Antihistamine Mepyramine Directly Inhibits KCNQ/M Channel and Depolarizes Rat Superior Cervical Ganglion Neurons

Available online at www.sciencedirect.com

Neuropharmacology 54 (2008) 629e639 www.elsevier.com/locate/neuropharm

Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons

Boyi Liu, Xuan Zhang, Chuan Wang, Guohong Zhang, Hailin Zhang*

Department of Pharmacology, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province 050017, China Received 31 July 2007; received in revised form 19 November 2007; accepted 20 November 2007

Abstract

The ﬁrst-generation antihistamines are widely prescribed medications that relieve allergic reactions and urticaria by blocking the peripheral histamine H1 receptor. Overdose of these drugs often results in serious neuronal toxic effects, including seizures, convulsions and worsening of epileptic symptoms. The KCNQ/M Kþ channel plays a crucial role in controlling neuron excitability. Here, we demonstrate that mepyramine and diphenhydramine, two structurally related ﬁrst-generation antihistamines, can act as potent KCNQ/M channel blockers. Extracellular application of these drugs quickly and reversibly reduced KCNQ2/Q3 currents heterologously expressed in HEK293 cells. The current inhibition was concentration and voltage dependent. The estimated IC50 (12.5 and 48.1 mM, respectively) is within the range of drug concentrations detected in poisoned patients (30e300 mM). Both drugs shifted the IeV curve of KCNQ2/Q3 channel to more depolarized potentials and altered channel gating properties by prolonging activation and shortening deactivation kinetics. Mepyramine also inhibited the individual homomeric KCNQ1e4 and heteromeric KCNQ3/Q5 currents. Moreover, mepyramine inhibited KCNQ2/Q3 current in an outside-out patch excised from HEK293 cells and the inhibitory effect was neither observed when it was applied intracellularly nor affected by blocking phospholipase C (PLC) activity, indicating an extracellular and direct channel blocking mechanism. Finally, in cultured rat superior cervical ganglion (SCG) neurons, mepyramine reduced the M type Kþ current in a concentration-dependent manner and led to marked membrane potential depolarization. It is likely that these effects may be involved in the adverse neuroexcitatory effects observed in patients experiencing an overdose of antihistamines. Ó 2007 Published by Elsevier Ltd.

Keywords: Mepyramine; Diphenhydramine; KCNQ/M channel; Membrane potential; Excitability; SCG neuron

1. Introduction addition to the sedative side effects occurring under therapeutic doses, overdoses of these drugs can lead to serious adverse ef- Histamine H1 receptor antagonists, often termed antihista- fects, such as worsening of epileptic symptoms, convulsions mines, are widely used drugs to relieve the symptoms of and fatal seizures (Olson et al., 1994; Yokoyama et al., 1993). upper respiratory tract infections, allergies and urticarial The first-generation antihistamines are small lipophilic conditions. In spite of the commonly occurring sedative side ef- molecules which can easily cross the bloodebrain barrier fect, the first-generation antihistamines, such as diphenhydra- (BBB), thus affecting the central nervous system (CNS) mine and hydroxyzine, are still widely prescribed medications (Katzung, 2003). The disturbance of central histaminergic to ameliorate the symptoms of some allergies, such as allergic receptors by first-generation antihistamines may explain the rhinitis and urticaria (Blaiss, 2004; Gelfand, 2005). Due to their neuronal toxicity resulting from overdose (Kamei et al., widespread use, these drugs are frequently involved in acciden- 2000; Yawata et al., 2004). Nevertheless, the detailed cellular tal or intentional poisonings (Taglialatela et al., 2000). In mechanisms are still not clear. In a previous study, we demonstrated that first-generation antihistamines, but not second or * Corresponding author. Tel.: þ86 031 1862 65562; fax: þ86 031 1860 third-generation antihistamines, inhibit inwardly rectifying po- 57291. tassium (Kir) channels, which are widely distributed in the E-mail address: [email protected] (H. Zhang). heart and CNS, serve important roles in the maintenance of

0028-3908/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.neuropharm.2007.11.012 630 B. Liu et al. / Neuropharmacology 54 (2008) 629e639 cell membrane potential and may be involved in the regulation for 30 min at 37 C. Then the ganglions were placed into trypsin solution of neuron excitability (Liu et al., 2007). (2.5 mg/ml) for 30 min at 37 C. The digested fragments were then rinsed The M type Kþ channel, whose molecular basis is consid- with 2 ml DMEM plus 10% fetal bovine serum three times, centrifuged and e dissociated by trituration. The ganglia were plated onto the glass coverslips ered to be KCNQ2 5, has been characterized in many types pre-coated with poly-D-lysine and incubated at 37 C. After the neurons had of peripheral and central neurons, including superior cervical attached to the coverslips, cell culture medium was changed to Neurobasal ganglion (SCG), dorsal root ganglion (DRG), hippocampal plus B27 supplement (Invitrogen). Neurons were cultured for 1 day and and cortical neurons (Owen et al., 1990; Passmore et al., used within 24 h. 2003; Peretz et al., 2005; Shah et al., 2002). The M current plays a key role in regulating various central and peripheral 2.4. Electrophysiology neuron excitabilities and stabilizing membrane potential For current measurements in SCG neurons and HEK293 cells, recordings (Delmas and Brown, 2005). Suppression of the M current were performed using the whole-cell and excised outside-out configurations through G protein coupled receptor (GPCR) activation or by of the patch-clamp technique. Signals were amplified using an Axopatch certain drugs leads to depolarization of membrane potential 200B patch-clamp amplifier (Axon Instruments) and filtered at 1 kHz. Patch and hyperexcitability of neurons (Brown and Yu, 2000). In ad- electrodes were pulled with a Flaming/Brown micropipette puller (Sutter In- e dition, a form of epilepsy, benign familial neonatal convulsion, struments) and fire-polished to a final resistance of 4 6MU when filled with internal solution. Data acquisition was achieved using pClamp 8.1 software. is thought to be closely related to loss-of-function mutations in The internal solution for HEK293 cell and rat SCG neuron recording was as

