmid to more negative values for both the endogenous current (from 69.8 71.5 mV to 72.9 7 1.5 mV, n ¼10, Po 0.05 vs. DMSO control group) and NaV1.6r (from 71.1 72.5 mV to 74.1 72.7 mV, n ¼11, P o0.05). The halfinactivation potentials were shifted from 72.4 71.2 mV to 78.6 7 2.0 mV (n ¼13, P o0.001), and from 69.4 71.3 mV to 80.7 7 1.5 mV (n ¼ 13, Po 0.001) for 30 μΜ RIS and 100 μΜ RIS, respectively.
The shift in the voltage dependence of steady-state inactivation indicates a preferential binding of RIS to the inactivated channel. We determined the apparent dissociation constant Ki of RIS for the inactivated state, based on a four channel state model (Bean et al., 1983). Using this model, Ki is calculated from the shift of the inactivation curves (see Section 2). Here, our data yielded Ki values of 12.3, 11.9 and 9.2 μΜ for 10, 30 and 100 μΜ RIS, respectively. Compounds that produce a shift in steady-state inactivation are also expected to slow recovery from inactivation (Bean et al., 1983; Jo and Bean, 2011). We examined the channel repriming using a double-pulse protocol (Fig. 3B). Two 5 ms depolarizing voltage steps to 0 mV with variable inter-pulse interval Δt at 90 mV were applied and the peak sodium current of the second pulse was expressed as a fraction of that of the first pulse. When examined at 10 mM, RIS did not significantly affect the mono-exponential time course of recovery for endogenous sodium currents (n¼10) or for NaV1.6r (Fig. 3C, n¼ 11). However, higher concentrations (30– 100 mM) of RIS produced a considerable slowing of NaV1.6r so that even after a 1 s interval, the amplitude of the second current reached only 90–95% of that of the first current (Fig. 3D). In quantitative terms, NaV1.6 recovered from inactivation along a mono-exponential curve with a time constant of 6.870.7 ms (n¼11) under control conditions. In the presence of 30 μΜ and 100 μΜ RIS, the time course of recovery required a bi-exponential fit. Whereas the majority of the current recovered almost as fast as under control conditions (30 μΜ RISː τfast ¼7.970.9 ms, n¼ 13; 100 μΜ RIS: 8.971.1 ms, n¼13), a percentage of channels displayed much slower repriming. For 30 μM RIS, 7.670.3% (percentages calculated from the amplitude factors of the bi-exponential fit) of current recovered with τslow ¼10447113 ms. For 100 μM RIS, the percentage of slowly recovering channels increased to 19.972.1% and τslow was 13487109 ms (Fig. 3D). The appearance of a slow component of recovery in the presence of higher drug concentrations suggests that, as the concentration was raised, more channels were in
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Fig. 3. Risperidone shifts steady-state inactivation and impairs recovery from inactivation. (A) Voltage protocol with conditioning pre-pulses (inset) was used to construct steady-state inactivation curves of endogenous sodium currents (red data points and curves) and NaV1.6r currents (black data points and curves) before (dotted lines) and during 10 μM RIS (solid lines). (B) Representative current traces from a double-pulse protocol with increasing time interval Δt that was used to determine recovery from inactivation. Sodium currents were evoked by brief voltage steps to 0 mV from a holding potential of 90 mV. Note that time axis was logarithmically transformed. Numbers above current responses indicate duration of Δt in ms. (C) Double-pulse protocol of (B) was used to study the effect of 10 mM RIS on repriming of endogenous and NaV1.6r channels. Same labeling as in (A). Data points were fitted by a mono-exponential function. A point-wise comparison between control and RIS for every inter-pulse interval was made as stated in Section 2. (D) High concentration of RIS (100 mM) significantly impaired repriming of NaV1.6r channels as indicated by the appearance of a second, much slower time constant of recovery. nPo 0.05, nnPo 0.01, and nnnP o 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
drug-bound state, from where repriming required additional time (Bean et al., 1983).
