- Email: [email protected]
Effects of fluoxetine on cloned Kv4.3 potassium channels
brain research 1500 (2013) 10–18
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Effects of fluoxetine on cloned Kv4.3 potassium channels Imju Jeonga, Jin-Sung Choib, Sang June Hahna,n a
Department of Physiology, Cell Death and Disease Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea b College of Pharmacy, Integrated Research Institute of Pharmaceutical, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do, Republic of Korea
ar t ic l e in f o
abs tra ct
Article history:
Fluoxetine is widely used for the treatment of depression. We examined the action of
Accepted 16 January 2013
fluoxetine on cloned Kv4.3 stably expressed in CHO cells using the whole-cell patch-clamp
Available online 21 January 2013
technique. Fluoxetine did not significantly produce a reduction in the peak amplitude of
Keywords:
Kv4.3, but increased the rate of current inactivation in a concentration-dependent manner.
Kv4.3
Thus, the effect of fluoxetine on Kv4.3 was measured from the integral of the current
Fluoxetine
during the depolarizing pulse. The integral of Kv4.3 was reduced by fluoxetine in a
SSRI
concentration-dependent manner with an IC50 of 11.8 mM. Using first-order kinetics
Antidepressant
analysis, the apparent association and dissociation rate constants were 1.5 mM1 s1 and
Open channel block
22.2 s1, respectively, with a KD of 14.2 mM, similar to the IC50 value calculated from the
Close-state inactivation
concentration-response curve. Under control conditions, the inactivation of Kv4.3 was best fit by a biexponential function. The fast and slow time constants were significantly decreased in the presence of fluoxetine. Time-to-peak and activation kinetics were significantly accelerated by fluoxetine. The block of Kv4.3 by fluoxetine became more prominent as the membrane potential became more depolarized, displaying a shallow voltage dependence (d ¼0.29) in the full activation voltage range. Fluoxetine did not affect the steady-state inactivation curves, but significantly accelerated the closed-state inactivation of Kv4.3. The block of Kv4.3 by fluoxetine was use-dependent during repetitive stimulation, which explained the slowing of the recovery from inactivation of Kv4.3. Our results indicate that fluoxetine blocks Kv4.3 by preferentially interacting with the open and accelerating closed-state inactivation of the channel. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Fluoxetine, a selective serotonin reuptake inhibitor, is widely used for treating depression with relatively few side effects (Wong and Bymaster, 1995; Wong et al., 1995). Although the exact mechanisms are unknown, fluoxetine has been clinically applied to the treatment of neuropathic pain, obesity n
and premenstrual dysphoric disorder (Brown et al., 2009; Caccia et al., 1992; Dharmshaktu et al., 2012; Garattini et al., 1992; Rani et al., 1996). Under these conditions, fluoxetine exerts diverse pharmacological actions in different systems, particularly with respect to its effect on ion channels; for instance, fluoxetine reportedly blocks a wide variety of ion channels with different potencies in different preparations
Corresponding author. Fax: þ82 2 532 9575. E-mail addresses: [email protected], [email protected] (S.J. Hahn).
0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.01.028
brain research 1500 (2013) 10–18
(Choi J.S. et al., 2003; Garcia-Colunga et al., 1997; Kennard et al., 2005; Kobayashi et al., 2003; Maertens et al., 1999; Nahon et al., 2005). Among ion channels, the effects of fluoxetine on voltage-gated potassium (Kv) channels are being widely studied. For example, we previously reported an open channel block by fluoxetine of Kv1.3, Kv1.4 and Kv3.1, which are stably expressed in Chinese hamster ovary (CHO) cells (Choi et al., 2001, 1999; Choi B.H. et al., 2003; Sung et al., 2008). In addition, fluoxetine preferentially blocked the open state of cloned HERG, Kv1.1 and Kv1.5 channels in CHO cells (Perchenet et al., 2001; Rajamani et al., 2006; Tytgat et al., 1997). Fluoxetine also inhibited the native Kv channels of various cells, such as Kv channels in outer hair cells, rat hippocampal neurons, cerebellar granule neurons, lens and corneal epithelia, and jejunal smooth muscle cells (Bian et al., 2002; Choi et al., 2004; Farrugia, 1996; Rae et al., 1995; Yeung et al., 1999). By using time-dependent activation and inactivation, Kv channels are classified into one of two types: delayed rectifier Kv and A-type or transient outward Kv currents (Rudy, 1988). A-type Kv currents, first observed in molluscan neurons, are Kv currents that possess rapid activating and inactivating kinetics following depolarization (Hagiwara et al., 1961; Rudy, 1988). Based on the voltagedependent kinetics of recovery from inactivation, A-type Kv current can be further distinguished into two phenotypes: Ito, fast and Ito, slow. Kv4.3 channels encode Ito, fast, whereas Kv1.4 encode Ito, slow (Niwa and Nerbonne, 2010; Patel and Campbell, 2005). In general, the N-type inactivation mechanism underlies a rapid inactivation process in A-type Kv currents, such as Kv1.4 and Kv3.4 channels (Rasmusson et al., 1998). Among A-type Kv currents, Kv4.3 do not follow typical N-type inactivation and have special inactivation gating mechanisms, including the putative concerted action of cytoplasmic N- and C-terminal regions (Jerng and Covarrubias, 1997). The Kv4.3 channel, Shal-type, is a rapidly activating and inactivating Kv channel, underlying A-type Kv currents in the brain and transient outward Kv currents in the heart (Birnbaum et al., 2004; Dixon et al., 1996). Kv4.3 currents activate in the subthreshold range of membrane potential and undergo inactivation at resting membrane potential in the neurons (Phuket and Covarrubias, 2009). We previously reported that fluoxetine produced a concentration-, and time-dependent block of cloned Kv1.4 currents in stably expressed CHO cell lines in a manner consistent with an open channel block (Choi B.H. et al., 2003). In the present study, we investigated the effects of fluoxetine on cloned Kv4.3 channels expressed in CHO cells using the whole-cell patch-clamp technique, and compared its mechanism of action with its effect on Kv1.4 channels. Our results suggested that fluoxetine blocked Kv4.3 by a different mechanism than that previously reported for Kv1.4 channels.
2.
Results
2.1.
Concentration-dependent block of Kv4.3 by fluoxetine
Fig. 1(A) displays the representative Kv4.3 currents recorded in 500 ms voltage steps to þ40 mV from a holding potential of 80 mV under whole-cell recording conditions in the absence
11
Fig. 1 – Concentration-dependent effect of fluoxetine on Kv4.3. (A) Representative traces of Kv4.3 currents under control conditions and in the presence of 3, 10, 30 and 100 lM fluoxetine, and (B) concentration curve for the block of Kv4.3 by fluoxetine. The line represents the best fit of the data to a Hill equation an IC50 value of 11.870.6 lM with a Hill coefficient of 1.370.1 (n ¼ 9). The data are represented as the means7S.E.
and presence of fluoxetine. Extracellular application of 3, 10, 30 and 100 mM of fluoxetine resulted in both a reduction in the peak amplitude and an acceleration in the decay rate of the inactivation of Kv4.3 in a concentration-dependent manner, indicating a time-dependent block. To better characterize the steady-state drug effects on the Kv4.3 current, we measured the reduction of the total charge (the integral of currents) during the entire time of depolarization. The relationship between the concentration of fluoxetine and the current integral was fitted by a Hill equation, which yielded an IC50 value of 11.870.6 mM, with a Hill coefficient of 1.370.1 (n¼9) (Fig. 1B).
2.2. Effects of fluoxetine on the inactivation and activation kinetics of Kv4.3 Under control conditions, the inactivation of Kv4.3 at þ40 mV was well fitted to a biexponential function with a fast time constant of 36.372.6 ms and a slow time constant of 217.5726.5 ms (n ¼9). The fast component of the inactivation
12
brain research 1500 (2013) 10–18
was predominant at þ40 mV under control conditions: the fast and slow component of inactivation represents 83.575.2 and 16.573.1% (n ¼9) of total inactivation, respectively. Following the addition of fluoxetine, the apparent inactivation of Kv4.3 was also best fitted to a biexponential function. Fluoxetine concentration-dependently decreased both the fast and slow time constants (Fig. 2A). The relative contribution of the fast component to the total inactivation of Kv4.3 was increased from 83.4% under control conditions to 83.8, 84.7, 90.4, and 93.5% in the presence of 3, 10, 30, and 100 mM of fluoxetine. Under control conditions, the activation time constant was 0.8670.02 ms (n ¼9). In the presence of fluoxetine, the time constants of activation were 0.8470.02, 0.6770.02 and 0.4470.05 (n¼9) for 3, 10, and 30 mM, respectively,
indicating that the kinetics of activation were significantly accelerated by the drug (Fig. 2B). Under control conditions, the time-to-peak was 3.9170.20 ms at þ40 mV (n¼ 9). In the presence of fluoxetine, the time-to-peak was reduced in a concentration-dependent manner, indicating that the timeto-peak was reduced secondary to the acceleration of activation and inactivation kinetics (Fig. 2C).
Fig. 2 – Effects of fluoxetine on the kinetics of activation and inactivation, and time to peak of activation of Kv4.3. (A) The fast (open symbols) and slow (filled symbols) components of the inactivation time constants were calculated from a biexponential function, (B) the dominant time constant of activation was determined by fitting a single exponential function to the latter 50% of activation, and (C) time-to-peak as a function of concentration of fluoxetine. Statistically significant difference from control. The data are represented as the means7S.E. (n ¼ 9).
Fig. 3 – Time-dependent effect of fluoxetine on Kv4.3. (A) Fluoxetine-sensitive currents were calculated by subtracting from the control current and then normalized by the control ((Icontrol/IDrug)/IControl). The time courses of the block were fitted to a biexponential function that yielded the concentration-dependent time constants, and (B) the inverse of drug-induced fast time constants was plotted vs. fluoxetine concentrations. The association and dissociation rate constants were obtained from the slope and intercept values of the fitted line, respectively. Data are means7S.E. (n¼ 9).
2.3.