KCNQ2 or KCNQ3 channel subunits (Schroeder et al., 1998). follows (in mM): 175 KCl, 5 MgCl2, 5 HEPES, 0.1 BAPTA, 3 K2ATP, 0.1 In a more recent paper, it is further proposed that M current NaGTP, pH 7.4 adjusted with KOH. The external solution for HEK293 cells inhibition can promote the transition to seizures in immature contained (in mM): 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 8 Glu- rat hippocampus (Qiu et al., 2007). cose, pH 7.4 adjusted with NaOH. For recordings from SCG neurons, the CaCl2 concentration was 5 mM. The perfusion system was a home made 100 ml per- Recently, it has been reported that first-generation antihist- fusion chamber through which solution flowed continuously at 1e2 ml/min. þ amines are able to inhibit a linopirdine-sensitive K current in Drugs were applied to cells by gravity via a BPS-8 valve control system (Sci- rat cortical neurons (Sato et al., 2005). In the present study, we entific Instruments). All recordings were carried out at room temperature. use pharmacological and electrophysiological approaches to show that mepyramine and diphenhydramine, two first-gener- 2.5. Chemicals ation antihistamines, can inhibit all KCNQ channels (KCNQ1e5). We further study the possible mechanism under- Mepyramine, diphenhydramine, cimetidine, oxo-M, linopirdine and U-73122 were all purchased from Sigma (St. Louis, MO). The antihistamine lying the inhibition and proceed to demonstrate that mepyr- solutions were all freshly prepared from stock solutions before each experi- amine can inhibit the native M current and depolarize ment and kept away from light exposure. membrane potential of rat SCG neurons. We hypothesize that these actions are likely to be involved in the adverse neu- 2.6. Data analysis and statistics roexcitatory effects observed in patients intoxicated by overdose with first-generation antihistamines. Currents were analyzed and fitted using Clampfit 9.0 (Axon Instrument) and Origin 7.0 (Originlab Corporation) software. To analyze the kinetics of 2. Methods KCNQ2/Q3 channel activation and deactivation, a single exponential fit was applied to the activation and deactivation currents. The current activation 2.1. cDNA constructs curves were generated by plotting the normalized tail current amplitudes against the step potentials and were fitted with a Boltzmann function according to the equation: y ¼ A/{1 þ exp[(V V )/k]}, where A is the amplitude of re- Plasmids encoding human KCNQ1, human KCNQ2, rat KCNQ3, human h m lationship, V is the voltage for half-maximal activation, V is the test potential KCNQ4 and human KCNQ5 (GenBank accession numbers: NM000218, h m and k is the slope. The concentration-response curve was fitted by logistic AF110020, AF091247, AF105202 and AF249278, respectively) were kindly equation: y ¼ A þ (A A )/(1 þ (x/x )p), where x is the drug concentration provided by Diomedes E. Logothetis (Mount Sinai School of Medicine, 2 1 2 0 and p is the Hill coefficient. New York, NY). Results were expressed as means SEM. Statistical analysis of differences between groups was carried out using Student’s t-test, paired t-test or 2.2. HEK293 cell culture and transfection one-way ANOVA. The differences were considered significant if p < 0.05.

HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics in a 3. Results humidified incubator at 37 C (5% CO2). Cells were seeded on poly-D-lysine-coated glass coverslips in a 24-multiwell plate and transfected when 3.1. Mepyramine and diphenhydramine inhibit KCNQ2/ e 60 70% confluence was reached. For transfection of 6 wells of cells, a mixture Q3 channel currents of 3 mg KCNQ, pEGFP-N1 cDNAs and 3 ml Lipofectamine 2000 reagent (In- vitrogen) were prepared in 1.2 ml of DMEM and incubated for 20 min according to manufacturer’s instruction. The mixture was then applied to the cell Upon transfection with KCNQ2 and KCNQ3 cDNAs, culture wells and incubated for 4e6 h. Recordings were made 24 h after cell HEK293 cells yielded a slowly activating, slowly deactivating transfection and cells were used within 48 h. and non-inactivating outward current with neuronal M current-like properties. Fig. 1A shows the typical current traces eli- 2.3. Rat SCG neuron culture cited from a cell recorded by conventional patch-clamp SCGs were isolated from 10e17 days old SpragueeDawley rats, cut into technique. The cell was held at 80 mV and depolarized to pieces, then transferred into collagenase solution (1 mg/ml) and incubated 20 mV, then hyperpolarized to 60 mV to induce the typical B. Liu et al. / Neuropharmacology 54 (2008) 629e639 631 deactivation tail current. Application of 100 mM mepyramine, cimetidine (100 mM), had no effect on channel currents which is a histamine H1 receptor antagonist of first-generation (n ¼ 6, data not shown). antihistamine, significantly inhibited both the activation current Fig. 1C shows the representative traces of KCNQ2/Q3 current and deactivation tail current of KCNQ2/Q3 channels (Fig. 1A). elicited by a train of voltage steps from 80 to þ30 mV in The inhibition developed rapidly and reached a steady state a 10 mV interval from a holding potential of 80 mV. A total soon after drug application. Removal of the drug from external of 100 mM mepyramine inhibited KCNQ2/Q3 current elicited solution resulted in a large extent recovery of the current from at each depolarizing voltage step. We then studied the voltage inhibition (Fig. 1A, inset). The inhibition of the activation and dependency of the inhibition by comparing the percentage deactivation tail current was 77.6 1.3% and 92.0 1.7%, re- inhibition of KCNQ2/Q3 currents activated from 30 to spectively (Fig. 1B, n ¼ 29). Similar results were also obtained þ30 mV. Inhibition of tail current reached 89.3 0.4% for test with another first-generation antihistamine, diphenhydramine. potentials of 30 mV but decreased to 66.1 0.7% when depo- Diphenhydramine (100 mM) reduced the KCNQ2/Q3 tail cur- larized to þ30 mV (Fig. 1D, n ¼ 9, p < 0.01, one-way ANOVA). rents in HEK293 cells by 66.9 5.9% (Fig. 1A,B, n ¼ 13). Therefore, these results indicate that mepyramine inhibited On the other hand, a histamine H2 receptor antagonist, KCNQ2/Q3 channel current in a voltage dependent manner.

