G protein-activated inwardly rectifying K+ channels (GIRK, also known as Kir3) are regulated by various G-protein-coupled receptors. Activation of GIRK channels plays an important role in reducing neuronal excitability in most brain regions and the heart rate. Ifenprodil, which is a clinically used cerebral vasodilator, interacts with several receptors, such as α1 adrenergic, N-methyl-D-aspartate, serotonin and σ receptors. However, the molecular mechanisms underlying the various clinically related effects of ifenprodil remain to be clarified. Here, we examined the effects of ifenprodil on GIRK channels by using Xenopus oocyte expression assays. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, ifenprodil reversibly reduced inward currents through the basal GIRK activity. The inhibition was concentration-dependent, but voltage- and time-independent, suggesting that ifenprodil may not act as an open channel blocker of the channels. In contrast, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to ifenprodil. Furthermore, GIRK current responses activated by the cloned κ-opioid receptor were similarly inhibited by ifenprodil. The inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly, and were not affected by extracellular pH, which changed the proportion of the uncharged to protonated ifenprodil, suggesting its action from the extracellular side. The GIRK currents induced by ethanol were also attenuated in the presence of ifenprodil. Our results suggest that direct inhibition of GIRK channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects.
Ifenprodil was originally developed as an α1 adrenergic receptor antagonist (Chenard et al, 1991), and has cerebral and peripheral vasodilator effects that stimulate blood circulation (Carron et al, 1971; Young et al, 1983). The drug has been clinically used in the treatment of cerebrovascular diseases and peripheral arterial obliterative disease (Carron et al, 1971; Young et al, 1983; Marquis et al, 1998). Furthermore, it is well known to be a new class of N-methyl-D-aspartate (NMDA) receptor antagonists that selectively inhibits NMDA receptors containing the NR2B subunit (Williams, 2001). Ifenprodil has been shown to have neuroprotective (Gotti et al, 1988), anticonvulsant (Thurgur and Church, 1998; Yourick et al, 1999) and antinociceptive effects (Chizh et al, 2001), and to have potential for the treatment of several neuropsychiatric disorders, such as Parkinson's disease (Nash et al, 2000), alcoholism (Malinowska et al, 1999; Napiórkowska-Pawlak et al, 2000; Narita et al, 2000), and drug addiction (Witkin and Acri, 1995; Suzuki et al, 1999). Inhibition of NMDA receptor channels by ifenprodil is thought to have important implications in these therapeutic effects (Williams, 2001). It has also been shown that ifenprodil interacts with several other receptors, namely, serotonin (5-hydroxytryptamine; 5-HT) subtype 1A, 5-HT2 and 5-HT3 receptors (Chenard et al, 1991; McCool and Lovinger, 1995), and σ receptors (Karbon et al, 1990) at nanomolar concentrations and that it inhibits voltage-gated Ca2+ channels at micromolar concentrations (Church et al, 1994; Bath et al, 1996). These effects might also be involved in the molecular mechanisms underlying some of the therapeutic effects and side effects of ifenprodil.
G protein-activated inwardly rectifying K+ (GIRK) channels (also known as Kir3 channels) are members of a family of inwardly rectifying K+ (Kir) channels that includes seven subfamilies (Doupnik et al, 1995; Reimann and Ashcroft, 1999). Four GIRK channel subunits have been identified in mammals (Dascal et al, 1993; Kubo et al, 1993b; Lesage et al, 1995; Wickman et al, 1997). Neuronal GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK2 subunits in most brain regions (Kobayashi et al, 1995; Lesage et al, 1995; Karschin et al, 1996; Liao et al, 1996) or homomultimers composed of GIRK2 subunits in the substantia nigra and ventral tegmental area (Inanobe et al, 1999), whereas atrial GIRK channels are heteromultimers composed of GIRK1 and GIRK4 subunits (Krapivinsky et al, 1995). A variety of G-protein-coupled receptors (GPCRs), such as M2 muscarinic, α2 adrenergic, D2 dopaminergic, 5-HT1A, μ-, δ- and κ-opioid, nociceptin/orphanin FQ and A1 adenosine receptors, activate GIRK channels (North, 1989; Ikeda et al, 1995, 1996, 1997) through the direct action of G-protein βγ-subunits released from pertussis toxin (PTX)-sensitive Gi/o proteins (Reuveny et al, 1994). On the other hand, GIRK channels are inhibited by PTX-insensitive GqPCRs, such as substance P, M1, and M3 muscarinic, α1 adrenergic, thyrotropin-releasing hormone, bombesin and endothelin receptors (Stanfield et al, 2002). In addition, ethanol activates GIRK channels independent of G-protein-coupled-signaling pathways (Kobayashi et al, 1999; Lewohl et al, 1999). Activation of GIRK channels causes membrane hyperpolarization (North, 1989). Also, GIRK2 knockout mice show spontaneous seizures (Signorini et al, 1997), whereas GIRK4 knockout mice show blunted heart rate regulation and mild tachycardia (Wickman et al, 1998; Bettahi et al, 2002). Thus the channels play an important role in the inhibitory regulation of neuronal excitability and heart rate. Therefore, modulators of GIRK channel activity may affect many brain and cardiac functions. Using the Xenopus oocyte expression system, we previously demonstrated that various antipsychotic drugs including haloperidol inhibited GIRK channels (Kobayashi et al, 1998, 2000, 2004a). Ifenprodil, a phenylethanolamine, is structurally related to the antipsychotic drug haloperidol (Williams, 2001), which weakly inhibits the channels (Kobayashi et al, 2000). Therefore, we hypothesized that ifenprodil may also interact with GIRK channels. In the present study, we examined the effects of ifenprodil on brain-type GIRK1/2 and GIRK2 channels and cardiac-type GIRK1/4 channels by using the Xenopus oocyte expression assay.