Time- dependent block of Kv4.3 by fluoxetine
The time course for the development of the block by fluoxetine is shown in Fig. 3. The time course for the fluoxetinesensitive currents ((Icontrol/IDrug)/IControl) was fit to a biexponential function that yielded the concentration-dependent fast and slow time constants (Fig. 3A). The fast components were taken as an approximation of the time course of the drug–channel interaction kinetics and were plotted as a function of time after the start of depolarization. A plot of
brain research 1500 (2013) 10–18
the reciprocal of fast time constants versus each concentration produced an apparent association rate constant (kþ1) of 1.570.1 mM1 s1 and a dissociation rate constant (kþ1) of 22.274.0 s1 (n¼9). Thus, the estimated KD (k1/kþ1) was 14.272.5 mM, which was in good agreement with the IC50 value of 11.8 mM that was calculated from the concentration–response curve (Fig. 3B).
2.4.
Voltage-dependent block of Kv4.3 by fluoxetine
Fig. 4 shows the current traces elicited by depolarizing pulses to potentials from 70 to þ60 mV in the absence (Fig. 4A) and presence (Fig. 4B) of 30 mM fluoxetine. The resultant current–voltage (I–V) relationships are presented in Fig. 4(C). To examine the voltage-dependence of the block, the relative integral of currents during the depolarizing pulses were plotted as a function of potential (Fig. 4D). In the presence of 30 mM fluoxetine, block increased steeply between 30 and 0 mV, coinciding with the voltage range for channel opening (F3,21 ¼3.94, Po0.05). The effect of block was slightly relieved over a voltage range of between þ10 and þ60 mV, where all Kv4.3 channels were open and showed a shallow voltage dependence. This voltage dependence was fitted to Woodhull’s equation and yielded d¼0.2970.02 (n¼6). Thus, fluoxetine produced a voltage-dependent block of Kv4.3, indicating an open channel block.
2.5. Effects of fluoxetine on the steady-state inactivation and closed-state inactivation of Kv4.3 The effect of fluoxetine on steady-state inactivation was examined using a standard two-pulse protocol (Fig. 5A).
13
Fig. 5(B) shows that the steady-state inactivation curve was slightly shifted in a hyperpolarizing direction after fluoxetine treatment, but this shift was not statistically significant. The mean values of V1/2 and k were 45.171.3 and 4.170.1 mV under control conditions and 47.571.6 and 4.470.2 mV (n ¼6) in the presence of fluoxetine. Because Kv4.3 channels are inactivated predominantly from the closed-state in the subthreshold voltage range (Beck and Covarrubias, 2001; Wang et al., 2005), closed-state inactivation kinetics was measured using a two-pulse protocol (Fig. 6A). Under control conditions, the time course for closed-state inactivation was fitted to a single exponential function with a time constant of 4.17 0.3 ms (n¼ 5) (Fig. 6B). In the presence of fluoxetine, closedstate inactivation was also fitted to a single exponential function and accelerated with a time constant of 3.17 0.2 ms (n ¼5, Po0.05).
2.6. Effects of fluoxetine on use-dependent block and the time course for recovery from the inactivation of Kv4.3 We examined the use dependence of the block of Kv4.3 by fluoxetine. Kv4.3 currents were elicited by a series of 10 depolarizing pulses from a holding potential of 80 mV to þ40 mV at 1 and 2 Hz stimulation frequencies (Fig. 7A). Under control conditions, the current amplitude was slightly reduced by 0.570.2 and 10.170.9% at 1 and 2 Hz, respectively (n¼ 7) (Fig. 7B). In the presence of fluoxetine, the block was significantly increased to 9.971.51% at 1 Hz. The block developed faster at 2 Hz than at 1 Hz and was significantly increased 28.573.3% (n¼ 7, Po0.05), suggesting a strong dependence on frequency. The time course of recovery from Kv4.3 inactivation was studied using a paired-pulse protocol
Fig. 4 – Voltage-dependent effect of fluoxetine on Kv4.3. Superimposed current traces were elicited by 500 ms depolarizing pulses from the holding potential of 80 mV to a potential between 70 and þ60 mV in increments of þ10 mV at 10 s intervals in the absence (A) and presence (B) of fluoxetine, (C) current–voltage relationship for peak amplitude of Kv4.3, and (D) relative currents from data obtained in the absence and presence of fluoxetine were plotted against the test potential. The data were taken from the integral of the depolarizing pulses. The dashed lines represent the activation curve under the control conditions (Jeong et al., 2011). The voltage dependence of the fractional block was fitted to a Woodhull equation (see Experimental procedures). The solid lines represent the linear fit. Statistically significant difference from data obtained at 30 mV (Po0.05). The data are represented as the means7S.E.
14
brain research 1500 (2013) 10–18
Fig. 5 – Effects of fluoxetine on the steady-state inactivation of Kv4.3. (A) The currents were elicited by 1 s prepulses that were varied from 110 mV to þ10 mV stepped by 10 mV and a 500 ms depolarizing pulse to þ40 mV in the absence and presence of fluoxetine, and (B) the steady-state inactivation curve of Kv4.3 shown as a plot of normalized peak current as a function of the conditioning pulse. The data are represented as the means7S.E. (n¼ 6).
Fig. 6 – Effects of fluoxetine on closed-state inactivation of Kv4.3 channels. (A) Kv4.3 currents were recorded at þ40 mV using a two-pulse protocol with a conditioning pulse to 60 mV of variable duration in the absence and presence of fluoxetine, and (B) the current amplitude resulting from the initial control pulse, was plotted against the duration of the conditioning pulse and fitted to a single exponential function. Data are means7S.E. (n ¼5).
(Fig. 8A). The time courses of recovery from inactivation can be fitted to a single exponential function with a time constant of 114.678.2 ms during control, and 305.2714.1 ms (n ¼6, Po0.05) after fluoxetine treatment (Fig. 8B). These results suggest that fluoxetine significantly slowed the recovery from inactivation of Kv4.3.
2003; Perchenet et al., 2001; Rajamani et al., 2006; Sung et al., 2008; Tytgat et al., 1997). Thus, our results suggest that fluoxetine interacts primarily with the open state of the Kv4.3 channels based on the following reasons. At the onset of depolarizing pulses, the initial time courses of channel activation and the peak amplitudes of Kv4.3 currents were not significantly affected. This result suggests that the interaction of fluoxetine with the channel occurs after the channel activation. Upon depolarization, however, the block of Kv4.3 increased in an exponential manner, and the time course of block development fit well with kinetic on- and off-rates, suggesting a simple model for open channel block (Jeong et al., 2011; Kim et al., 2007; Snyders and Yeola, 1995). In addition, the block increased in the voltage range of activation with a steep phase, and additional voltage dependence of the block was observed over the full range of activation with a shallow phase (Snyders et al., 1992; Wang et al., 1995). A fractional electrical distance (d) of 0.29 for fluoxetine was obtained. This value was similar to the d values of 0.17 to 0.38 obtained in previous reports for fluoxetine block of Kv1.1, Kv1.3, Kv1.5 and Kv3.1 channels (Choi et al., 1999; Perchenet
3.
Discussion
In the present study, we found that fluoxetine blocked the Kv4.3 current in concentration-, time-, voltage-, and usedependent manners. The main finding of this study was that fluoxetine decreased the peak amplitude of Kv4.3 currents in a concentration-dependent manner, but the peak current was less affected than the steady-state current. Accordingly, fluoxetine induced an acceleration of current inactivation of Kv4.3. These results are similar to the effects of fluoxetine on delayed rectifier and A-type Kv currents from several preparations, and are consistent with the mechanism of an open channel block (Choi et al., 2001, 1999, 2004; Choi B.H. et al.,
brain research 1500 (2013) 10–18
Fig. 7 – (A) Use-dependent effect of fluoxetine Kv4.3 (A) Ten consecutive depolarizing pulses to 40 mV were applied at 1 and 2 Hz in the absence and the presence of fluoxetine, and (B) the peak amplitudes of current at each pulse were normalized by the initial amplitudes and plotted as a function of the pulse number. The data are represented as the means7S.E (n ¼ 7). et al., 2001; Sung et al., 2008; Tytgat et al., 1997). However, fluoxetine reportedly blocked Kv1.4 channels by preferentially binding in the open state but produced a voltage-independent block of Kv1.4 (Choi B.H. et al., 2003). The block of Kv4.3 by fluoxetine was dependent on the stimulation frequency: use-dependent block can also be interpreted as the result of open channel block (Butterworth and Strichartz, 1990; Wang et al., 1995). Thus, Kv4.3 channels in the open state have a much higher affinity compared with channels in the resting state. Fluoxetine slows the recovery kinetics from inactivation, indicating that the dissociation rate of fluoxetine is lower than the transition rate between the open and the resting state under control conditions (Choi et al., 2000). Furthermore, fluoxetine produced a use-dependent block of Kv4.3, which reflected the slow recovery kinetics of Kv4.3 in the presence of fluoxetine. However, the application of fluoxetine on Kv4.3 did not change the steady-state inactivation curves, which suggests that there was no change of affinity in the inactivated state. A-type Kv currents have rapidly activating and inactivating kinetics (Hagiwara et al., 1961). Based on the kinetics of
15
Fig. 8 – Effects of fluoxetine on the time course of recovery from the inactivation of Kv4.3. (A) The recovery from inactivation of Kv4.3 was assessed with a two-pulse protocol with varying interpulse intervals, and (B) the current elicited by the second pulse was normalized to the current elicited by the first pulse and is shown as a function of recovery time. Time courses of recovery from inactivation were fitted with a single exponential function. The data are represented as the means7S.E. (n ¼6).
recovery from inactivation, A-type Kv currents can be classified into 2 phenotypes: Ito, fast and Ito, slow. Kv4.3 has been linked to somatodendritic A-type Kv currents in nervous cells and to transient outward Kv currents in the heart with underlying Ito, fast. Kv1.4 channels are thought to underlie Ito, slow. These channels are widely expressed in neuronal cells of the brain (Lujan et al., 2003). In contrast to Kv1.4 which undergoes an N-type inactivation, Kv4.3 is inactivated by concerted action of the cytoplasmic N- and C-termini (Jerng and Covarrubias, 1997). Whereas Kv1.4 channels inactivate from an open state during depolarizing potentials, Kv4.3 is known to preferentially inactivate via a closed-state inactivation in the subthreshold range of membrane potential (Beck and Covarrubias, 2001; Wang et al., 2005). In the present study, fluoxetine accelerated the closed-state inactivation of Kv4.3. Because the potency of a drug is dependent on its membrane potential, which determines the state of the channels, the acceleration of closed-state inactivation by fluoxetine increases the potency at subthreshold potentials.