Fig. 1. Mepyramine and diphenhydramine inhibit KCNQ2/Q3 channel current in HEK293 cells. (A) Superimposed KCNQ2/Q3 channel currents recorded from a HEK293 cell transfected with KCNQ2/Q3 cDNAs before and after application of 100 mM mepyramine (mepy) and diphenhydramine (diphen). Cells were held at 80 mV and depolarized to 20 mV, then hyperpolarized to 60 mV to induce the typical deactivation tail current. The dotted line indicates the zero current level and the inset graph shows the reversible inhibitory effect of 100 mM mepyramine on current. (B) Summary of the percent inhibition of KCNQ2/Q3 tail current by 100 mM mepyramine and diphenhydramine. (C) Representative current traces recorded from a typical HEK293 cell under the control condition and in the presence of 100 mM mepyramine. Cell was held at 80 mV and stepped to þ30 mV in 10 mV interval for 1-s duration, followed by a 0.75-s step to 60 mV. (D) Percent inhibitions of KCNQ2/Q3 current by 100 mM mepyramine over the voltage ranges from 30 mV to þ30 mV. Error bars indicate SEM. 632 B. Liu et al. / Neuropharmacology 54 (2008) 629e639

3.2. Mepyramine alters KCNQ2/Q3 channel kinetics and and changed the slope from 16.0 0.9 to 14.5 1.4 shifts the voltage dependence of channel activation (Fig. 2D, n ¼ 7, p < 0.01). In addition, diphenhydramine also produced a significant rightward shift of the activation We then studied the effects of mepyramine on the kinetics curve of KCNQ2/Q3 current, from 15.2 1.1 mV to of KCNQ2/Q3 currents. Application of 100 mM mepyramine 6.6 0.9 mV (Fig. 2E, n ¼ 7, p < 0.01). significantly slowed channel activation and accelerated channel deactivation kinetics, respectively (Fig. 2A,B). The activa- 3.3. Effect of mepyramine on homomeric KCNQ1e4 and tion and deactivation currents were both fitted to a single heteromeric KCNQ3/Q5 channel currents exponential function. The time constant for the activation current was 88.5 10.9 ms for the control condition and We next compared the sensitivities of different KCNQ 117.5 9.6 ms for mepyramine-treated current (Fig. 2C, channels to mepyramine. All five channels of the KCNQ fam- p < 0.01, n ¼ 12). For the deactivation current, the time con- ily (KCNQ1e5) were individually expressed in HEK293 cells. stant was 69.8 8.7 ms for the control condition and Surprisingly, KCNQ5 channel showed little or no detectable 57.2 8.8 ms for mepyramine-treated current (Fig. 2C, current under the expression conditions of the present study, p < 0.05, n ¼ 6). a phenomenon which is in agreement with a previous report Next, we studied the effects of mepyramine on voltage-de- (Xiong et al., 2007). However, the coexpression of KCNQ5 pendent activation of KCNQ2/Q3 current. For this study, tail with KCNQ3 channels could lead to robust channel currents current amplitudes at 60 mV resulting from different test po- (Fig. 3E). As shown in Fig. 3C,E, the current resulting from tentials were normalized and fitted by Boltzmann function, as heteromeric KCNQ3/Q5 channels is considerably faster in described in the methods. A total of 100 mM mepyramine activation (101.5 8.8 ms vs. 209.3 7.4 ms, n ¼ 7) and de- shifted the voltage for half-maximal activation (V50)of activation (43.8 6.5 ms vs. 183.0 22.1 ms, n ¼ 7) kinetics KCNQ2/Q3 currents towards more depolarized potentials, than KCNQ3 channel current alone, ruling out the possibility from 16.9 0.9 mV to 7.8 1.4 mV (n ¼ 7, p < 0.01) of a simple recording from KCNQ3 homomers. Thus, this