MATERIALS AND METHODS
Preparation of Specific mRNAs
Plasmids containing the entire coding sequences for the mouse GIRK1, GIRK2, and GIRK4 channel subunits and the κ-opioid receptor (κOR) were obtained by using the polymerase chain reaction method as described previously (Ikeda et al, 1995; Kobayashi et al, 1995, 2000). In addition, cDNAs for rat Kir1.1 in pSPORT and mouse Kir2.1 in pcDNA1 were provided by Dr Steven C Hebert and Dr Lily Y Jan, respectively. These plasmids were linearized by digestion with the appropriate enzyme as described previously (Ho et al, 1993; Kubo et al, 1993a; Kobayashi et al, 2000); and the specific mRNAs were synthesized in vitro by using the mMESSAGE mMACHINE™ In vitro Transcription Kit (Ambion, Austin, TX, USA).
Adult female Xenopus laevis frogs were purchased from Copacetic (Soma, Aomori, Japan) and maintained in the laboratory until used. Frogs were anesthetized by immersion in water containing 0.15% tricaine (Sigma Chemical Co., St Louis, MO, USA). A small incision was made in the abdomen to remove several ovarian lobes from the frogs, which were humanely killed after the final collection. Oocytes (Stages V and VI) were isolated manually from the ovary and maintained in Barth's solution (Kobayashi et al, 2002). Xenopus laevis oocytes were injected with mRNA(s) for GIRK1/GIRK2 or GIRK1/GIRK4 combinations (each ∼0.4 ng), GIRK2 (∼5 ng), Kir1.1 (∼5 ng) or Kir2.1 (∼0.5 ng), and/or κOR (∼10 ng). The oocytes were incubated at 19°C in Barth's solution, and defolliculated following treatment with 0.8 mg ml−1 collagenase as described previously (Kobayashi et al, 2002). Whole-cell currents of the oocytes were recorded from 2 to 10 days after the injection with a conventional two-electrode voltage clamp (Kobayashi et al, 1999; Ikeda et al, 2003). The membrane potential was held at −70 mV, unless otherwise specified. Microelectrodes were filled with 3 M KCl. The oocytes were placed in a 0.05 ml narrow chamber and superfused continuously with a high-potassium (hK) solution (composition in mM: KCl 96, NaCl 2, MgCl2 1, CaCl2 1.5 and HEPES 5, pH 7.4 with KOH) or a K+-free high-sodium (ND98) solution (composition in mM: NaCl 98, MgCl2 1, CaCl2 1.5 and HEPES 5, pH 7.4 with NaOH) at a flow rate of 2.5 ml min−1. Kir channels allow K+ ions to enter the cells much more readily than does K+ permeation in the outward direction (Kubo et al, 1993a). In the hK solution used to readily analyze Kir currents by enhancing the magnitude of currents, the K+ equilibrium potential (EK) was close to 0 mV, and inward K+ current flow through GIRK channels was observed at negative holding potentials, as shown in previous studies (Dascal et al, 1993; Lewohl et al, 1999; Kobayashi et al, 2004b). For examining the effect of intracellular ifenprodil, 23 nl of 10 mM ifenprodil or 30 mM lidocaine N-ethyl bromide (QX-314) dissolved in distilled water was administered to an oocyte through an additional pipette by pressure injection using a Nanoliter injector (World Precision Instruments, Sarasota, FL, USA) as described previously (Kobayashi et al, 2003), and the oocyte currents were then continuously recorded for approximately 30–40 min. As the volume of a Xenopus oocyte used is ∼1 μl, the intracellular concentration of ifenprodil or QX-314 was presumed as ∼225 or ∼674 μM, respectively. Data were fitted to a standard logistic equation by using KaleidaGraph (Synergy Software, Reading, PA, USA) for analysis of concentration–response relationships. The EC50 value, which is the concentration of a drug that produces 50% of the maximal current response for that drug; the IC25 and IC50 values, which are the concentrations of a drug that reduces control current responses by 25 and 50%, respectively; and the Hill coefficient (nH) were obtained from the concentration–response relationships.