16
brain research 1500 (2013) 10–18
Generally, A-type Kv currents play an important role in setting the resting membrane potential, and determining the action potential duration and neuronal firing rates in the central nervous system (Bardoni and Belluzzi, 1993; Malin and Nerbonne, 2000). Fluoxetine is a selective inhibitor of the 5-hydroxytryptamine (HT) reuptake transporter and results in increased concentrations of 5-HT levels in the synapses of neuronal cells, which explains its therapeutic action as an antidepressant (Wong and Bymaster, 1995; Wong et al., 1995). Pharmacological blockade of A-type Kv currents by 4aminopyrine increased the spontaneous basal release of [3H]5-HT from rat hippocampal slices (Schechter, 1997). In both the previous and present studies, fluoxetine blocked Kv1.4 and Kv4.3 channels expressed in CHO cells in a concentration-dependent manner (Choi B.H. et al., 2003). Furthermore, fluoxetine and its major metabolite, norfluoxetine, potently blocked A-type Kv currents in primary cultured rat hippocampal neurons (Choi et al., 2004). Thus, a block of Kv4.3 channels by fluoxetine could contribute to the antidepressant effect by increasing the 5-HT concentrations in the synaptic cleft of neuronal cells. In addition to its antidepressant action, fluoxetine possesses an analgesic effect and is widely used in the treatment of neuropathic pain (Dharmshaktu et al., 2012; Rani et al., 1996). Kv4.3 and Kv1.4 channels are thought to encode A-type Kv currents in dorsal root ganglion (DRG) neurons and are involved in the generation of nociceptive impulses in peripheral DRG neurons (Birnbaum et al., 2004; Hsieh, 2008; Phuket and Covarrubias, 2009). Thus, it is interesting to investigate whether fluoxetine may exert its analgesic effects in DRG neurons via the block of these channels. The therapeutic plasma concentrations of fluoxetine range approximately from 0.1 to 3.4 mM (Altamura et al., 1994; Orsulak et al., 1988). In the present study, fluoxetine blocked Kv4.3 currents with an IC50 of 11.8 mM, which is 3-fold higher than the plasma concentrations in human beings. The lowest concentration of fluoxetine (3 mM) in the present study significantly blocked the Kv4.3 currents (about 18%), and this concentration falls within the range of the therapeutic plasma concentration. In addition, the brain concentration of fluoxetine seems to be 20 times higher than the corresponding blood levels because of the high lipophilicity of the drug (Caccia et al., 1992; Karson et al., 1993). Thus, the blocking effect of fluoxetine on Kv4.3 could be clinically relevant. In conclusion, fluoxetine blocks the open state of Kv4.3 and accelerates the close-state inactivation of Kv4.3 expressed in CHO cells. The block of Kv4.3 by fluoxetine should therefore be taken into account for its pharmacological effect in the treatment of depression.
supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine (Invitrogen), 0.1 mM hypoxanthine (Invitrogen), 0.01 mM thymidine (Invitrogen), 0.3 mg/ml G418 (Invitrogen), and 1% of a 100X antibiotic antimycotic mixture (Invitrogen) at 37 1C with 5% CO2-enriched air. The cells treated with trypsin–EDTA solution were plated on glass coverslips (12 mm diameter; Fisher Scientific, Pittsburgh, PA, USA) and placed in 35 mm Petri dishes. Patch-clamp recordings were obtained 12–24 h later. For voltage-clamp recordings, the coverslips containing adherent CHO cells were mounted on the glass bottom of the recording chamber (RC13; Warner Instrument, Hamden, CT, USA) and were perfused with an extracellular bath solution.
4.2.
Kv4.3 currents were recorded using a whole-cell configuration of the patch-clamp technique with an Axopatch 200B patchclamp amplifier (Molecular Devices, Sunnyvale, CA, USA) at room temperature (22–24 1C). Patch electrodes were pulled from soft glass capillaries (PG10165-4; World Precision Instruments, Sarasota, FL, USA) on a programmable horizontal microelectrode puller (P-97; Sutter Instrument Co., Novato, CA, USA). When filled with the pipette solution, the electrode resistance was maintained at 2–4 MO. Data acquisition and analysis were performed on an IBM-compatible Pentium computer running pClamp 10.0 software (Molecular Devices) using a Digidata 1322A interface (Molecular Devices). Leak subtraction was not applied to the data. In the whole-cell configuration, the average series resistance was 2.970.2M O (n ¼58). The effective series resistances were usually compensated by 80% if the Kv4.3 current exceeded 1 nA. Voltage drops were based on the calculated series resistance and were less than 5 mV.
4.3.
Experimental procedures
4.1.
Cell culture
The CHO cells were transfected with cDNA encoding Kv4.3 channels using the Lipofectamine reagent (Invitrogen, Grand Island, NY, USA), as previously described in detail (Ahn et al., 2006). A stable cell line expressing Kv4.3 channels was maintained in Iscove’s modified Dulbecco’s medium (Invitrogen)
Solutions and drugs
To record Kv4.3 current, the extracellular bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, which was adjusted to pH 7.3 using NaOH. The intracellular pipette solution contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 EGTA, which was adjusted to pH 7.3 using KOH. Fluoxetine (Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) to yield the stock solution. The stock solution was then diluted with the bath solution to obtain the desired concentration. The concentration of DMSO in the final dilution was less than 0.1%, and this concentration had no effect on Kv4.3 currents.
4.4.
4.
Electrophysiology
Data analysis
Data analysis was performed using Origin 8.0 software (Origin Lab Corp., Northampton, MA, USA). The concentrationdependent block of Kv4.3 current by fluoxetine at þ40 mV was fitted to the Hill equation: f ¼ 1=½1 þ ðIC50 =Þ½DÞn
ð1Þ
where f is the fractional block for the fluoxetine concentration [D], IC50 is the concentration of fluoxetine that produces a half-maximum current block, and n is the Hill coefficient.
brain research 1500 (2013) 10–18
Kv4.3 currents were elicited by applying 500 ms depolarizing pulses from a holding potential of 80 to þ40 mV every 10 s. The drug-induced time constants were used as an approximation of the drug–channel interaction kinetics (Slawsky and Castle, 1994; Snyders and Yeola, 1995) according to the following equations: 1=tBlock ¼ kþ1 ½D þ k1
ð2aÞ
Kd ¼ k1 =kþ1
ð2bÞ
where tBlock is the drug-induced time constant, [D] denotes the drug concentration, and kþ1 and k1 are the apparent rate constants of binding and unbinding for the drug, respectively. We measured the fractional block at each potential and fitted the data to the Woodhull equation, as follows (Woodhull, 1973): f ¼ ½D=f½D þ Kd ð0Þ x expð-zdFV=RTÞg
ð3Þ
where Kd(0) is the apparent affinity at 0 mV (the reference voltage), z is the charge valence of the drug, d is the fractional electrical distance (i.e., the fraction of the transmembrane electric field sensed by a single charge at the receptor site), F is Faraday’s constant, R is the gas constant, and T is the absolute temperature. In the present study, 25.4 mV was used as the RT/F value at 22 1C. The steady-state inactivation curves were obtained by normalizing the peak current amplitudes using a two-pulse protocol. The current was produced by a 500 ms depolarizing pulse to þ40 mV, whereas 1 s preconditioning pulses were varied from 110 to þ10 mV in increments of 10 mV at 10 s intervals in the absence and presence of fluoxetine. The curves were fitted to the Boltzmann equation: ðI2Ic Þ=ðImax 2Ic Þ ¼ 1=½1 þ expðV2V1=2 Þ=k
ð4Þ
in which Imax represents the current measured at the most hyperpolarized preconditioning pulse, and Ic represents a non-inactivating current at the most depolarized preconditioning pulse. V, V1/2, and k are the test potential, the point where channels are half-inactivated, and the slope, respectively. The non-inactivating residual current was removed by subtracting it from the actual value. A two-pulse protocol was used to determine the time course of recovery of the Kv4.3 currents from inactivation: the first pre-pulse of a 500 ms depolarizing pulse of þ40 mV from a holding potential of 80 mV was followed by a second identical pulse after increasing the inter-pulse intervals between 5 and 10,000 ms at 80 mV. The dominant time constant of activation was determined by fitting a single exponential function to the latter 50% of activation (Snyders et al., 1993). We studied the kinetics of activation, which reflects the transition from the closed to the open state, by measuring the time to peak.
4.5.
Statistics
The data are summarized as the means7S.E. The paired Student’s t-test and one-way analysis of variance were for comparisons of multiple groups followed by Bonferroni’s test was used for statistical analysis. Differences were considered significant at Po0.05.