Fig. 2. Mepyramine alters KCNQ2/Q3 channel kinetics and shifts the voltage dependence of channel activation. (A) Superimposed activation traces under control condition and in the presence of 100 mM mepyramine recorded from a typical HEK293 cell. Scaling the current trace to the peak outward current under control conditions revealed a pronounced prolongation of channel activation by mepyramine. (B) Tail current before and after application of mepyramine. Currents were scaled up to the peak current under the control condition and revealed a shortening in channel deactivation. (C) Summary of the effects of 100 mM mepyramine on activation and deactivation time constants (tau) of KCNQ2/Q3 channel currents. Activation and deactivation currents were both ﬁtted with a single exponential function. **p < 0.01 compared with control, *p < 0.05 compared with control. (D) and (E) Relative current-voltage relationship for KCNQ2/Q3 current under control conditions and in the presence of 100 mM mepyramine or diphenhydramine. Tail currents elicited from each stepping voltages (from 80 mV to þ40 mV) were normalized to the peak current at þ40 mV. The dashed line is the curve ﬁt of the current treated with 100 mM mepyramine or diphenhydramine, normalized to the peak current at þ40 mV under the control condition. Error bars indicate SEM. B. Liu et al. / Neuropharmacology 54 (2008) 629e639 633 property has ascertained a functional expression of KCNQ5 solution resulted in a substantial recovery of the currents when co-assembled with the KCNQ3 channel. from the inhibition and the currents usually recovered within Application of 100 mM mepyramine strongly inhibited 1 min (Fig. 3AeE). To further elucidate the detailed actions homomeric KCNQ1e4 and heteromeric KCNQ3/Q5 currents. of mepyramine on KCNQ channels, we constructed the con- The tail currents recorded from 20 mV depolarizing step centration-response relationships for mepyramine inhibition were reduced by 63.5 3.9% (n ¼ 11), 92.0 1.5% of all homomeric KCNQ1e4 as well as for heteromeric (n ¼ 12), 94.1 3.8% (n ¼ 10), 88.1 2.4% (n ¼ 19) and KCNQ2/Q3 and KCNQ3/Q5 currents. As shown in 98.5 0.7% (n ¼ 9) for KCNQ1e4 channels and KCNQ3/ Fig. 4A,B, application of mepyramine inhibited the tail cur- Q5 channels. Removing mepyramine from the external rents of these channels in a concentration-dependent manner.

Fig. 3. Effects of mepyramine on KCNQ1e4 and heteromeric KCNQ3/Q5 channel currents in HEK293 cells. (A)e(E) Representative current traces of KCNQ1e4 and heteromeric KCNQ3/Q5 currents under control conditions, in the presence of mepyramine and after drug removal are shown. The voltage protocol is shown in the inset at the top. Tail currents were measured at 60 mV. 634 B. Liu et al. / Neuropharmacology 54 (2008) 629e639

Table 1 summarizes the IC50 and nH values obtained from the to see if direct introduction of mepyramine into the cell would concentration-response relationships. As is clear from Fig. 4A result in KCNQ2/Q3 current inhibition in HEK293 cells. and Table 1, KCNQ3 shows the highest sensitivity to mepyr- Fig. 5A shows the summary data of the KCNQ2/Q3 tail cur- amine among homomeric KCNQ1e4 channels. rent amplitudes under control conditions (n ¼ 7) and with A concentration-response relationship was also established 300 mM mepyramine in the recording pipette (n ¼ 7). It should for diphenhydramine induced inhibition of KCNQ2/Q3 cur- be noted that the KCNQ2/Q3 current underwent a nearly 40% rents (Fig. 4B, dashed line). The concentration of diphenhy- decrease, even under control conditions, during the 15 min of dramine needed to produce half-maximal inhibition (IC50)of whole cell recording. This is the characteristic ‘‘run down’’ KCNQ2/Q3 currents was estimated to be 48.1 5.1 mM. property of KCNQ channel currents due to the loss of some intracellular components during whole cell recording (Suh and Hille, 2002). The relative remaining current amplitudes 3.4. Mepyramine directly inhibits KCNQ2/Q3 channel at the end of the recording period (time 150) were current from outside of the cell membrane 56.0 9.0% for the control condition and 54.8 11.6% with 300 mM mepyramine in the recording pipette. These It is well established that the first-generation antihistamines two values were not significantly different ( p > 0.05). There- are lipophilic molecules that can easily cross the plasma mem- fore, this result indicates that mepyramine may possibly brane (Katzung, 2003). Thus, the active sites of mepyramine inhibit the channel from the outside of cell membrane. action on KCNQ channels could possibly be located in the in- The most clearly established modulation of KCNQ/M tracellular space of the cell. To address this issue, we included channel is the inhibition mediated through the PLC-signaling mepyramine in the recording pipette for whole-cell recording pathway, which involves PLC stimulation by GPCR activation and the subsequent hydrolysis of membrane PIP2 (Delmas and Brown, 2005; Delmas et al., 2004). In order to get a better understanding of the mechanism underlying the inhibition, we decided to investigate if the effect of mepyramine on KCNQ channels requires PLC activity. For this study, the effect of mepyramine was compared with that of muscarinic M1 receptor activation, a well established system for modulation of KCNQ2/Q3 currents by PLC activity. U-73122, a PLC inhibitor, was used to block the PLC activity. cDNA encoding the M1 receptor was co-transfected with KCNQ2/Q3 channel in HEK293 cells. In non-treated cells, 100 mM mepyramine and 10 mM oxo-M, a specific M1 receptor agonist, markedly sup- pressed the KCNQ2/Q3 channel currents, by 86.6 7.3% (n ¼ 5) and 94.2 3.0% (n ¼ 5), respectively (Fig. 5C). As expected, the inhibitory effect of oxo-M was significantly reduced to 27.7 8.0% (Fig. 5D, n ¼ 5, p < 0.01 vs. inhibition in the absence of U-73122) when treated with 5 mM U-73122, a concentration known for adequate PLC inhibition (Horowitz et al., 2005). In contrast, the inhibitory effect of mepyramine was slightly reduced to 71.2 3.8% when treated with 5 mM U-73122, which was insignificant compared with the inhibition in the absence of U-73122 (Fig. 5D, n ¼ 5, p > 0.05). These data strongly suggest that PLC activity is not involved in mepyramine induced KCNQ2/Q3 current inhibition. Next, the question of whether the blockage of KCNQ channels by mepyramine occurs in a direct way was addressed. For these experiments, excised outside-out patch recording in HEK293 cells transiently expressing KCNQ2/Q3 channels was used. The currents were elicited by depolarization to 20 mV followed by repolarization back to 60 mV. The inset in Fig. 5B shows such an experiment with multi-channel Fig. 4. Concentration dependences of inhibition of homomeric KCNQ1e4 outside-out patch indicating the effect of mepyramine on (A), heteromeric KCNQ2/Q3 and KCNQ3/Q5 currents (B) in HEK293 cells. KCNQ2/3 channels. Mepyramine was successfully tested on 5 Data sets of mepyramine and diphenhydramine were presented as the superim- excised outside-out patches. Of these, 100 mM mepyramine in- posed solid and dashed lines, respectively. The lines represent the fit of experimental data to the logistic equation, as described in the method. Current hibited KCNQ2/Q3 currents by 90.1 2.8% (n ¼ 5). The ef- responses were measured with the tail current at 60 mV, preceded by an fect of mepyramine was rapid in onset and rapidly reversed activating step at 20 mV. Error bars indicate SEM. after washout (Fig. 5B). From the results above, we propose B. Liu et al. / Neuropharmacology 54 (2008) 629e639 635