Statistical Analysis of Results
The values obtained are expressed as the mean±SEM, and n is the number of oocytes tested. Statistical analysis of differences between groups was carried out by using paired t-test, Student's t-test, one-way ANOVA or two-way factorial ANOVA followed by Bonferroni/Dunn post hoc test. A probability of 0.05 was taken as the level of statistical significance.
Ifenprodil tartrate and trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50488H), a selective κ-opioid-receptor agonist, were purchased from Research Biochemicals Inc. (Natick, MA, USA). Ifenprodil was dissolved in distilled water or dimethyl sulfoxide (DMSO), and U50488H was dissolved in distilled water. The stock solutions of all of the compounds were stored at −30°C until used. Ethanol was purchased from Wako Pure Chemical Industries (Osaka, Japan). Each compound was added to the perfusion solution in appropriate amounts immediately before the experiments.
Inhibition of GIRK Channels by Ifenprodil
To investigate whether ifenprodil interacts with brain-type GIRK1/2 and GIRK2 channels and cardiac-type GIRK1/4 channels, we conducted Xenopus oocyte expression assays. In oocytes co-injected with GIRK1 and GIRK2 mRNAs, basal GIRK currents (Kobayashi et al, 2003), which are known to depend on free G-protein βγ-subunits present in the oocytes because of the inherent activity of G-proteins (Dascal, 1997), were observed under the conditions of a hK solution containing 96 mM K+ and negative membrane potentials (1961.9±248.6 nA at −70 mV, n=5, Figure 1a). Application of 3 μM ifenprodil immediately and reversibly caused a reduction of the inward currents through the expressed GIRK channels in the hK solution (Figure 1a). The current responses were abolished in the presence of 3 mM Ba2+, which blocks the Kir channel family including GIRK channels (n=3). The 3 mM Ba2+-sensitive current components in oocytes expressing GIRK channels are considered to correspond to the magnitudes of GIRK1/2 currents (Kobayashi et al, 2004b). In uninjected oocytes, ifenprodil, even at the highest concentration used, or 3 mM Ba2+ caused no significant response (Figure 1c; n=4), suggesting no effect of ifenprodil or Ba2+ on intrinsic oocyte channels. The Ba2+-insensitive current components in oocytes expressing GIRK channels were not significantly different from those in uninjected oocytes as previously shown (Kobayashi et al, 2000), indicating that the current components insensitive to 3 mM Ba2+ were composed of intrinsic oocyte currents independent of the GIRK currents. Moreover, in oocytes co-injected with GIRK1 and GIRK2 mRNAs, ifenprodil produced no significant response in the K+-free ND98 solution containing 98 mM Na+ instead of the hK solution (n=3; data not shown), suggesting that the ifenprodil-sensitive current components show K+ selectivity. In addition, application of DMSO, the solvent vehicle, at the highest concentration (0.3%) used, had no significant effect on the current responses (n=4; data not shown). These results suggest that ifenprodil inhibited GIRK1/2 channels. Similarly, in oocytes injected with either GIRK1 and GIRK4 mRNAs (Figure 1b) or GIRK2 mRNA, basal GIRK currents were observed under the same conditions; and the current components sensitive to 3 mM Ba2+ were 1197.7±203.9 nA (n=9) or 915.0±223.4 nA (n=4) at −70 mV, respectively. Ifenprodil inhibited basal GIRK1/4 and GIRK2 currents (Figures 1b and 3), suggesting that ifenprodil also inhibited GIRK1/4 channels and GIRK2 channels.