4. Discussion We have studied the effects of RIS on endogenous sodium currents of cultured N1E-115 cells, which are mainly mediated by sodium channel subtypes NaV1.1, 1.2, and 1.3 (Benzinger et al., 1999; Jo and Bean, 2011), and on a TTX-insensitive variant of NaV1.6, which was transfected into the same cell line. All these channel subtypes are widely expressed in central and peripheral neurons (Candenas et al., 2006). Notably, NaV1.6 is thought to act as an axonal “detonator” owing to its comparably low activation potential and its strategic location at the axon initial segment, the trigger site for action potentials, where it is present at high density (Royeck et al., 2008). The use of the TTX-resistant NaV1.6r channel variant is an established method to record selectively NaV1.6 mediated currents in cells with endogenous TTX-sensitive channels (Herzog et al., 2003; Wittmack et al., 2005). Resistance to TTX is introduced by a point mutation in the TTX binding region (Y371S). We do not expect the effect of RIS on the mutant channel to be different from the effect on wildtype NaV1.6, because the established local anesthetic binding site does not overlap with the TTX binding site (Catterall, 2012). RIS inhibited voltage-gated sodium channels in a use- and state-dependent fashion. The dissociation constant for resting NaV1.6r was Kr ¼45.8 μΜ. RIS (10 μM) shifted the steady-state inactivation curve to more negative potentials. In principle, small shifts in voltage dependence might arise from a voltage error due to residual series resistance. RIS inhibits the peak sodium current
and could therefore reduce the voltage drop across residual series resistance. This would cause a shift in the apparent voltage dependence of sodium current. However, such a shift was not observed for activation curves in the presence of r100 μM RIS (66% peak current inhibition). Furthermore, reducing the voltage drop across residual series resistance would be expected to shift the steady-state inactivation curve to more positive potentials (Marty and Neher, 1995; Traynelis, 1998). The mean dissociation constant (Ki ¼ 11.17 1.0 μΜ) was calculated from the dosedependent shift of the steady-state inactivation curves. The resulting state dependence (K r =K i ¼ 4:1) indicated a four times higher affinity to the inactivated states. At higher concentrations (30–100 μΜ) RIS caused a percentage of channels to recover from inactivation with slower kinetics, while the majority of current recovered with kinetics similar to control conditions. This observation is compatible with the notion that a percentage of channels binds RIS while in open or inactivated states and drug-bound channels reprime more slowly. Similar effects on recovery are typically obtained with local anesthetics (Bean et al., 1983). Our study demonstrates that RIS is able to inhibit neuronal NaV activity. Although psychotic patients treated with RIS typically show plasma concentrations in the range of 0.1 μΜ, the pronounced accumulation of the drug in synaptic vesicles (see Introduction) has been calculated to entail intravesicular concentrations of about 7 μM (Tischbirek et al., 2012). During strong neuronal activity, when RIS is synaptically co-released with neurotransmitters, pre- and postsynaptic NaV located in the vicinity of synapses will be hence exposed to low micromolar concentrations, which we show here to be able to exert a considerable impact on sodium channel gating. Notably, the usedependent inhibition of NaV by RIS does not only arise from the
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way in which its delivery is contingent upon synaptic activity. Our experiments indicate that use dependence is also an important intrinsic feature of the interaction of the drug with the channel. A characteristic property of clinically used NaV blockers is their preferential binding to sodium channels in their inactivated state, whereas the affinity to resting channels is much lower. This state dependence is expressed as the ratio between the apparent dissociation constants of the drug for the resting and the inactivated state (Kr/Ki). In a recent study, we found that the state dependence of the typical antipsychotic drug haloperidol for NaV1.6 was 57 (Tischbirek et al., 2012). Another typical antipsychotic, chlorpromazine, inhibited the endogenous sodium currents of N1E-115 cells in a similar experimental setup with a state dependence of 13 (Ogata et al., 1990). In a survey that probed a number of widely used antipsychotics, antidepressants, anticonvulsants and local anesthetics that all act on NaV, values for their state dependence were in the same order of magnitude (Lenkey et al., 2010). Here, we found that RIS behaves differently. With a state dependence of only 4.1, RIS displays an unusually low binding