Table 1

Summary of the half-maximal blocking concentration (IC50), percent inhibition by highest drug concentration tested (% max), Hill coefﬁcient (nH) and number of cells tested (n) for inhibitory effects of mepyramine on KCNQ/M channels in HEK293 cells and rat SCG neurons KCNQ1 KCNQ2 KCNQ3 KCNQ4 KCNQ2/Q3 KCNQ3/Q5 M channel

IC50 (mM) 59.2 1.7 20.7 4.0 9.1 0.5 24.5 1.1 12.5 1.8 4.2 0.3 21.5 0.7 % max 86.5 5.7% 99.1 0.5% 99.3 0.3% 98.8 0.5% 98.6 0.6% 98.5 0.7% 96.8 1.8% nH 0.8 0.02 1.0 0.2 1.4 0.1 1.4 0.1 0.9 0.1 1.5 0.2 0.8 0.02 n 5e12 5e15 5e11 4e19 6e20 4e96e12 that the inhibition of KCNQ2/Q3 channel by mepyramine is due this study, M currents from rat SCG neurons were recorded to a direct channel blocking effect derived from extracellular using the whole-cell patch clamp technique. M currents were sites. measured from the slow deactivating tail currents at 60 mV proceeded by activating step voltages from 60 to 3.5. Mepyramine inhibits M current in rat SCG neurons þ30 mV, which could minimize contamination from other conductances including some other Kþ currents. A family of KCNQ channels are generally believed to underlie the deactivating tail currents is shown in Fig. 6A. As shown, appli- molecular composition of the neuronal M type Kþ channel, cation of mepyramine remarkably reduced all tail currents. which serves to stabilize membrane potential and regulates The inhibitions were 76.7 2.6% and 69.6 4.2% for tail neuronal excitability. Considering the actions of mepyramine currents measured at 20 and þ30 mV, respectively (n ¼ 7). on all KCNQ channels, we were interested in checking the Fig. 6B shows the concentration dependent inhibition of M potential impact of mepyramine on native M current. For current produced by mepyramine. The character of M current

Fig. 5. The inhibitory effect of mepyramine on KCNQ2/Q3 current is PLC independent and it acts directly from extracellular side. (A) Effect of intracellular application of 300 mM mepyramine on HEK293 cells. The diffusion process of mepyramine was recorded for 15 min after breaking into the cell (time 0 min). The tail currents in each experiment were normalized to the current recorded at time 0 min (100%). (B) Effect of mepyramine on an excised outside-out patch. (C) and (D) Effects of 100 mM mepyramine and 10 mM oxo-M on KCNQ2/Q3 currents in the absence and presence of the PLC inhibitor, U-73122 (5 mM), respectively. Representative current traces from each experiment are indicated in the insets. Error bars indicate SEM. 636 B. Liu et al. / Neuropharmacology 54 (2008) 629e639

Fig. 6. Mepyramine inhibits M current in rat SCG neurons. (A) M current recorded from a typical rat SCG neuron. Neurons were held at 60 mV, stepped to þ30 mV and stepped back to 60 mV to record the typical tail current. Tail currents under control conditions and in the presence of mepyramine were artiﬁcially magniﬁed to give a clear view and are shown on the right panel. (B) Concentration-dependent inhibition of M current induced by mepyramine. Neurons were held at 20 mV and then hyperpolarized to 60 mV to record the tail current. 30 mM linopirdine (LP) was applied at the end of recording in order to determine M current contribution. The inset denotes the corresponding current traces. (C) Concentration-response relationship for inhibition of M current by mepyramine. (D) Membrane potential of a SCG neuron in the presence of 100 mM mepyramine and 10 mM oxo-M. (E) Membrane potential of a SCG neuron when 100 mM mepyramine was applied in the continued presence of 30 mM linopirdine. Error bars indicate SEM.