We further investigated the inhibitory effect of ifenprodil on GIRK channels in more detail. The instantaneous GIRK1/4 currents elicited by the voltage step to −100 mV from a holding potential of 0 mV were diminished in the presence of 3 μM ifenprodil (Figure 2a). The percentage inhibition of the steady-state GIRK current at the end of the voltage step by ifenprodil was not significantly different from that of the instantaneous current (paired t-test, p>0.05; n=4 at −60, −80, −100, and −120 mV). These results suggest that the channels were inhibited by ifenprodil primarily at the holding potential of 0 mV and in a time-independent manner during each voltage pulse. Furthermore, similar results were obtained for GIRK1/2 channels (n=4; data not shown).
Like 3 mM Ba2+-sensitive currents corresponding to basal GIRK currents, ifenprodil-sensitive currents in oocytes expressing GIRK channels increased with negative membrane potentials, and the current–voltage relationships showed strong inward rectification (Figure 2b), a characteristic of GIRK currents.
The percentage inhibition of GIRK currents by 3 μM ifenprodil was measured at membrane potentials between −100 and −20 mV. For GIRK1/2 and GIRK1/4 channels, the percentage inhibition showed no significant difference across the voltages (p>0.05, one-way ANOVA; Figure 2c), thus suggesting that the inhibition of GIRK channels by ifenprodil was voltage-independent.
Ifenprodil Concentration-Dependently Inhibits GIRK Channels, but not Kir1.1 and Kir2.1 Channels
We next investigated the concentration–response relationship of the inhibitory effects of ifenprodil on GIRK channels expressed in Xenopus oocytes, compared with the current components sensitive to 3 mM Ba2+, which fully blocks basal GIRK currents (Kobayashi et al, 2004b). Figure 3 shows that the inhibition of GIRK1/2, GIRK2, and GIRK1/4 channels by ifenprodil was concentration-dependent with distinctive potency and effectiveness at nanomolar concentrations or more. Table 1 shows the EC50 and nH values obtained from the concentration–response relationships for ifenprodil and the percentage inhibition of the GIRK currents by ifenprodil at the highest concentrations tested. To further compare the effects of ifenprodil on GIRK channels, we also calculated the drug concentrations required to inhibit the GIRK currents by 25 or 50% (Table 1). The rank order of the inhibition of GIRK channels by ifenprodil at 0.3–3 μM was as follows: GIRK1/4>GIRK1/2⩾GIRK2 channels (p<0.05, significant interaction between the channel effect and the effect of ifenprodil, two-way factorial ANOVA; and p<0.05, significant differences between the effects of ifenprodil on GIRK1/2 and GIRK2 channels and those on GIRK1/4 channels at 0.3–3 μM, Bonferroni/Dunn post hoc test). However, there were no significant differences in the inhibitory effects on these GIRK channels by ifenprodil at each concentration from 10 to 300 μM (p>0.05, Bonferroni/Dunn post hoc test).
Furthermore, we examined whether ifenprodil could interact with Kir1.1, an ATP-regulated Kir channel, and Kir2.1, a constitutively active Kir channel, in other Kir channel subfamilies. In oocytes expressing Kir1.1 or Kir2.1 channels, application of ifenprodil at 100 μM had no significant effect on the inward currents through the channels in the hK solution (Ba2+-sensitive current components at −70 mV: 503.8±62.0 nA for Kir1.1 and 936.6±213.6 nA for Kir2.1, n=8; Figure 3).
Similar Inhibition of GIRK Channels by Ifenprodil at pH 7.4 and 9
At physiological pH or below, ifenprodil exists mainly in a protonated form, and the proportion of the uncharged form increases with an increase in pH, because ifenprodil has two pKa values of 9.05 and 9.66. We examined whether changes in extracellular pH affect the inhibition by ifenprodil of GIRK channels expressed in oocytes prepared from the same donor. No significant effect of pH on the inhibition was observed in the concentration–response relationships for ifenprodil in oocytes expressing GIRK1/2 channels (p>0.5, two-way factorial ANOVA; p>0.05 at each concentration, Bonferroni/Dunn post hoc test, Figure 4). These results suggest that the inhibition may be mediated by both forms of ifenprodil with almost the same effectiveness. It also appears unlikely that the inhibition by ifenprodil was caused by hydrophobic interactions with GIRK channels within the membrane bilayer.