17
Acknowledgments We thank Dr. Yuji Imaizumi (Department of Molecular and Cellular Pharmacology, Nagoya City University, Nagoya, Japan) for the Kv4.3 cDNA.
r e f e r e nc e s
Ahn, H.S., Kim, S.E., Jang, H.J., Kim, M.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2006. Interaction of riluzole with the closed inactivated state of Kv4.3 channels. J. Pharmacol. Exp. Ther. 319, 323–331. Altamura, A.C., Moro, A.R., Percudani, M., 1994. Clinical pharmacokinetics of fluoxetine. Clin. Pharmacokinet. 26, 201–214. Bardoni, R., Belluzzi, O., 1993. Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices. J. Neurophysiol. 69, 2222–2231. Beck, E.J., Covarrubias, M., 2001. Kv4 channels exhibit modulation of closed-state inactivation in inside-out patches. Biophys. J. 81, 867–883. Bian, J.T., Yeh, J.Z., Aistrup, G.L., Narahashi, T., Moore, E.J., 2002. Inhibition of Kþ currents of outer hair cells in guinea pig cochlea by fluoxetine. Eur. J. Pharmacol. 453, 159–166. Birnbaum, S.G., Varga, A.W., Yuan, L.L., Anderson, A.E., Sweatt, J.D., Schrader, L.A., 2004. Structure and function of Kv4-family transient potassium channels. Physiol. Rev. 84, 803–833. Brown, J., PM, O.B., Marjoribanks, J., Wyatt, K., 2009. Selective serotonin reuptake inhibitors for premenstrual syndrome. Cochrane Database Syst. Rev., CD001396. Butterworth, J.F.t., Strichartz, G.R., 1990. Molecular mechanisms of local anesthesia: a review. Anesthesiology 72, 711–734. Caccia, S., Bizzi, A., Coltro, G., Fracasso, C., Frittoli, E., Mennini, T., Garattini, S., 1992. Anorectic activity of fluoxetine and norfluoxetine in rats: relationship between brain concentrations and in-vitro potencies on monoaminergic mechanisms. J. Pharm. Pharmacol. 44, 250–254. Choi, B.H., Choi, J.S., Jeong, S.W., Hahn, S.J., Yoon, S.H., Jo, Y.H., Kim, M.S., 2000. Direct block by bisindolylmaleimide of rat Kv1.5 expressed in Chinese hamster ovary cells. J. Pharmacol. Exp. Ther. 293, 634–640. Choi, B.H., Choi, J.S., Yoon, S.H., Rhie, D.J., Min, D.S., Jo, Y.H., Kim, M.S., Hahn, S.J., 2001. Effects of norfluoxetine, the major metabolite of fluoxetine, on the cloned neuronal potassium channel Kv3.1. Neuropharmacology 41, 443–453. Choi, B.H., Choi, J.S., Ahn, H.S., Kim, M.J., Rhie, D.J., Yoon, S.H., Min, D.S., Jo, Y.H., Kim, M.S., Hahn, S.J., 2003. Fluoxetine blocks cloned neuronal A-type Kþ channels Kv1.4. Neuroreport 14, 2451–2455. Choi, J.S., Hahn, S.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., 1999. Mechanism of fluoxetine block of cloned voltage-activated potassium channel Kv1.3. J. Pharmacol. Exp. Ther. 291, 1–6. Choi, J.S., Choi, B.H., Ahn, H.S., Kim, M.J., Rhie, D.J., Yoon, S.H., S., Min do, Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2003. Mechanism of block by fluoxetine of 5-hydroxytryptamine3 (5-HT3)-mediated currents in NCB-20 neuroblastoma cells. Biochem. Pharmacol. 66, 2125–2132. Choi, J.S., Choi, B.H., Ahn, H.S., Kim, M.J., Han, T.H., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Hahn, S.J., 2004. Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons. Brain Res. 1018, 201–207. Dharmshaktu, P., Tayal, V., Kalra, B.S., 2012. Efficacy of antidepressants as analgesics: a review. J. Clin. Pharmacol. 52, 6–17. Dixon, J.E., Shi, W., Wang, H.S., McDonald, C., Yu, H., Wymore, R.S., Cohen, I.S., McKinnon, D., 1996. Role of the Kv4.3 Kþ channel in
18
brain research 1500 (2013) 10–18
ventricular muscle. A molecular correlate for the transient outward current. Circ. Res. 79, 659–668. Farrugia, G., 1996. Modulation of ionic currents in isolated canine and human jejunal circular smooth muscle cells by fluoxetine. Gastroenterology 110, 1438–1445. Garattini, S., Bizzi, A., Caccia, S., Mennini, T., 1992. Progress report on the anorectic effects of dexfenfluramine, fluoxetine and sertraline. Int. J. Obes. Relat. Metab. Disord. 16 (Suppl. 3), S43–S50. Garcia-Colunga, J., Awad, J.N., Miledi, R., 1997. Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac). Proc. Natl. Acad. Sci. USA 94, 2041–2044. Hagiwara, S., Kusano, K., Saito, N., 1961. Membrane changes of Onchidium nerve cell in potassium-rich media. J. Physiol. 155, 470–489. Hsieh, C.P., 2008. Redox modulation of A-type Kþ currents in pain-sensing dorsal root ganglion neurons. Biochem. Biophys. Res. Commun. 370, 445–449. Jeong, I., Choi, B.H., Hahn, S.J., 2011. Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation. Br. J. Pharmacol. 163, 510–520. Jerng, H.H., Covarrubias, M., 1997. Kþ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains. Biophys. J. 72, 163–174. Karson, C.N., Newton, J.E., Livingston, R., Jolly, J.B., Cooper, T.B., Sprigg, J., Komoroski, R.A., 1993. Human brain fluoxetine concentrations. J. Neuropsychiatry Clin. Neurosci. 5, 322–329. Kennard, L.E., Chumbley, J.R., Ranatunga, K.M., Armstrong, S.J., Veale, E.L., Mathie, A., 2005. Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine. Br. J. Pharmacol. 144, 821–829. Kim, S.E., Ahn, H.S., Choi, B.H., Jang, H.J., Kim, M.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2007. Open channel block of A-type, Kv4.3, and delayed rectifier Kþ channels, Kv1.3 and Kv3.1, by sibutramine. J. Pharmacol. Exp. Ther. 321, 753–762. Kobayashi, T., Washiyama, K., Ikeda, K., 2003. Inhibition of G protein-activated inwardly rectifying Kþ channels by fluoxetine (Prozac). Br. J. Pharmacol. 138, 1119–1128. Lujan, R., de Cabo de la Vega, C., Dominguez del Toro, E., Ballesta, J.J., Criado, M., Juiz, J.M., 2003. Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat. J. Chem. Neuroanat. 26, 209–224. Maertens, C., Wei, L., Voets, T., Droogmans, G., Nilius, B., 1999. Block by fluoxetine of volume-regulated anion channels. Br. J. Pharmacol. 126, 508–514. Malin, S.A., Nerbonne, J.M., 2000. Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F: molecular dissection of IA. J. Neurosci. 20, 5191–5199. Nahon, E., Israelson, A., Abu-Hamad, S., Varda, S.B., 2005. Fluoxetine (Prozac) interaction with the mitochondrial voltage-dependent anion channel and protection against apoptotic cell death. FEBS Lett. 579, 5105–5110. Niwa, N., Nerbonne, J.M., 2010. Molecular determinants of cardiac transient outward potassium current (Ito) expression and regulation. J. Mol. Cell Cardiol. 48, 12–25. Orsulak, P.J., Kenney, J.T., Debus, J.R., Crowley, G., Wittman, P.D., 1988. Determination of the antidepressant fluoxetine and its metabolite norfluoxetine in serum by reversed-phase HPLC with ultraviolet detection. Clin. Chem. 34, 1875–1878. Patel, S.P., Campbell, D.L., 2005. Transient outward potassium current, ‘Ito’, phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms. J. Physiol. 569, 7–39. Perchenet, L., Hilfiger, L., Mizrahi, J., Clement-Chomienne, O., 2001. Effects of anorexinogen agents on cloned voltage-gated Kþ channel hKv1.5. J. Pharmacol. Exp Ther. 298, 1108–1119.
Phuket, T.R., Covarrubias, M., 2009. Kv4 channels underlie the subthreshold-operating A-type K-current in nociceptive dorsal root ganglion neurons. Front. Mol. Neurosci. 2, 3. Rae, J.L., Rich, A., Zamudio, A.C., Candia, O.A., 1995. Effect of Prozac on whole cell ionic currents in lens and corneal epithelia. Am. J. Physiol. 269, C250–C256. Rajamani, S., Eckhardt, L.L., Valdivia, C.R., Klemens, C.A., Gillman, B.M., Anderson, C.L., Holzem, K.M., Delisle, B.P., Anson, B.D., Makielski, J.C., January, C.T., 2006. Drug-induced long QT syndrome: hERG Kþ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine. Br. J. Pharmacol. 149, 481–489. Rani, P.U., Naidu, M.U., Prasad, V.B., Rao, T.R., Shobha, J.C., 1996. An evaluation of antidepressants in rheumatic pain conditions. Anesth. Analg. 83, 371–375. Rasmusson, R.L., Morales, M.J., Wang, S., Liu, S., Campbell, D.L., Brahmajothi, M.V., Strauss, H.C., 1998. Inactivation of voltagegated cardiac Kþ channels. Circ. Res. 82, 739–750. Rudy, B., 1988. Diversity and ubiquity of K channels. Neuroscience 25, 729–749. Schechter, L.E., 1997. The potassium channel blockers 4aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices. J. Pharmacol. Exp. Ther 282, 262–270. Slawsky, M.T., Castle, N.A., 1994. Kþ channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes. J. Pharmacol. Exp. Ther 269, 66–74. Snyders, D.J., Tamkun, M.M., Bennett, P.B., 1993. A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression. J. Gen. Physiol. 101, 513–543. Snyders, D.J., Yeola, S.W., 1995. Determinants of antiarrhythmic drug action. Electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel. Circ. Res 77, 575–583. Snyders, J., Knoth, K.M., Roberds, S.L., Tamkun, M.M., 1992. Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel. Mol. Pharmacol. 41, 322–330. Sung, M.J., Ahn, H.S., Hahn, S.J., Choi, B.H., 2008. Open channel block of Kv3.1 currents by fluoxetine. J. Pharmacol. Sci. 106, 38–45. Tytgat, J., Maertens, C., Daenens, P., 1997. Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1). Br. J. Pharmacol. 122, 1417–1424. Wang, S., Bondarenko, V.E., Qu, Y.J., Bett, G.C., Morales, M.J., Rasmusson, R.L., Strauss, H.C., 2005. Time- and voltagedependent components of Kv4.3 inactivation. Biophys. J. 89, 3026–3041. Wang, Z., Fermini, B., Nattel, S., 1995. Effects of flecainide, quinidine, and 4-aminopyridine on transient outward and ultrarapid delayed rectifier currents in human atrial myocytes. J. Pharmacol. Exp. Ther. 272, 184–196. Wong, D.T., Bymaster, F.P., 1995. Development of antidepressant drugs. Fluoxetine (Prozac) and other selective serotonin uptake inhibitors. Adv. Exp. Med. Biol. 363, 77–95. Wong, D.T., Bymaster, F.P., Engleman, E.A., 1995. Prozac (fluoxetine, Lilly 110140), the first selective serotonin uptake inhibitor and an antidepressant drug: twenty years since its first publication. Life Sci. 57, 411–441. Woodhull, A.M., 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687–708. Yeung, S.Y., Millar, J.A., Mathie, A., 1999. Inhibition of neuronal KV potassium currents by the antidepressant drug, fluoxetine. Br. J. Pharmacol. 128, 1609–1615.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Effects of fluoxetine on cloned Kv4.3 potassium channels Imju Jeonga, Jin-Sung Choib, Sang June Hahna,n a
Department of Physiology, Cell Death and Disease Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea b College of Pharmacy, Integrated Research Institute of Pharmaceutical, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do, Republic of Korea
ar t ic l e in f o
abs tra ct
Article history:
Fluoxetine is widely used for the treatment of depression. We examined the action of
Accepted 16 January 2013
fluoxetine on cloned Kv4.3 stably expressed in CHO cells using the whole-cell patch-clamp
Available online 21 January 2013
technique. Fluoxetine did not significantly produce a reduction in the peak amplitude of
Keywords:
Kv4.3, but increased the rate of current inactivation in a concentration-dependent manner.