inhibition is similar to that of heteromeric KCNQ2/Q3 current, respectively (Table 1). Thus, the above data provide direct exhibiting a rapid and reversible inhibition by mepyramine. evidence that mepyramine is capable of inhibiting the M cur- Linopirdine, which is a specific blocker to M current that rent in native neurons. completely abolishes M current at 30 mM(Aiken et al., Since the M current is the major contributor to membrane 1995), was used as a reasonable estimation of the contribu- potential in many types of neurons, including SCG neurons, tion of M current in each neuron at the end of recording. we thus proceeded to study the effect of mepyramine on the The inset denotes the corresponding current traces recorded. resting membrane potential of SCG neurons. The neurons A concentration-response relationship was constructed for were constantly current clamped at zero and the membrane mepyramine inhibition of M current (Fig. 6C). The estimated potential was recorded. As shown in Fig. 6D, treatment with IC50 and nH values are 21.5 0.7 mM and 0.8 0.02, 100 mM mepyramine produced a significant and reversible B. Liu et al. / Neuropharmacology 54 (2008) 629e639 637 depolarization of neuron membrane potential, from a rat SCG neuron upon injection of depolarizing current under 51.0 5.6 mV to 38.9 7.3 mV (n ¼ 11, p < 0.01). As currentclamp.Asexpected,theexcitabilityoftheneuronwassig- a comparison, 10 mM oxo-M also produced a marked depolar- nificantlyincreasedwhenexposedto15 mMlinopirdine,duetoM ization with similar magnitude as mepyramine, from current suppression. The action potential firing frequency (APF) 50.6 2.4 mV to 41.7 3.1 mV (n ¼ 11, Fig. 6D). In or- during injection of 100 pA depolarizing current was der to establish the relationship between membrane depolar- 2.4 0.4 AP/s, whereas after linopirdine, it was 9.1 1.6 AP/ ization and M current inhibition, we tested the effect of s(n ¼ 10, p < 0.01, Fig. 7B). To our surprise, application of linopirdine on mepyramine-induced membrane depolarization. 100 mMmepyraminehad no significanteffect on the evokedneu- As shown in Fig. 6E, the M current specific blocker, linopir- ron action potentials, despite its potent inhibitory effect on M dine (30 mM), which induced a large membrane depolarization current and the resting membrane potential (Fig. 7C,D). The on its own (from 50.7 1.4 mV to 33.2 3.3 mV, n ¼ 6), APF was 2.4 0.7 AP/s under the control condition and completely abolished the depolarization effect of mepyramine. 1.3 0.2 AP/s in the presence of 100 mM mepyramine, reveal- This result supports the notion that the depolarization of SCG ing no statistical significance (n ¼ 12, p > 0.05). neuron membrane potential induced by mepyramine is due to M current inhibition. 4. Discussion

3.6. Effect of mepyramine on evoked neuronal action In the present study, we demonstrated that mepyramine, the potentials first-generation antihistamine, inhibited KCNQ1e4, heteromeric KCNQ2/Q3, KCNQ3/Q5 and neuronal M type Kþ cur- The primary physiological relevance of M current is to rents in a concentration dependent manner. Mepyramine dampen neuronal spiking discharges. Thus inhibition of the M significantly altered the gating properties and the voltage current results in neuronal hyperexcitability. The above de- dependent activation of the heteromeric KCNQ2/Q3 channel. scribed effects of first-generation antihistamines on KCNQ/M Furthermore, it is noteworthy that mepyramine, through M currents suggest that neurons will become more excitable in the current inhibition, produced a noticeable depolarization of presence of these antihistamines, which may underlie their toxic neuron membrane potential. neuronal effects observed clinically. To test this, we used 15 mM The first-generation antihistamines are widely sold medi- linopirdine, a concentration which has been reported to cause cines used to treat many common allergic reactions. Due to a similar amplitude (75e80%) of M current inhibition as their widespread usage, these drugs are often involved in poi- 100 mM mepyramine (Lamas et al., 1997), as a positive control. soning cases (Scharman et al., 2006). Intoxication with over- Fig. 7A illustrates the induced action potential spikes fired by doses of these drugs often leads to serious adverse effects,