Effect of Ifenprodil on GIRK Channels Activated by a G-Protein-Coupled Receptor or Ethanol
Moreover, we examined the effects of ifenprodil on GIRK channels activated by a G-protein-coupled receptor. In oocytes co-expressing GIRK1/2 channels and κORs (Kobayashi et al, 2004c), application of 100 nM U50488H, a selective κ-opioid-receptor agonist, induced inward GIRK currents, and application of ifenprodil alone inhibited basal GIRK currents consistently at the concentrations tested (Figure 5a). The effects of ifenprodil on GIRK channels activated by the κOR were evaluated by measuring the amplitude of the U50488H-induced current response during application of ifenprodil at different concentrations. The current responses to 100 nM U50488H were reversibly inhibited by ifenprodil with an IC50 value of 3.4±1.2 μM and an nH value of 1.03±0.09 (n=5, Figure 5a and b). The percentage inhibition by ifenprodil was similar to that of basally active GIRK1/2 channels (p>0.05 at each concentration, Student's t-test), suggesting interaction of ifenprodil with GIRK channels but not the κOR. In addition, the U50488H-induced GIRK currents were not significantly affected by intracellularly applied ifenprodil (90.4±12.7% of untreated control current, paired t-test, p>0.1, n=4, Figure 5c), whereas such GIRK currents were significantly inhibited by intracellularly applied QX-314 as reported previously (Zhou et al, 2001; Kobayashi et al, 2003). The results indicate that intracellular ifenprodil could not inhibit GIRK channels and G-proteins mediated by κOR activation. Taken together, it is suggested that extracellular ifenprodil directly inhibits GIRK channels activated by the κOR.
GIRK channels are also shown to be activated by ethanol independently of PTX-sensitive G-proteins (Kobayashi et al, 1999; Lewohl et al, 1999). Results of previous single-channel analyses with excised outside-out and cell-attached patch-clamp configurations suggested that ethanol activates GIRK channels directly without interacting with G-protein-signaling pathways and intracellular second messengers (Kobayashi et al, 1999). So we next examined the effect of ifenprodil on GIRK channel activation by ethanol. In oocytes expressing GIRK1/2 channels, the GIRK currents induced by ethanol were attenuated in the presence of ifenprodil, with an IC50 value of 6.1±1.8 μM and an nH value of 0.69±0.09, in a reversible manner (88.9±2.7% inhibition at 100 μM, n=6; Figure 6). In addition, since the carboxyl terminal domains of GIRK channels are crucial for the ethanol sensitivity of the channel (Lewohl et al, 1999; Zhou et al, 2001), we examined whether intracellular ifenprodil affects ethanol activation of GIRK channels. However, the ethanol-induced GIRK currents were not significantly affected by intracellularly applied ifenprodil (91.7±8.0% of untreated control current, paired t-test, p>0.1, n=4, Figure 6c). These results, therefore, suggest that extracellular ifenprodil inhibits GIRK channels activated by ethanol.
Characteristics of GIRK Channel Inhibition by Ifenprodil
The present study demonstrated that ifenprodil inhibited brain-type GIRK1/2 and GIRK2 channels and cardiac-type GIRK1/4 channels at nanomolar concentrations or more in a distinctive manner. The inhibition of GIRK channels by ifenprodil was concentration-dependent, but voltage-independent and time-independent with a primarily significant effect on the instantaneous current and a steady percentage inhibition during each voltage pulse. Our results also suggest that ifenprodil acted at the channels from the extracellular side of the cell membrane. On the other hand, blockade by extracellular Ba2+ and Cs+, typical of Kir channel blockers that occlude the pore of the open channel, shows a concentration-dependence, a strong voltage-dependence, and a time-dependence with a comparatively small effect on the instantaneous current but a marked inhibition on the steady-state current at the end of voltage pulses (Lesage et al, 1995). These observations suggest that ifenprodil probably causes a conformational change in the GIRK channels, but does not act as an open channel blocker of the channels, as Ba2+ and Cs+ do. The action mechanism may also be involved in the incomplete blockade of GIRK currents by ifenprodil. In the present study, ifenprodil similarly inhibited GIRK currents induced by basally free G-protein βγ subunits present in oocytes, by G-proteins mediated by κOR activation, or by ethanol. Further studies using single channel experiments may be useful for understanding the mechanism of the action of ifenprodil on GIRK channels.
In addition, the potency of inhibition by ifenprodil of GIRK1/4 channels was higher than that of GIRK1/2 and GIRK2 channels. Although the rank order of the effectiveness by ifenprodil at the highest concentrations tested was GIRK2>GIRK1/2⩾GIRK1/4 channels, the differences were not statistically significant. Moreover, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to ifenprodil. Further studies using GIRK/Kir1.1 and GIRK/Kir2.1 chimeric channels and mutant GIRK channels may clarify the critical sites mediating the effects of ifenprodil on GIRK channels. Furthermore, high-resolution structure analysis of GIRK channels may allow characterization of the binding sites. Additionally, although haloperidol is structurally related to ifenprodil (Williams, 2001), haloperidol weakly inhibits GIRK1/2 and GIRK1/4 channels in a similar manner (Kobayashi et al, 2000). The different effectiveness of these drugs on GIRK channels may be due to the different chemical structures between them or to their different binding sites on GIRK channels. Studies on the relationship between the structures of GIRK channels and the structure of ifenprodil may provide the basis for designing candidates for potent GIRK inhibitors.