Kv4.3
Thus, the effect of fluoxetine on Kv4.3 was measured from the integral of the current
Fluoxetine
during the depolarizing pulse. The integral of Kv4.3 was reduced by fluoxetine in a
SSRI
concentration-dependent manner with an IC50 of 11.8 mM. Using first-order kinetics
Antidepressant
analysis, the apparent association and dissociation rate constants were 1.5 mM1 s1 and
Open channel block
22.2 s1, respectively, with a KD of 14.2 mM, similar to the IC50 value calculated from the
Close-state inactivation
concentration-response curve. Under control conditions, the inactivation of Kv4.3 was best fit by a biexponential function. The fast and slow time constants were significantly decreased in the presence of fluoxetine. Time-to-peak and activation kinetics were significantly accelerated by fluoxetine. The block of Kv4.3 by fluoxetine became more prominent as the membrane potential became more depolarized, displaying a shallow voltage dependence (d ¼0.29) in the full activation voltage range. Fluoxetine did not affect the steady-state inactivation curves, but significantly accelerated the closed-state inactivation of Kv4.3. The block of Kv4.3 by fluoxetine was use-dependent during repetitive stimulation, which explained the slowing of the recovery from inactivation of Kv4.3. Our results indicate that fluoxetine blocks Kv4.3 by preferentially interacting with the open and accelerating closed-state inactivation of the channel. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Fluoxetine, a selective serotonin reuptake inhibitor, is widely used for treating depression with relatively few side effects (Wong and Bymaster, 1995; Wong et al., 1995). Although the exact mechanisms are unknown, fluoxetine has been clinically applied to the treatment of neuropathic pain, obesity n
and premenstrual dysphoric disorder (Brown et al., 2009; Caccia et al., 1992; Dharmshaktu et al., 2012; Garattini et al., 1992; Rani et al., 1996). Under these conditions, fluoxetine exerts diverse pharmacological actions in different systems, particularly with respect to its effect on ion channels; for instance, fluoxetine reportedly blocks a wide variety of ion channels with different potencies in different preparations
Corresponding author. Fax: þ82 2 532 9575. E-mail addresses: [email protected], [email protected] (S.J. Hahn).
0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.01.028
brain research 1500 (2013) 10–18
(Choi J.S. et al., 2003; Garcia-Colunga et al., 1997; Kennard et al., 2005; Kobayashi et al., 2003; Maertens et al., 1999; Nahon et al., 2005). Among ion channels, the effects of fluoxetine on voltage-gated potassium (Kv) channels are being widely studied. For example, we previously reported an open channel block by fluoxetine of Kv1.3, Kv1.4 and Kv3.1, which are stably expressed in Chinese hamster ovary (CHO) cells (Choi et al., 2001, 1999; Choi B.H. et al., 2003; Sung et al., 2008). In addition, fluoxetine preferentially blocked the open state of cloned HERG, Kv1.1 and Kv1.5 channels in CHO cells (Perchenet et al., 2001; Rajamani et al., 2006; Tytgat et al., 1997). Fluoxetine also inhibited the native Kv channels of various cells, such as Kv channels in outer hair cells, rat hippocampal neurons, cerebellar granule neurons, lens and corneal epithelia, and jejunal smooth muscle cells (Bian et al., 2002; Choi et al., 2004; Farrugia, 1996; Rae et al., 1995; Yeung et al., 1999). By using time-dependent activation and inactivation, Kv channels are classified into one of two types: delayed rectifier Kv and A-type or transient outward Kv currents (Rudy, 1988). A-type Kv currents, first observed in molluscan neurons, are Kv currents that possess rapid activating and inactivating kinetics following depolarization (Hagiwara et al., 1961; Rudy, 1988). Based on the voltagedependent kinetics of recovery from inactivation, A-type Kv current can be further distinguished into two phenotypes: Ito, fast and Ito, slow. Kv4.3 channels encode Ito, fast, whereas Kv1.4 encode Ito, slow (Niwa and Nerbonne, 2010; Patel and Campbell, 2005). In general, the N-type inactivation mechanism underlies a rapid inactivation process in A-type Kv currents, such as Kv1.4 and Kv3.4 channels (Rasmusson et al., 1998). Among A-type Kv currents, Kv4.3 do not follow typical N-type inactivation and have special inactivation gating mechanisms, including the putative concerted action of cytoplasmic N- and C-terminal regions (Jerng and Covarrubias, 1997). The Kv4.3 channel, Shal-type, is a rapidly activating and inactivating Kv channel, underlying A-type Kv currents in the brain and transient outward Kv currents in the heart (Birnbaum et al., 2004; Dixon et al., 1996). Kv4.3 currents activate in the subthreshold range of membrane potential and undergo inactivation at resting membrane potential in the neurons (Phuket and Covarrubias, 2009). We previously reported that fluoxetine produced a concentration-, and time-dependent block of cloned Kv1.4 currents in stably expressed CHO cell lines in a manner consistent with an open channel block (Choi B.H. et al., 2003). In the present study, we investigated the effects of fluoxetine on cloned Kv4.3 channels expressed in CHO cells using the whole-cell patch-clamp technique, and compared its mechanism of action with its effect on Kv1.4 channels. Our results suggested that fluoxetine blocked Kv4.3 by a different mechanism than that previously reported for Kv1.4 channels.
2.
Results
2.1.
Concentration-dependent block of Kv4.3 by fluoxetine
Fig. 1(A) displays the representative Kv4.3 currents recorded in 500 ms voltage steps to þ40 mV from a holding potential of 80 mV under whole-cell recording conditions in the absence
11
Fig. 1 – Concentration-dependent effect of fluoxetine on Kv4.3. (A) Representative traces of Kv4.3 currents under control conditions and in the presence of 3, 10, 30 and 100 lM fluoxetine, and (B) concentration curve for the block of Kv4.3 by fluoxetine. The line represents the best fit of the data to a Hill equation an IC50 value of 11.870.6 lM with a Hill coefficient of 1.370.1 (n ¼ 9). The data are represented as the means7S.E.
and presence of fluoxetine. Extracellular application of 3, 10, 30 and 100 mM of fluoxetine resulted in both a reduction in the peak amplitude and an acceleration in the decay rate of the inactivation of Kv4.3 in a concentration-dependent manner, indicating a time-dependent block. To better characterize the steady-state drug effects on the Kv4.3 current, we measured the reduction of the total charge (the integral of currents) during the entire time of depolarization. The relationship between the concentration of fluoxetine and the current integral was fitted by a Hill equation, which yielded an IC50 value of 11.870.6 mM, with a Hill coefficient of 1.370.1 (n¼9) (Fig. 1B).
2.2. Effects of fluoxetine on the inactivation and activation kinetics of Kv4.3 Under control conditions, the inactivation of Kv4.3 at þ40 mV was well fitted to a biexponential function with a fast time constant of 36.372.6 ms and a slow time constant of 217.5726.5 ms (n ¼9). The fast component of the inactivation
12
brain research 1500 (2013) 10–18
was predominant at þ40 mV under control conditions: the fast and slow component of inactivation represents 83.575.2 and 16.573.1% (n ¼9) of total inactivation, respectively. Following the addition of fluoxetine, the apparent inactivation of Kv4.3 was also best fitted to a biexponential function. Fluoxetine concentration-dependently decreased both the fast and slow time constants (Fig. 2A). The relative contribution of the fast component to the total inactivation of Kv4.3 was increased from 83.4% under control conditions to 83.8, 84.7, 90.4, and 93.5% in the presence of 3, 10, 30, and 100 mM of fluoxetine. Under control conditions, the activation time constant was 0.8670.02 ms (n ¼9). In the presence of fluoxetine, the time constants of activation were 0.8470.02, 0.6770.02 and 0.4470.05 (n¼9) for 3, 10, and 30 mM, respectively,
indicating that the kinetics of activation were significantly accelerated by the drug (Fig. 2B). Under control conditions, the time-to-peak was 3.9170.20 ms at þ40 mV (n¼ 9). In the presence of fluoxetine, the time-to-peak was reduced in a concentration-dependent manner, indicating that the timeto-peak was reduced secondary to the acceleration of activation and inactivation kinetics (Fig. 2C).
Fig. 2 – Effects of fluoxetine on the kinetics of activation and inactivation, and time to peak of activation of Kv4.3. (A) The fast (open symbols) and slow (filled symbols) components of the inactivation time constants were calculated from a biexponential function, (B) the dominant time constant of activation was determined by fitting a single exponential function to the latter 50% of activation, and (C) time-to-peak as a function of concentration of fluoxetine. Statistically significant difference from control. The data are represented as the means7S.E. (n ¼ 9).
Fig. 3 – Time-dependent effect of fluoxetine on Kv4.3. (A) Fluoxetine-sensitive currents were calculated by subtracting from the control current and then normalized by the control ((Icontrol/IDrug)/IControl). The time courses of the block were fitted to a biexponential function that yielded the concentration-dependent time constants, and (B) the inverse of drug-induced fast time constants was plotted vs. fluoxetine concentrations. The association and dissociation rate constants were obtained from the slope and intercept values of the fitted line, respectively. Data are means7S.E. (n¼ 9).
2.3.