preference for the inactivated state. As a consequence, RIS produced only a minor shift of steady-state channel availability towards more negative potentials. Also, repriming of sodium channels after depolarization-induced inactivation was not appreciably affected by RIS. A significant slowing of recovery from inactivation was only observed when we applied RIS at concentrations (Z 30 mM) well exceeding the calculated level at synaptic release sites. So far, all neuropsychiatric drugs that were found to inhibit NaV displayed a pronounced state dependence (Lenkey et al., 2010; Tischbirek et al., 2012; Ogata et al., 1990). By contrast, the extent of use dependence appeared more variable and, of note, this feature was completely absent in several drugs. For example, the anticonvulsant carbamazepine shows a 44 times higher affinity for inactivated than for resting sodium channels, but its block does not occur in a use-dependent fashion (Lenkey et al., 2010; Ragsdale et al., 1991; see also Lang et al. (1993)). Thus, compared to other NaV-targeting neuropsychiatric drugs, the blocking profile of RIS appears unusual in that it exerts a powerful use-dependent (dynamic) block that is accompanied by little state-dependent (tonic) inhibition. It remains to be determined whether this profile is a particular characteristic of RIS or a more general feature of atypical antipsychotic drugs, because data on the effect of atypical APD on NaV are still very sparse. It is widely believed that a pronounced state dependence is an essential requirement for a NaV blocker to be considered a safe drug: the greater its state dependence, the more reliable the prediction that the drug leaves normal firing largely intact and suppresses mainly aberrant discharges. Do our data then question the safety of RIS? In view of the particular mechanism of drug release that requires strong synaptic activation, it seems unlikely that the ambient concentration of RIS in the overall extracellular space will reach levels that appreciably interfere with normal firing. Remember that RIS inhibited resting channels with an IC50 of 46 mM, whereas its concentration in synaptic vesicles was assumed to attain 7 mM at therapeutic plasma levels of about 0.1 mM. Furthermore, owing to its selective accumulation in intracellular vesicles, pharmacologically effectual concentrations of RIS will only be transiently reached at and around highly active synapses. Thus, rather than having all axonal and somatodendritic sodium channels exposed to a fairly equal drug level, considerable channel block should be restricted to a confined space around synaptic “hotspots”. Whereas our experiments in a heterologous expression system firmly establish an intriguing and unusual role of RIS in NaV gating, this approach inevitably leaves a number of questions open regarding the impact that these effects will have in intact neuronal
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networks. Let us consider, for example, an inhibitory axo-axonic synapse that impinges upon the initial segment of a pyramidal cell axon. Assuming that RIS will be co-released with GABA, one might expect that RIS supports the inhibitory action of GABA by blocking the NaV1.6 “detonator” of the initial segment, but the drug might also cause a feed-back inhibition of GABA release by blocking presynaptic NaV, thereby curtailing synaptic inhibition. What effect will prevail? Another question relates to glutamatergic synapses, where RIS might interfere with high frequency activity required for synaptic plasticity, and thus impair essential mechanisms of cognitive functions, learning and memory. However, in view of the glutamate hypothesis of schizophrenia, which posits that interneuron dysfunction leads to disinhibition of glutamatergic neurons and excessive glutamate release (Moghaddam and Javitt, 2012), the use-dependent block of overactive glutamatergic synapses might emerge as a therapeutic action of risperidone. Only with issues like these resolved, we will be in a position to address two more fundamental questions: first, do the effects of RIS on NaV contribute to its therapeutic benefits or do they rather mediate adverse reactions, and, second, are the distinct blocking properties of RIS at NaV, that we have explored here and compared to those of haloperidol, linked to the different psychopharmacological profiles of the two APD in psychotic patients?
Acknowledgments We thank Iwona Izydorczyk for technical assistance. T.G. was supported by a grant from the Interdisciplinary Centre for Clinical Research Erlangen (IZKF). C.A. and T.H. were supported by intramural grants of the University of Erlangen-Nürnberg.