Clinical and Pharmacological Implications
The human plasma concentrations of ifenprodil are reported to be approximately 0.1 μM. after a single administration of its clinical dosage (Aventis Pharma's data). In animals, the radioactive ifenprodil in the brain and heart after its intramuscular administration was approximately 5–8 times and 5–10 times higher, respectively, than that in blood (Nakagawa et al, 1975). Therefore, the present findings suggest that GIRK channels in the brain and heart may be inhibited by ifenprodil at clinically relevant concentrations in these tissues. Activation of GIRK channels in physiological conditions induces K+ efflux, leading to membrane hyperpolarization (North, 1989), whereas inhibition of GIRK channels leads to a depolarization of the membrane potential, resulting in an increase in cell excitability (Kuzhikandathil and Oxford, 2002). Therefore, in clinical use ifenprodil might affect various brain and heart functions via the inhibition of GIRK channels, which are expressed widely in the nervous system and the atrium (Kobayashi et al, 1995; Karschin et al, 1996).
GIRK2 knockout mice show spontaneous seizures and are more susceptible to seizures induced by pentylenetetrazol, a GABAA receptor antagonist, than wild-type mice (Signorini et al, 1997). In addition, the resting membrane potentials of neurons in GIRK knockout mice were depolarized compared to those in wild-type mice (Lüscher et al, 1997; Torrecilla et al, 2002). High doses of ifenprodil potentiated seizures induced by some convulsants including pentylenetetrazol (Mizusawa et al, 1976), although ifenprodil has been shown to have anticonvulsant effects (Thurgur and Church, 1998; Yourick et al, 1999), probably due to inhibition of NMDA receptor channels (Williams, 2001) and Ca2+ channels (Church et al, 1994; Bath et al, 1996). In spite of its anticonvulsant property, potent blockade of neuronal GIRK channels by ifenprodil may contribute to the increased susceptibility to seizure by causing an increase in neuronal excitability.
Interestingly, GIRK2 knockout mice show reduced anxiety with an increase in motor activity in three tests for anxiety: the elevated plus-maze, light/dark box, and canopy test (Blednov et al, 2001). Ifenprodil had an anxiolytic property with an increase in locomotion in MF1 mice in the elevated plus-maze test (Fraser et al, 1996), although it had no anxiolytic effect in the light/dark exploratory test and caused no change in locomotor activity in Wistar rats (Mikolajczak et al, 2003). This discrepancy might have been caused by differences in the behavioral tests including difference in the ratio of the two light/dark compartments in the apparatus and/or in animal species. A clinical study showed that ifenprodil improved anxiety, a decrease in spontaneity, and melancholy in patients with sequelae of cerebrovascular diseases (Otomo et al, 1976). Therefore, inhibition of neuronal GIRK channels by ifenprodil might partly contribute to the clinical effects on anxiety and decreased activity, which are observed in some neuropsychiatric disorders as well.
In the heart, acetylcholine opens atrial GIRK channels via activation of the M2 muscarinic acetylcholine receptor, and ultimately causes slowing of the heart rate (Brown and Birnbaumer, 1990). Sinus tachycardia during treatment with ifenprodil is observed along with its hypotensive effect (Carron et al, 1971; Young et al, 1983; Yajima et al, 1987). Ifenprodil exhibits no significant affinity for the muscarinic acetylcholine receptor (Chenard et al, 1991). The present study demonstrated that ifenprodil, at submicromolar concentrations or more, inhibited cardiac-type GIRK1/4 channels, which are abundantly present in the atrium of the heart (Krapivinsky et al, 1995). Therefore, atrial GIRK channels may also be inhibited by ifenprodil in clinical practice. GIRK1 or GIRK4 knockout mice show mild tachycardia (Bettahi et al, 2002). Additionally, the hypotensive effect of ifenprodil may induce compensational activation of the sympathetic nervous system, which plays an important role in the stimulatory regulation of the heart rate. Taken together, our data suggest that sinus tachycardia during treatment with ifenprodil may be partly related to inhibition of atrial GIRK channels.