Time- dependent block of Kv4.3 by fluoxetine
The time course for the development of the block by fluoxetine is shown in Fig. 3. The time course for the fluoxetinesensitive currents ((Icontrol/IDrug)/IControl) was fit to a biexponential function that yielded the concentration-dependent fast and slow time constants (Fig. 3A). The fast components were taken as an approximation of the time course of the drug–channel interaction kinetics and were plotted as a function of time after the start of depolarization. A plot of
brain research 1500 (2013) 10–18
the reciprocal of fast time constants versus each concentration produced an apparent association rate constant (kþ1) of 1.570.1 mM1 s1 and a dissociation rate constant (kþ1) of 22.274.0 s1 (n¼9). Thus, the estimated KD (k1/kþ1) was 14.272.5 mM, which was in good agreement with the IC50 value of 11.8 mM that was calculated from the concentration–response curve (Fig. 3B).
2.4.
Voltage-dependent block of Kv4.3 by fluoxetine
Fig. 4 shows the current traces elicited by depolarizing pulses to potentials from 70 to þ60 mV in the absence (Fig. 4A) and presence (Fig. 4B) of 30 mM fluoxetine. The resultant current–voltage (I–V) relationships are presented in Fig. 4(C). To examine the voltage-dependence of the block, the relative integral of currents during the depolarizing pulses were plotted as a function of potential (Fig. 4D). In the presence of 30 mM fluoxetine, block increased steeply between 30 and 0 mV, coinciding with the voltage range for channel opening (F3,21 ¼3.94, Po0.05). The effect of block was slightly relieved over a voltage range of between þ10 and þ60 mV, where all Kv4.3 channels were open and showed a shallow voltage dependence. This voltage dependence was fitted to Woodhull’s equation and yielded d¼0.2970.02 (n¼6). Thus, fluoxetine produced a voltage-dependent block of Kv4.3, indicating an open channel block.
2.5. Effects of fluoxetine on the steady-state inactivation and closed-state inactivation of Kv4.3 The effect of fluoxetine on steady-state inactivation was examined using a standard two-pulse protocol (Fig. 5A).
13
Fig. 5(B) shows that the steady-state inactivation curve was slightly shifted in a hyperpolarizing direction after fluoxetine treatment, but this shift was not statistically significant. The mean values of V1/2 and k were 45.171.3 and 4.170.1 mV under control conditions and 47.571.6 and 4.470.2 mV (n ¼6) in the presence of fluoxetine. Because Kv4.3 channels are inactivated predominantly from the closed-state in the subthreshold voltage range (Beck and Covarrubias, 2001; Wang et al., 2005), closed-state inactivation kinetics was measured using a two-pulse protocol (Fig. 6A). Under control conditions, the time course for closed-state inactivation was fitted to a single exponential function with a time constant of 4.17 0.3 ms (n¼ 5) (Fig. 6B). In the presence of fluoxetine, closedstate inactivation was also fitted to a single exponential function and accelerated with a time constant of 3.17 0.2 ms (n ¼5, Po0.05).
2.6. Effects of fluoxetine on use-dependent block and the time course for recovery from the inactivation of Kv4.3 We examined the use dependence of the block of Kv4.3 by fluoxetine. Kv4.3 currents were elicited by a series of 10 depolarizing pulses from a holding potential of 80 mV to þ40 mV at 1 and 2 Hz stimulation frequencies (Fig. 7A). Under control conditions, the current amplitude was slightly reduced by 0.570.2 and 10.170.9% at 1 and 2 Hz, respectively (n¼ 7) (Fig. 7B). In the presence of fluoxetine, the block was significantly increased to 9.971.51% at 1 Hz. The block developed faster at 2 Hz than at 1 Hz and was significantly increased 28.573.3% (n¼ 7, Po0.05), suggesting a strong dependence on frequency. The time course of recovery from Kv4.3 inactivation was studied using a paired-pulse protocol
Fig. 4 – Voltage-dependent effect of fluoxetine on Kv4.3. Superimposed current traces were elicited by 500 ms depolarizing pulses from the holding potential of 80 mV to a potential between 70 and þ60 mV in increments of þ10 mV at 10 s intervals in the absence (A) and presence (B) of fluoxetine, (C) current–voltage relationship for peak amplitude of Kv4.3, and (D) relative currents from data obtained in the absence and presence of fluoxetine were plotted against the test potential. The data were taken from the integral of the depolarizing pulses. The dashed lines represent the activation curve under the control conditions (Jeong et al., 2011). The voltage dependence of the fractional block was fitted to a Woodhull equation (see Experimental procedures). The solid lines represent the linear fit. Statistically significant difference from data obtained at 30 mV (Po0.05). The data are represented as the means7S.E.
14
brain research 1500 (2013) 10–18
Fig. 5 – Effects of fluoxetine on the steady-state inactivation of Kv4.3. (A) The currents were elicited by 1 s prepulses that were varied from 110 mV to þ10 mV stepped by 10 mV and a 500 ms depolarizing pulse to þ40 mV in the absence and presence of fluoxetine, and (B) the steady-state inactivation curve of Kv4.3 shown as a plot of normalized peak current as a function of the conditioning pulse. The data are represented as the means7S.E. (n¼ 6).
Fig. 6 – Effects of fluoxetine on closed-state inactivation of Kv4.3 channels. (A) Kv4.3 currents were recorded at þ40 mV using a two-pulse protocol with a conditioning pulse to 60 mV of variable duration in the absence and presence of fluoxetine, and (B) the current amplitude resulting from the initial control pulse, was plotted against the duration of the conditioning pulse and fitted to a single exponential function. Data are means7S.E. (n ¼5).
(Fig. 8A). The time courses of recovery from inactivation can be fitted to a single exponential function with a time constant of 114.678.2 ms during control, and 305.2714.1 ms (n ¼6, Po0.05) after fluoxetine treatment (Fig. 8B). These results suggest that fluoxetine significantly slowed the recovery from inactivation of Kv4.3.
2003; Perchenet et al., 2001; Rajamani et al., 2006; Sung et al., 2008; Tytgat et al., 1997). Thus, our results suggest that fluoxetine interacts primarily with the open state of the Kv4.3 channels based on the following reasons. At the onset of depolarizing pulses, the initial time courses of channel activation and the peak amplitudes of Kv4.3 currents were not significantly affected. This result suggests that the interaction of fluoxetine with the channel occurs after the channel activation. Upon depolarization, however, the block of Kv4.3 increased in an exponential manner, and the time course of block development fit well with kinetic on- and off-rates, suggesting a simple model for open channel block (Jeong et al., 2011; Kim et al., 2007; Snyders and Yeola, 1995). In addition, the block increased in the voltage range of activation with a steep phase, and additional voltage dependence of the block was observed over the full range of activation with a shallow phase (Snyders et al., 1992; Wang et al., 1995). A fractional electrical distance (d) of 0.29 for fluoxetine was obtained. This value was similar to the d values of 0.17 to 0.38 obtained in previous reports for fluoxetine block of Kv1.1, Kv1.3, Kv1.5 and Kv3.1 channels (Choi et al., 1999; Perchenet
3.
Discussion
In the present study, we found that fluoxetine blocked the Kv4.3 current in concentration-, time-, voltage-, and usedependent manners. The main finding of this study was that fluoxetine decreased the peak amplitude of Kv4.3 currents in a concentration-dependent manner, but the peak current was less affected than the steady-state current. Accordingly, fluoxetine induced an acceleration of current inactivation of Kv4.3. These results are similar to the effects of fluoxetine on delayed rectifier and A-type Kv currents from several preparations, and are consistent with the mechanism of an open channel block (Choi et al., 2001, 1999, 2004; Choi B.H. et al.,
brain research 1500 (2013) 10–18
Fig. 7 – (A) Use-dependent effect of fluoxetine Kv4.3 (A) Ten consecutive depolarizing pulses to 40 mV were applied at 1 and 2 Hz in the absence and the presence of fluoxetine, and (B) the peak amplitudes of current at each pulse were normalized by the initial amplitudes and plotted as a function of the pulse number. The data are represented as the means7S.E (n ¼ 7). et al., 2001; Sung et al., 2008; Tytgat et al., 1997). However, fluoxetine reportedly blocked Kv1.4 channels by preferentially binding in the open state but produced a voltage-independent block of Kv1.4 (Choi B.H. et al., 2003). The block of Kv4.3 by fluoxetine was dependent on the stimulation frequency: use-dependent block can also be interpreted as the result of open channel block (Butterworth and Strichartz, 1990; Wang et al., 1995). Thus, Kv4.3 channels in the open state have a much higher affinity compared with channels in the resting state. Fluoxetine slows the recovery kinetics from inactivation, indicating that the dissociation rate of fluoxetine is lower than the transition rate between the open and the resting state under control conditions (Choi et al., 2000). Furthermore, fluoxetine produced a use-dependent block of Kv4.3, which reflected the slow recovery kinetics of Kv4.3 in the presence of fluoxetine. However, the application of fluoxetine on Kv4.3 did not change the steady-state inactivation curves, which suggests that there was no change of affinity in the inactivated state. A-type Kv currents have rapidly activating and inactivating kinetics (Hagiwara et al., 1961). Based on the kinetics of
15
Fig. 8 – Effects of fluoxetine on the time course of recovery from the inactivation of Kv4.3. (A) The recovery from inactivation of Kv4.3 was assessed with a two-pulse protocol with varying interpulse intervals, and (B) the current elicited by the second pulse was normalized to the current elicited by the first pulse and is shown as a function of recovery time. Time courses of recovery from inactivation were fitted with a single exponential function. The data are represented as the means7S.E. (n ¼6).
recovery from inactivation, A-type Kv currents can be classified into 2 phenotypes: Ito, fast and Ito, slow. Kv4.3 has been linked to somatodendritic A-type Kv currents in nervous cells and to transient outward Kv currents in the heart with underlying Ito, fast. Kv1.4 channels are thought to underlie Ito, slow. These channels are widely expressed in neuronal cells of the brain (Lujan et al., 2003). In contrast to Kv1.4 which undergoes an N-type inactivation, Kv4.3 is inactivated by concerted action of the cytoplasmic N- and C-termini (Jerng and Covarrubias, 1997). Whereas Kv1.4 channels inactivate from an open state during depolarizing potentials, Kv4.3 is known to preferentially inactivate via a closed-state inactivation in the subthreshold range of membrane potential (Beck and Covarrubias, 2001; Wang et al., 2005). In the present study, fluoxetine accelerated the closed-state inactivation of Kv4.3. Because the potency of a drug is dependent on its membrane potential, which determines the state of the channels, the acceleration of closed-state inactivation by fluoxetine increases the potency at subthreshold potentials.