Fig. 7. Mepyramine does not increase the excitability of SCG neurons. (A) Action potentials were evoked by 100 pA depolarizing current for 2-s durationas indicated and recorded under current-clamp in rat SCG neurons. Bath application of 15 mM linopirdine (B), washout (C) and finally 100 mM mepyramine treatment (D) are as indicated. 638 B. Liu et al. / Neuropharmacology 54 (2008) 629e639 including tremor, seizure and convulsion. It has been reported of the neurons. In addition to the inhibitory effects on the M that higher doses of diphenhydramine, promethazine and me- current described in the present study, we (unpublished data) pyramine can produce convulsions in epileptic patients and and others (Kuo et al., 2000) observed suppression of Naþ cur- in young children (Yokoyama and Iinuma, 1996). A recent sur- rent in rat SCG and DRG neurons by 100 mM mepyramine and vey on diphenhydramine intoxication in patients revealed that diphenhydramine. Since activation of the Naþ channel is the the plasma drug concentration ranged from 2 to 34.8 mMin major component of an action potential, thus inhibition of non-fatal cases and even reached as much as 460 mMin both Naþ channel and M type Kþ by mepyramine may explain some fatal cases (Pragst et al., 2006). The drug concentration the paradoxical result that membrane potential depolarization in the CNS should be similar to that in the blood, considering was not accompanied by a significant increase in action poten- that the first-generation antihistamines are small lipophilic tial numbers. Nevertheless, the possible contribution of M cur- molecules and can easily penetrate the BBB (Katzung, 2003). rent inhibition to the clinically observed neuronal toxicity due In our current study, mepyramine and diphenhydramine began to first-generation antihistamine intoxication should not be to inhibit KCNQ/M channel at a concentration of about 3 mM compromised by the failure of mepyramine to increase the and the IC50 was 12.5 and 48.1 mM, respectively. This implies excitability of a single isolated neuron. Recently, it has been that M current blocking is very likely to occur in the nervous reported that functional pre-synaptic M channels are present systems of patients intoxicated with these drugs. in unmyelinated glutamatergic hippocampal axons (Vervaeke Our results suggest that KCNQ2/Q3 current inhibition in- et al., 2006). It is generally known that presynaptic membrane duced by mepyramine is most probably due to a direct channel depolarization will trigger the release of glutamate or other blocking effect from the outside. As shown in Fig. 1A, the in- excitatory neurotransmitters by a Ca2þ dependent mechanism hibition occurred immediately after mepyramine was applied from vesicles in presynaptic terminals (Meldrum, 2000), and quickly reached the steady state. The current can be rapidly which play an important role in the molecular mechanism un- and fully restored after drug washout, suggesting a direct inhib- derlying epilepsy (Chapman, 2000). Indeed, during seizures in itory action on the channels. These features are different from ambulatory patients, there is a marked, bilateral and transient those commonly observed in GPCR induced KCNQ/M channel increase in the level of extracellular hippocampal glutamate, inhibition (Suh and Hille, 2005). Not surprisingly, the actions which is largest in the epileptic hemisphere (During and Spen- of mepyramine do not require the PLC signaling pathway, an cer, 1993; Wilson et al., 1996). Here we suggest that mem- exclusive mechanism followed by GPCR inhibition of brane potential depolarization produced by mepyramine may KCNQ/M currents (Delmas and Brown, 2005). Furthermore, be involved in the molecular basis for the adverse neuroexci- mepyramine was capable of blocking KCNQ2/Q3 channel in tatory effects observed in patients intoxicated by first-genera- excised outside-out patches but was ineffective when intro- tion antihistamines. duced intracellularly. Taken together, these results suggest In conclusion, mepyramine and diphenhydramine, two first- that mepyramine can directly block KCNQ2/Q3 channels and generation antihistamines, show a potent inhibitory effect on the inhibition is likely to take place from the extracellular KCNQ and M channels in HEK293 cells and SCG neurons. side, an interesting reminder of the classic M current blocker KCNQ/M channel inhibition and the subsequent marked depo- linopirdine (Costa and Brown, 1997; Lamas et al., 1997). larization of neuron membrane potential may provide new in- The five members of the KCNQ family (KCNQ1e5) dis- sights into the understanding of neurological adverse effects play distinct patterns of expression. KCNQ1 is restricted to observed in patients intoxicated by overdose with first-genera- the heart, peripheral epithelial and smooth muscle cells, tion antihistamines. whereas the other four KCNQ channels are exclusively distributed throughout the central and peripheral nervous systems, Acknowledgements constituting homomeric and heteromeric channels of different composition in distinct nervous tissues (Dalby-Brown et al., This work was supported by a NSFC grant 30270361, grant 2006). It has generally been accepted that the classical M cur- from Ministry of Science and Technology of China rents of ganglion neurons are mostly constituted by hetero- (2003CCA00300) and a National 863 project meric KCNQ2/Q3 channels. It is also known that KCNQ4 (2006AA02Z183). HZ is a beneficiary of the National Science and KCNQ5 can form heteromeric channels with KCNQ3 as Fund for Distinguished Young Scholars of China (No. well. Furthermore, when associated as homomeric channels, 30325038). We thank Qingzhong Jia, Zhiying Zhao, Junjie all KCNQ channels (even the cardiac KCNQ1), can form func- Bei, Li Liu and Fan Zhang for their kind help. tional M channels (Delmas and Brown, 2005). Therefore, the present study demonstrating that KCNQ1e4 and two heteromeric channels KCNQ2/Q3, KCNQ3/Q5 are inhibited by me- References pyramine may help describe the neuronal toxicity caused by the first-generation antihistamine intoxication in more detail. Aiken, S.P., Lampe, B.J., Murphy, P.A., Brown, B.S., 1995. Reduction of spike Our data clearly showed that mepyramine greatly depolar- frequency adaptation and blockade of M-current in rat CA1 pyramidal neurons by linopirdine (DuP 996), a neurotransmitter release enhancer. British ized the SCG neuron membrane potential by inhibiting the M Journal of Pharmacology 115, 1163e1168. current. However, unlike some classical inhibitory modulators Blaiss, M., 2004. Current concepts and therapeutic strategies for allergic of the M current, mepyramine did not increase the excitability rhinitis in school-age children. Clinical Therapeutics 26, 1876e1889. B. Liu et al. / Neuropharmacology 54 (2008) 629e639 639

Brown, B.S., Yu, S.P., 2000. Modulation and genetic identiﬁcation of the M KCNQ2/Q3 potassium channel openers, depress cortical neuron activity channel. Progress in Biophysics & Molecular Biology 73, 135e166. and exhibit anticonvulsant properties. Molecular Pharmacology 67, Chapman, A.G., 2000. Glutamate and epilepsy. Journal of Nutrition 130, 1053e1066. 1043Se1045S. Pragst, F., Herre, S., Bakdash, A., 2006. Poisonings with diphenhydramineda Costa, A.M., Brown, B.S., 1997. Inhibition of M-current in cultured rat survey of 68 clinical and 55 death cases. Forensic Science International superior cervical ganglia by linopirdine: mechanism of action studies. 161, 189e197. Neuropharmacology 36, 1747e1753. Qiu, C., Johnson, B.N., Tallent, M.K., 2007. K(þ) M-current regulates the Dalby-Brown, W., Hansen, H.H., Korsgaard, M.P., Mirza, N., Olesen, S.P., transition to seizures in immature and adult hippocampus. Epilepsia 48, 2006. K(v)7 channels: function, pharmacology and channel modulators. 2047e2058.