Ifenprodil influenced ethanol-related behavioral changes in animals, such as suppression of amnestic effects and withdrawal signs including convulsions (Malinowska et al, 1999; Napiórkowska-Pawlak et al, 2000; Narita et al, 2000). Ethanol activates GIRK channels (Kobayashi et al, 1999; Lewohl et al, 1999). The present study demonstrated that ifenprodil inhibited GIRK1/2 currents induced by ethanol. Interestingly, GIRK2 knockout mice show reduced ethanol-induced conditioned taste aversion and conditioned place preference (Hill et al, 2003), and are less sensitive to some of acute ethanol effects, including anxiolysis, habituated locomotor stimulation and handling-induced convulsions after an acute administration of ethanol, than wild-type mice (Blednov et al, 2001). Taken together, ifenprodil might suppress GIRK-related ethanol effects.
Morphine, a commonly used potent analgesic, preferentially binds to the μ-opioid receptor, and exerts various pharmacological effects, including analgesia, euphoria, and dependence (Gutstein and Akil, 2001). The μ-opioid receptor is coupled to G-protein-mediated signal transductions involving GIRK channels, adenylyl cyclase, Ca2+ channels, and phospholipase C (Ikeda et al, 2002). Although morphine produces a conditioned place preference in animals, indicating its rewarding effect, pretreatment with ifenprodil suppresses the rewarding effect produced by morphine (Suzuki et al, 1999). However, ifenprodil exhibits no significant affinity for the opioid receptors (Chenard et al, 1991). The present study demonstrated that ifenprodil inhibited G-protein-mediated GIRK currents. It may be important to determine whether GIRK channel function contributes to the rewarding effect of morphine. Interestingly, GIRK knockout mice show reduced self-administration of cocaine (Morgan et al, 2003). In a clinical report, desipramine, which acts as an inhibitor of GIRK channels as well as of norepinephrine transporters (Kobayashi et al, 2004b), facilitated initial abstinence from cocaine (Gawin et al, 1989). Thus, selective GIRK inhibitors might be potential agents for the treatment of abusers of cocaine. Further studies on the effects of ifenprodil on GIRK knockout mice might clarify the roles of the GIRK-mediated effects of ifenprodil in addiction to morphine and cocaine.
Bath CP, Farrell LN, Gilmore J, Ward MA, Hicks CA, O'Neill MJ et al (1996). The effects of ifenprodil and eliprodil on voltage-dependent Ca2+ channels and in gerbil global cerebral ischaemia. Eur J Pharmacol 299: 103–112.
Bettahi I, Marker CL, Roman MI, Wickman K (2002). Contribution of the Kir3.1 subunit to the muscarinic-gated atrial potassium channel IKACh . J Biol Chem 277: 48282–48288.
Blednov YA, Stoffel M, Chang SR, Harris RA (2001). Potassium channels as targets for ethanol: studies of G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2) null mutant mice. J Pharmacol Exp Ther 298: 521–530.
Brown AM, Birnbaumer L (1990). Ionic channels and their regulation by G protein subunits. Annu Rev Physiol 52: 197–213.
Carron C, Jullien A, Bucher B (1971). Synthesis and pharmacological properties of a series of 2-piperidino alkanol derivatives. Arzneim Forsch 21: 1992–1998.
Chenard BL, Shalaby IA, Koe BK, Rounau RT, Butler TW, Prochiniak MA et al (1991). Separation of α1 adrenergic and N-methyl-D-aspartate antagonist activity in a series of ifenprodil compounds. J Med Chem 34: 3085–3090.
Chizh BA, Headley PM, Tzschentke TM (2001). NMDA receptor antagonists as analgesics: focus on the NR2B subtype. Trends Pharmacol Sci 22: 636–642.
Church J, Fletcher EJ, Baxter K, MacDonald JF (1994). Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurons: comparison with N-methyl-D-aspartate receptor antagonist actions. Br J Pharmacol 113: 499–507.
Dascal N (1997). Signalling via the G protein-activated K+ channels. Cell Signal 9: 551–573.
Dascal N, Schreibmayer W, Lim NF, Wang W, Chavkin C, DiMagno L et al (1993). Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc Natl Acad Sci USA 90: 10235–10239.
Doupnik CA, Davidson N, Lester HA (1995). The inward rectifier potassium channel family. Curr Opin Neurobiol 5: 268–277.
Fraser CM, Cooke MJ, Fisher A, Thompsom ID, Stone TW (1996). Interactions between ifenprodil and dizocilpine on mouse behaviour in models of anxiety and working memory. Eur Neurophermacol 6: 311–316.