16
brain research 1500 (2013) 10–18
Generally, A-type Kv currents play an important role in setting the resting membrane potential, and determining the action potential duration and neuronal firing rates in the central nervous system (Bardoni and Belluzzi, 1993; Malin and Nerbonne, 2000). Fluoxetine is a selective inhibitor of the 5-hydroxytryptamine (HT) reuptake transporter and results in increased concentrations of 5-HT levels in the synapses of neuronal cells, which explains its therapeutic action as an antidepressant (Wong and Bymaster, 1995; Wong et al., 1995). Pharmacological blockade of A-type Kv currents by 4aminopyrine increased the spontaneous basal release of [3H]5-HT from rat hippocampal slices (Schechter, 1997). In both the previous and present studies, fluoxetine blocked Kv1.4 and Kv4.3 channels expressed in CHO cells in a concentration-dependent manner (Choi B.H. et al., 2003). Furthermore, fluoxetine and its major metabolite, norfluoxetine, potently blocked A-type Kv currents in primary cultured rat hippocampal neurons (Choi et al., 2004). Thus, a block of Kv4.3 channels by fluoxetine could contribute to the antidepressant effect by increasing the 5-HT concentrations in the synaptic cleft of neuronal cells. In addition to its antidepressant action, fluoxetine possesses an analgesic effect and is widely used in the treatment of neuropathic pain (Dharmshaktu et al., 2012; Rani et al., 1996). Kv4.3 and Kv1.4 channels are thought to encode A-type Kv currents in dorsal root ganglion (DRG) neurons and are involved in the generation of nociceptive impulses in peripheral DRG neurons (Birnbaum et al., 2004; Hsieh, 2008; Phuket and Covarrubias, 2009). Thus, it is interesting to investigate whether fluoxetine may exert its analgesic effects in DRG neurons via the block of these channels. The therapeutic plasma concentrations of fluoxetine range approximately from 0.1 to 3.4 mM (Altamura et al., 1994; Orsulak et al., 1988). In the present study, fluoxetine blocked Kv4.3 currents with an IC50 of 11.8 mM, which is 3-fold higher than the plasma concentrations in human beings. The lowest concentration of fluoxetine (3 mM) in the present study significantly blocked the Kv4.3 currents (about 18%), and this concentration falls within the range of the therapeutic plasma concentration. In addition, the brain concentration of fluoxetine seems to be 20 times higher than the corresponding blood levels because of the high lipophilicity of the drug (Caccia et al., 1992; Karson et al., 1993). Thus, the blocking effect of fluoxetine on Kv4.3 could be clinically relevant. In conclusion, fluoxetine blocks the open state of Kv4.3 and accelerates the close-state inactivation of Kv4.3 expressed in CHO cells. The block of Kv4.3 by fluoxetine should therefore be taken into account for its pharmacological effect in the treatment of depression.
supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine (Invitrogen), 0.1 mM hypoxanthine (Invitrogen), 0.01 mM thymidine (Invitrogen), 0.3 mg/ml G418 (Invitrogen), and 1% of a 100X antibiotic antimycotic mixture (Invitrogen) at 37 1C with 5% CO2-enriched air. The cells treated with trypsin–EDTA solution were plated on glass coverslips (12 mm diameter; Fisher Scientific, Pittsburgh, PA, USA) and placed in 35 mm Petri dishes. Patch-clamp recordings were obtained 12–24 h later. For voltage-clamp recordings, the coverslips containing adherent CHO cells were mounted on the glass bottom of the recording chamber (RC13; Warner Instrument, Hamden, CT, USA) and were perfused with an extracellular bath solution.
4.2.
Kv4.3 currents were recorded using a whole-cell configuration of the patch-clamp technique with an Axopatch 200B patchclamp amplifier (Molecular Devices, Sunnyvale, CA, USA) at room temperature (22–24 1C). Patch electrodes were pulled from soft glass capillaries (PG10165-4; World Precision Instruments, Sarasota, FL, USA) on a programmable horizontal microelectrode puller (P-97; Sutter Instrument Co., Novato, CA, USA). When filled with the pipette solution, the electrode resistance was maintained at 2–4 MO. Data acquisition and analysis were performed on an IBM-compatible Pentium computer running pClamp 10.0 software (Molecular Devices) using a Digidata 1322A interface (Molecular Devices). Leak subtraction was not applied to the data. In the whole-cell configuration, the average series resistance was 2.970.2M O (n ¼58). The effective series resistances were usually compensated by 80% if the Kv4.3 current exceeded 1 nA. Voltage drops were based on the calculated series resistance and were less than 5 mV.
4.3.
Experimental procedures
4.1.
Cell culture
The CHO cells were transfected with cDNA encoding Kv4.3 channels using the Lipofectamine reagent (Invitrogen, Grand Island, NY, USA), as previously described in detail (Ahn et al., 2006). A stable cell line expressing Kv4.3 channels was maintained in Iscove’s modified Dulbecco’s medium (Invitrogen)
Solutions and drugs
To record Kv4.3 current, the extracellular bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, which was adjusted to pH 7.3 using NaOH. The intracellular pipette solution contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 EGTA, which was adjusted to pH 7.3 using KOH. Fluoxetine (Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) to yield the stock solution. The stock solution was then diluted with the bath solution to obtain the desired concentration. The concentration of DMSO in the final dilution was less than 0.1%, and this concentration had no effect on Kv4.3 currents.
4.4.
4.
Electrophysiology
Data analysis
Data analysis was performed using Origin 8.0 software (Origin Lab Corp., Northampton, MA, USA). The concentrationdependent block of Kv4.3 current by fluoxetine at þ40 mV was fitted to the Hill equation: f ¼ 1=½1 þ ðIC50 =Þ½DÞn
ð1Þ
where f is the fractional block for the fluoxetine concentration [D], IC50 is the concentration of fluoxetine that produces a half-maximum current block, and n is the Hill coefficient.
brain research 1500 (2013) 10–18
Kv4.3 currents were elicited by applying 500 ms depolarizing pulses from a holding potential of 80 to þ40 mV every 10 s. The drug-induced time constants were used as an approximation of the drug–channel interaction kinetics (Slawsky and Castle, 1994; Snyders and Yeola, 1995) according to the following equations: 1=tBlock ¼ kþ1 ½D þ k1
ð2aÞ
Kd ¼ k1 =kþ1
ð2bÞ
where tBlock is the drug-induced time constant, [D] denotes the drug concentration, and kþ1 and k1 are the apparent rate constants of binding and unbinding for the drug, respectively. We measured the fractional block at each potential and fitted the data to the Woodhull equation, as follows (Woodhull, 1973): f ¼ ½D=f½D þ Kd ð0Þ x expð-zdFV=RTÞg
ð3Þ
where Kd(0) is the apparent affinity at 0 mV (the reference voltage), z is the charge valence of the drug, d is the fractional electrical distance (i.e., the fraction of the transmembrane electric field sensed by a single charge at the receptor site), F is Faraday’s constant, R is the gas constant, and T is the absolute temperature. In the present study, 25.4 mV was used as the RT/F value at 22 1C. The steady-state inactivation curves were obtained by normalizing the peak current amplitudes using a two-pulse protocol. The current was produced by a 500 ms depolarizing pulse to þ40 mV, whereas 1 s preconditioning pulses were varied from 110 to þ10 mV in increments of 10 mV at 10 s intervals in the absence and presence of fluoxetine. The curves were fitted to the Boltzmann equation: ðI2Ic Þ=ðImax 2Ic Þ ¼ 1=½1 þ expðV2V1=2 Þ=k
ð4Þ
in which Imax represents the current measured at the most hyperpolarized preconditioning pulse, and Ic represents a non-inactivating current at the most depolarized preconditioning pulse. V, V1/2, and k are the test potential, the point where channels are half-inactivated, and the slope, respectively. The non-inactivating residual current was removed by subtracting it from the actual value. A two-pulse protocol was used to determine the time course of recovery of the Kv4.3 currents from inactivation: the first pre-pulse of a 500 ms depolarizing pulse of þ40 mV from a holding potential of 80 mV was followed by a second identical pulse after increasing the inter-pulse intervals between 5 and 10,000 ms at 80 mV. The dominant time constant of activation was determined by fitting a single exponential function to the latter 50% of activation (Snyders et al., 1993). We studied the kinetics of activation, which reflects the transition from the closed to the open state, by measuring the time to peak.
4.5.
Statistics
The data are summarized as the means7S.E. The paired Student’s t-test and one-way analysis of variance were for comparisons of multiple groups followed by Bonferroni’s test was used for statistical analysis. Differences were considered significant at Po0.05.
17
Acknowledgments We thank Dr. Yuji Imaizumi (Department of Molecular and Cellular Pharmacology, Nagoya City University, Nagoya, Japan) for the Kv4.3 cDNA.