Current Topics in Medicinal Chemistry 6, 999e1023. Sato, I., Munakata, M., Iinuma, K., 2005. Histamine H1 antagonists block Delmas, P., Brown, D.A., 2005. Pathways modulating neural KCNQ/M (Kv7) M-currents in dissociated rat cortical neurons. Brain Research 1057, 81e87. potassium channels. Nature Reviews. Neuroscience 6, 850e862. Scharman, E.J., Erdman, A.R., Wax, P.M., Chyka, P.A., Caravati, E.M., Delmas, P., Crest, M., Brown, D.A., 2004. Functional organization of PLC Nelson, L.S., Manoguerra, A.S., Christianson, G., Olson, K.R., signaling microdomains in neurons. Trends in Neurosciences 27, 41e47. Woolf, A.D., Keyes, D.C., Booze, L.L., Troutman, W.G., 2006. Diphenhy- During, M.J., Spencer, D.D., 1993. Extracellular hippocampal glutamate and dramine and dimenhydrinate poisoning: an evidence-based consensus spontaneous seizure in the conscious human brain. Lancet 341, guideline for out-of-hospital management. Clinical Toxicology (Phila) 1607e1610. 44, 205e223. Gelfand, E.W., 2005. Pediatric allergic rhinitis: factors affecting treatment Schroeder, B.C., Kubisch, C., Stein, V., Jentsch, T.J., 1998. Moderate loss of choice. Ear, Nose and Throat Journal 84, 163e168. function of cyclic-AMP-modulated KCNQ2/KCNQ3 Kþ channels causes Horowitz, L.F., Hirdes, W., Suh, B.C., Hilgemann, D.W., Mackie, K., Hille, B., epilepsy. Nature 396, 687e690. 2005. Phospholipase C in living cells: activation, inhibition, Ca2þ require- Shah, M.M., Mistry, M., Marsh, S.J., Brown, D.A., Delmas, P., 2002. Mole- ment, and regulation of M current. Journal of General Physiology 126, cular correlates of the M-current in cultured rat hippocampal neurons. 243e262. Journal of Physiology 544, 29e37. Kamei, C., Ohuchi, M., Sugimoto, Y., Okuma, C., 2000. Mechanism respon- Suh, B.C., Hille, B., 2002. Recovery from muscarinic modulation of M current

sible for epileptogenic activity by ﬁrst-generation H1-antagonists in rats. channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron Brain Research 887, 183e186. 35, 507e520. Katzung, B.G., 2003. Histamine, Serotonin, and the Ergot Alkaloids. Basic Suh, B.C., Hille, B., 2005. Regulation of ion channels by phosphatidylinositol and Clinical Pharmacology. McGraw-Hill, San Francisco, pp. 263e268. 4,5-bisphosphate. Current Opinion in Neurobiology 15, 370e378. Kuo, C.C., Huang, R.C., Lou, B.S., 2000. Inhibition of Na(þ) current by Taglialatela, M., Timmerman, H., Annunziato, L., 2000. Cardiotoxic potential diphenhydramine and other diphenyl compounds: molecular determinants and CNS effects of ﬁrst-generation antihistamines. Trends in Pharmacolo- of selective binding to the inactivated channels. Molecular Pharmacology gical Sciences 21, 52e56. 57, 135e143. Vervaeke, K., Gu, N., Agdestein, C., Hu, H., Storm, J.F., 2006. Kv7/KCNQ/ Lamas, J.A., Selyanko, A.A., Brown, D.A., 1997. Effects of a cognition- M-channels in rat glutamatergic hippocampal axons and their role in regu- enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) lation of excitability and transmitter release. Journal of Physiology 576, and some other voltage- and ligand-gated membrane currents in rat sym- 235e256. pathetic neurons. European Journal of Neuroscience 9, 605e616. Wilson, C.L., Maidment, N.T., Shomer, M.H., Behnke, E.J., Ackerson, L., Liu, B., Jia, Z., Geng, X., Bei, J., Zhao, Z., Jia, Q., Zhang, H., 2007. Selective Fried, I., Engel Jr., J., 1996. Comparison of seizure related amino acid re- inhibition of Kir currents by antihistamines. European Journal of Pharma- lease in human epileptic hippocampus versus a chronic, kainate rat model cology 558, 21e26. of hippocampal epilepsy. Epilepsy Research 26, 245e254. Meldrum, B.S., 2000. Glutamate as a neurotransmitter in the brain: review of Xiong, Q., Sun, H., Li, M., 2007. Zinc pyrithione-mediated activation of physiology and pathology. Journal of Nutrition 130, 1007Se1015S. voltage-gated KCNQ potassium channels rescues epileptogenic mutants. Olson, K.R., Kearney, T.E., Dyer, J.E., Benowitz, N.L., Blanc, P.D., 1994. Nature Chemical Biology 3, 287e296. Seizures associated with poisoning and drug overdose. American Journal Yawata, I., Tanaka, K., Nakagawa, Y., Watanabe, Y., Murashima, Y.L., of Emergency Medicine 12, 392e395. Nakano, K., 2004. Role of histaminergic neurons in development of Owen, D.G., Marsh, S.J., Brown, D.A., 1990. M-current noise and putative M- epileptic seizures in EL mice. Brain Research. Molecular Brain Research channels in cultured rat sympathetic ganglion cells. Journal of Physiology 132, 13e17. 431, 269e290. Yokoyama, H., Iinuma, K., Yanai, K., Watanabe, T., Sakurai, E.,

Passmore, G.M., Selyanko, A.A., Mistry, M., Al-Qatari, M., Marsh, S.J., Onodera, K., 1993. Proconvulsant effect of ketotifen, a histamine H1 Matthews, E.A., Dickenson, A.H., Brown, T.A., Burbidge, S.A., antagonist, conﬁrmed by the use of d-chlorpheniramine with monitoring Main, M., Brown, D.A., 2003. KCNQ/M currents in sensory neurons: electroencephalography. Methods and Findings in Experimental and signiﬁcance for pain therapy. Journal of Neuroscience 23, 7227e7236. Clinical Pharmacology 15, 183e188. Peretz, A., Degani, N., Nachman, R., Uziyel, Y., Gibor, G., Shabat, D., Yokoyama, H., Iinuma, K., 1996. Histamine and seizures: implications for the Attali, B., 2005. Meclofenamic acid and diclofenac, novel templates of treatment of epilepsy. CNS Drugs 5, 321e330.