References Aparasu, R.R., Bhatara, V., 2006. Antipsychotic use and expenditure in the United States. Psychiatr. Serv. 57, 1693. Bean, B.P., Cohen, C.J., Tsien, R.W., 1983. Lidocaine block of cardiac sodium channels. J. Gen. Physiol. 81, 613–642. Benzinger, G.R., Tonkovich, G.S., Hanck, D.A., 1999. Augmentation of recovery from inactivation by site-3 Na channel toxins. A single-channel and whole-cell study of persistent currents. J. Gen. Physiol. 113, 333–346. Bolotina, V., Courtney, K.R., Khodorov, B., 1992. Gate-dependent blockade of sodium channels by phenothiazine derivatives: structure-activity relationships. Mol. Pharmacol. 42, 423–431. Candenas, L., Seda, M., Noheda, P., Buschmann, H., Cintado, C.G., Martin, J.D., Pinto, F.M., 2006. Molecular diversity of voltage-gated sodium channel alpha and beta subunit mRNAs in human tissues. Eur. J. Pharmacol. 541, 9–16. Catterall, W.A., 2012. Voltage-gated sodium channels at 60: structure, function and pathophysiology. J. Physiol. 590, 2577–2589. Crumb , W.J., Ekins, S., Sarazan, R.D., Wikel, J.H., Wrighton, S.A., Carlson, C., Beasley , C.M., 2006. Effects of antipsychotic drugs on I(to), I (Na), I (sus), I (K1), and hERG: QT prolongation, structure activity relationship, and network analysis. Pharm. Res. 23, 1133–1143. Herzog, R.I., Cummins, T.R., Ghassemi, F., Dib-Hajj, S.D., Waxman, S.G., 2003. Distinct repriming and closed-state inactivation kinetics of NaV1.6 and NaV1.7 sodium channels in mouse spinal sensory neurons. J. Physiol. 551, 741–750. Huth, T., Schmidtmayer, J., Alzheimer, C., Hansen, U.P., 2008. Four-mode gating model of fast inactivation of sodium channel NaV1.2a. Pflug. Arch. 457, 103–119. Ito, K., Nishimura, Y., Uji, Y., Yamamoto, T., 1997. Haloperidol effects on Na current in acutely isolated rat retinal ganglion cells. Japan. J. Ophthalmol. 41, 221–225. Jo, S., Bean, B.P., 2011. Inhibition of neuronal voltage-gated sodium channels by brilliant blue G. Mol. Pharmacol. 80, 247–257. Lang, D.G., Wang, C.M., Cooper, B.R., 1993. Lamotrigine, phenytoin and carbamazepine interactions on the sodium current present in N4TG1 mouse neuroblastoma cells. J. Pharmacol. Exp. Ther. 266, 829–835. Leipold, E., Hansel, A., Borges, A., Heinemann, S.H., 2006. Subtype specificity of scorpion beta-toxin Tz1 interaction with voltage-gated sodium channels is determined by the pore loop of domain 3. Mol. Pharmacol. 70, 340–347. Lenkey, N., Karoly, R., Lukacs, P., Vizi, E.S., Sunesen, M., Fodor, L., Mike, A., 2010. Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study. PLOS One 5, e15568. Marty, A., Neher, E., 1995. Tight-seal whole-cell recording. In: Sakmann, B., Neher, E. (Eds.), Single-Channel Recording. Plenum, New York
106
J.M. Brauner et al. / European Journal of Pharmacology 728 (2014) 100–106
Moghaddam, B., Javitt, D., 2012. From revolution to evolution: the glutamate hypothesis of schizophrenia and its implication for treatment. Neuropsychopharmacology 37, 4–15. Ogata, N., Yoshii, M., Narahashi, T., 1990. Differential block of sodium and calcium channels by chlorpromazine in mouse neuroblastoma cells. J. Physiol. 420, 165–183. Ragsdale, D.S., Scheuer, T., Catterall, W.A., 1991. Frequency and voltage-dependent inhibition of type IIA Na þ channels, expressed in a mammalian cell line, by local anesthetic, antiarrhythmic, and anticonvulsant drugs. Mol. Pharmacol. 40, 756–765. Royeck, M., Horstmann, M.T., Remy, S., Reitze, M., Yaari, Y., Beck, H., 2008. Role of axonal NaV1.6 sodium channels in action potential initiation of CA1 pyramidal neurons. J. Neurophysiol. 100, 2361–2380. Schotte, A., Janssen, P.F., Gommeren, W., Luyten, W.H., Van, G.P., Lesage, A.S., De, L.K., Leysen, J.E., 1996. Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding. Psychopharmacology 124, 57–73. Schwabe, U., Paffrath, D., 2012. Arzneiverordnungs Report. Springer, Heidelberg.
Tischbirek, C.H., Wenzel, E.M., Zheng, F., Huth, T., Amato, D., Trapp, S., Denker, A., Welzel, O., Lueke, K., Svetlitchny, A., Rauh, M., Deusser, J., Schwab, A., Rizzoli, S.O., Henkel, A.W., Muller, C.P., Alzheimer, C., Kornhuber, J., Groemer, T.W., 2012. Usedependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs. Neuron 74, 830–844. Traynelis, S.F., 1998. Software-based correction of single compartment series resistance errors. J. Neurosci. Methods 86, 25–34. Wakamori, M., Kaneda, M., Oyama, Y., Akaike, N., 1989. Effects of chlordiazepoxide, chlorpromazine, diazepam, diphenylhydantoin, flunitrazepam and haloperidol on the voltage-dependent sodium current of isolated mammalian brain neurons. Brain Res. 494, 374–378. Wittmack, E.K., Rush, A.M., Hudmon, A., Waxman, S.G., Dib-Hajj, S.D., 2005. Voltage-gated sodium channel NaV1.6 is modulated by p38 mitogen-activated protein kinase. J. Neurosci. 25, 6621–6630. Yang, T.T., Wang, S.J., 2005. Effects of haloperidol and clozapine on glutamate release from nerve terminals isolated from rat prefrontal cortex. Synapse 56, 12–20.