r e f e r e nc e s
Ahn, H.S., Kim, S.E., Jang, H.J., Kim, M.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2006. Interaction of riluzole with the closed inactivated state of Kv4.3 channels. J. Pharmacol. Exp. Ther. 319, 323–331. Altamura, A.C., Moro, A.R., Percudani, M., 1994. Clinical pharmacokinetics of fluoxetine. Clin. Pharmacokinet. 26, 201–214. Bardoni, R., Belluzzi, O., 1993. Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices. J. Neurophysiol. 69, 2222–2231. Beck, E.J., Covarrubias, M., 2001. Kv4 channels exhibit modulation of closed-state inactivation in inside-out patches. Biophys. J. 81, 867–883. Bian, J.T., Yeh, J.Z., Aistrup, G.L., Narahashi, T., Moore, E.J., 2002. Inhibition of Kþ currents of outer hair cells in guinea pig cochlea by fluoxetine. Eur. J. Pharmacol. 453, 159–166. Birnbaum, S.G., Varga, A.W., Yuan, L.L., Anderson, A.E., Sweatt, J.D., Schrader, L.A., 2004. Structure and function of Kv4-family transient potassium channels. Physiol. Rev. 84, 803–833. Brown, J., PM, O.B., Marjoribanks, J., Wyatt, K., 2009. Selective serotonin reuptake inhibitors for premenstrual syndrome. Cochrane Database Syst. Rev., CD001396. Butterworth, J.F.t., Strichartz, G.R., 1990. Molecular mechanisms of local anesthesia: a review. Anesthesiology 72, 711–734. Caccia, S., Bizzi, A., Coltro, G., Fracasso, C., Frittoli, E., Mennini, T., Garattini, S., 1992. Anorectic activity of fluoxetine and norfluoxetine in rats: relationship between brain concentrations and in-vitro potencies on monoaminergic mechanisms. J. Pharm. Pharmacol. 44, 250–254. Choi, B.H., Choi, J.S., Jeong, S.W., Hahn, S.J., Yoon, S.H., Jo, Y.H., Kim, M.S., 2000. Direct block by bisindolylmaleimide of rat Kv1.5 expressed in Chinese hamster ovary cells. J. Pharmacol. Exp. Ther. 293, 634–640. Choi, B.H., Choi, J.S., Yoon, S.H., Rhie, D.J., Min, D.S., Jo, Y.H., Kim, M.S., Hahn, S.J., 2001. Effects of norfluoxetine, the major metabolite of fluoxetine, on the cloned neuronal potassium channel Kv3.1. Neuropharmacology 41, 443–453. Choi, B.H., Choi, J.S., Ahn, H.S., Kim, M.J., Rhie, D.J., Yoon, S.H., Min, D.S., Jo, Y.H., Kim, M.S., Hahn, S.J., 2003. Fluoxetine blocks cloned neuronal A-type Kþ channels Kv1.4. Neuroreport 14, 2451–2455. Choi, J.S., Hahn, S.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., 1999. Mechanism of fluoxetine block of cloned voltage-activated potassium channel Kv1.3. J. Pharmacol. Exp. Ther. 291, 1–6. Choi, J.S., Choi, B.H., Ahn, H.S., Kim, M.J., Rhie, D.J., Yoon, S.H., S., Min do, Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2003. Mechanism of block by fluoxetine of 5-hydroxytryptamine3 (5-HT3)-mediated currents in NCB-20 neuroblastoma cells. Biochem. Pharmacol. 66, 2125–2132. Choi, J.S., Choi, B.H., Ahn, H.S., Kim, M.J., Han, T.H., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Hahn, S.J., 2004. Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons. Brain Res. 1018, 201–207. Dharmshaktu, P., Tayal, V., Kalra, B.S., 2012. Efficacy of antidepressants as analgesics: a review. J. Clin. Pharmacol. 52, 6–17. Dixon, J.E., Shi, W., Wang, H.S., McDonald, C., Yu, H., Wymore, R.S., Cohen, I.S., McKinnon, D., 1996. Role of the Kv4.3 Kþ channel in
18
brain research 1500 (2013) 10–18
ventricular muscle. A molecular correlate for the transient outward current. Circ. Res. 79, 659–668. Farrugia, G., 1996. Modulation of ionic currents in isolated canine and human jejunal circular smooth muscle cells by fluoxetine. Gastroenterology 110, 1438–1445. Garattini, S., Bizzi, A., Caccia, S., Mennini, T., 1992. Progress report on the anorectic effects of dexfenfluramine, fluoxetine and sertraline. Int. J. Obes. Relat. Metab. Disord. 16 (Suppl. 3), S43–S50. Garcia-Colunga, J., Awad, J.N., Miledi, R., 1997. Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac). Proc. Natl. Acad. Sci. USA 94, 2041–2044. Hagiwara, S., Kusano, K., Saito, N., 1961. Membrane changes of Onchidium nerve cell in potassium-rich media. J. Physiol. 155, 470–489. Hsieh, C.P., 2008. Redox modulation of A-type Kþ currents in pain-sensing dorsal root ganglion neurons. Biochem. Biophys. Res. Commun. 370, 445–449. Jeong, I., Choi, B.H., Hahn, S.J., 2011. Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation. Br. J. Pharmacol. 163, 510–520. Jerng, H.H., Covarrubias, M., 1997. Kþ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains. Biophys. J. 72, 163–174. Karson, C.N., Newton, J.E., Livingston, R., Jolly, J.B., Cooper, T.B., Sprigg, J., Komoroski, R.A., 1993. Human brain fluoxetine concentrations. J. Neuropsychiatry Clin. Neurosci. 5, 322–329. Kennard, L.E., Chumbley, J.R., Ranatunga, K.M., Armstrong, S.J., Veale, E.L., Mathie, A., 2005. Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine. Br. J. Pharmacol. 144, 821–829. Kim, S.E., Ahn, H.S., Choi, B.H., Jang, H.J., Kim, M.J., Rhie, D.J., Yoon, S.H., Jo, Y.H., Kim, M.S., Sung, K.W., Hahn, S.J., 2007. Open channel block of A-type, Kv4.3, and delayed rectifier Kþ channels, Kv1.3 and Kv3.1, by sibutramine. J. Pharmacol. Exp. Ther. 321, 753–762. Kobayashi, T., Washiyama, K., Ikeda, K., 2003. Inhibition of G protein-activated inwardly rectifying Kþ channels by fluoxetine (Prozac). Br. J. Pharmacol. 138, 1119–1128. Lujan, R., de Cabo de la Vega, C., Dominguez del Toro, E., Ballesta, J.J., Criado, M., Juiz, J.M., 2003. Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat. J. Chem. Neuroanat. 26, 209–224. Maertens, C., Wei, L., Voets, T., Droogmans, G., Nilius, B., 1999. Block by fluoxetine of volume-regulated anion channels. Br. J. Pharmacol. 126, 508–514. Malin, S.A., Nerbonne, J.M., 2000. Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F: molecular dissection of IA. J. Neurosci. 20, 5191–5199. Nahon, E., Israelson, A., Abu-Hamad, S., Varda, S.B., 2005. Fluoxetine (Prozac) interaction with the mitochondrial voltage-dependent anion channel and protection against apoptotic cell death. FEBS Lett. 579, 5105–5110. Niwa, N., Nerbonne, J.M., 2010. Molecular determinants of cardiac transient outward potassium current (Ito) expression and regulation. J. Mol. Cell Cardiol. 48, 12–25. Orsulak, P.J., Kenney, J.T., Debus, J.R., Crowley, G., Wittman, P.D., 1988. Determination of the antidepressant fluoxetine and its metabolite norfluoxetine in serum by reversed-phase HPLC with ultraviolet detection. Clin. Chem. 34, 1875–1878. Patel, S.P., Campbell, D.L., 2005. Transient outward potassium current, ‘Ito’, phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms. J. Physiol. 569, 7–39. Perchenet, L., Hilfiger, L., Mizrahi, J., Clement-Chomienne, O., 2001. Effects of anorexinogen agents on cloned voltage-gated Kþ channel hKv1.5. J. Pharmacol. Exp Ther. 298, 1108–1119.
Phuket, T.R., Covarrubias, M., 2009. Kv4 channels underlie the subthreshold-operating A-type K-current in nociceptive dorsal root ganglion neurons. Front. Mol. Neurosci. 2, 3. Rae, J.L., Rich, A., Zamudio, A.C., Candia, O.A., 1995. Effect of Prozac on whole cell ionic currents in lens and corneal epithelia. Am. J. Physiol. 269, C250–C256. Rajamani, S., Eckhardt, L.L., Valdivia, C.R., Klemens, C.A., Gillman, B.M., Anderson, C.L., Holzem, K.M., Delisle, B.P., Anson, B.D., Makielski, J.C., January, C.T., 2006. Drug-induced long QT syndrome: hERG Kþ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine. Br. J. Pharmacol. 149, 481–489. Rani, P.U., Naidu, M.U., Prasad, V.B., Rao, T.R., Shobha, J.C., 1996. An evaluation of antidepressants in rheumatic pain conditions. Anesth. Analg. 83, 371–375. Rasmusson, R.L., Morales, M.J., Wang, S., Liu, S., Campbell, D.L., Brahmajothi, M.V., Strauss, H.C., 1998. Inactivation of voltagegated cardiac Kþ channels. Circ. Res. 82, 739–750. Rudy, B., 1988. Diversity and ubiquity of K channels. Neuroscience 25, 729–749. Schechter, L.E., 1997. The potassium channel blockers 4aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices. J. Pharmacol. Exp. Ther 282, 262–270. Slawsky, M.T., Castle, N.A., 1994. Kþ channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes. J. Pharmacol. Exp. Ther 269, 66–74. Snyders, D.J., Tamkun, M.M., Bennett, P.B., 1993. A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression. J. Gen. Physiol. 101, 513–543. Snyders, D.J., Yeola, S.W., 1995. Determinants of antiarrhythmic drug action. Electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel. Circ. Res 77, 575–583. Snyders, J., Knoth, K.M., Roberds, S.L., Tamkun, M.M., 1992. Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel. Mol. Pharmacol. 41, 322–330. Sung, M.J., Ahn, H.S., Hahn, S.J., Choi, B.H., 2008. Open channel block of Kv3.1 currents by fluoxetine. J. Pharmacol. Sci. 106, 38–45. Tytgat, J., Maertens, C., Daenens, P., 1997. Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1). Br. J. Pharmacol. 122, 1417–1424. Wang, S., Bondarenko, V.E., Qu, Y.J., Bett, G.C., Morales, M.J., Rasmusson, R.L., Strauss, H.C., 2005. Time- and voltagedependent components of Kv4.3 inactivation. Biophys. J. 89, 3026–3041. Wang, Z., Fermini, B., Nattel, S., 1995. Effects of flecainide, quinidine, and 4-aminopyridine on transient outward and ultrarapid delayed rectifier currents in human atrial myocytes. J. Pharmacol. Exp. Ther. 272, 184–196. Wong, D.T., Bymaster, F.P., 1995. Development of antidepressant drugs. Fluoxetine (Prozac) and other selective serotonin uptake inhibitors. Adv. Exp. Med. Biol. 363, 77–95. Wong, D.T., Bymaster, F.P., Engleman, E.A., 1995. Prozac (fluoxetine, Lilly 110140), the first selective serotonin uptake inhibitor and an antidepressant drug: twenty years since its first publication. Life Sci. 57, 411–441. Woodhull, A.M., 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687–708. Yeung, S.Y., Millar, J.A., Mathie, A., 1999. Inhibition of neuronal KV potassium currents by the antidepressant drug, fluoxetine. Br. J. Pharmacol. 128, 1609–1615.