Gawin FH, Kleber HD, Byck R, Rounsaville BJ, Kosten TR, Jatlow PI et al (1989). Desipramine facilitation of initial cocaine abstinence. Arch Gen Psychiatr 46: 117–121.
Gotti B, Duverger D, Bertin J, Carter C, Dupont R, Frost J et al (1988). Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. I. evidence for efficacy in models of focal cerebral ischemia. J Pharmacol Exp Ther 247: 1211–1221.
Gutstein HB, Akil H (2001). Opioid analgesics. In Hardman JG, Limbird LE, Gilman AG (eds). Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th edn. McGraw-Hill: New York. pp 569–619.
Hill KG, Alva H, Blednov YA, Cunningham CL (2003). Reduced ethanol-induced conditioned taste aversion and conditioned place preference in GIRK2 null mutant mice. Psychopharmacology 169: 108–114.
Ho K, Nichols CG, Lederer WJ, Lytton J, Vassilev PM, Kanazirska MV et al (1993). Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 362: 31–38.
Ikeda K, Kobayashi K, Kobayashi T, Ichikawa T, Kumanishi T, Kishida H et al (1997). Functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated K+ (GIRK) channel. Mol Brain Res 45: 117–126.
Ikeda K, Kobayashi T, Ichikawa T, Usui H, Abe S, Kumanishi T (1996). Comparison of the three mouse G-protein-activated K+ (GIRK) channels and functional couplings of the opioid receptors with the GIRK1 channel. Ann NY Acad Sci 801: 95–109.
Ikeda K, Kobayashi T, Ichikawa T, Usui H, Kumanishi T (1995). Functional couplings of the δ- and the κ-opioid receptors with the G-protein-activated K+ channel. Biochem Biophys Res Commun 208: 302–308.
Ikeda K, Kobayashi T, Kumanishi T, Yano R, Sora I, Niki H (2002). Molecular mechanisms of analgesia induced by opioids and ethanol: is the GIRK channel one of the keys? Neurosci Res 44: 121–131.
Ikeda K, Yoshii M, Sora I, Kobayashi T (2003). Opioid receptor coupling to GIRK channels. In vitro studies using a Xenopus oocyte expression system and in vivo studies on weaver mutant mice. Methods Mol Med 84: 53–64.
Inanobe A, Yoshimoto Y, Horio Y, Morishige K-I, Hibino H, Matsumoto S et al (1999). Characterization of G-protein-gated K+ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra. J Neurosci 19: 1006–1017.
Karbon EW, Patch RJ, Pontecorvo MJ, Ferkany JW (1990). Ifenprodil potently interacts with [3H](+)-3-PPP-labeled σ binding sites in guinea pig brain membranes. Eur J Pharmacol 193: 247–248.
Karschin C, Diβmann E, Stuhmer W, Karschin A (1996). IRK(1-3) and GIRK(1-4) inwardly rectifying K+ channel mRNAs are differentially expressed in the adult rat brain. J Neurosci 16: 3559–3570.
Kobayashi T, Ikeda K, Ichikawa T, Abe S, Togashi S, Kumanishi T (1995). Molecular cloning of a mouse G-protein-activated K+ channel (mGIRK1) and distinct distributions of three GIRK (GIRK1, 2 and 3) mRNAs in mouse brain. Biochem Biophys Res Commun 208: 1166–1173.
Kobayashi T, Ikeda K, Kojima H, Niki H, Yano R, Yoshioka T et al (1999). Ethanol opens G-protein-activated inwardly rectifying K+ channels. Nat Neurosci 2: 1091–1097.
Kobayashi T, Ikeda K, Kumanishi T (1998). Effects of clozapine on the δ- and κ-opioid receptors and the G-protein-activated K+ (GIRK) channel expressed in Xenopus oocytes. Br J Pharmacol 123: 421–426.
Kobayashi T, Ikeda K, Kumanishi T (2000). Inhibition by various antipsychotic drugs of the G-protein-activated inwardly rectifying K+ (GIRK) channels expressed in Xenopus oocytes. Br J Pharmacol 129: 1716–1722.
Kobayashi T, Ikeda K, Kumanishi T (2002). Functional characterization of an endogenous Xenopus oocyte adenosine receptor. Br J Pharmacol 135: 313–322.
Kobayashi T, Washiyama K, Ikeda K (2003). Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac). Br J Pharmacol 138: 1119–1128.
Kobayashi T, Washiyama K, Ikeda K (2004a). Modulators of G protein-activated inwardly rectifying K+ channels: potentially therapeutic agents for addictive drug users. Ann NY Acad Sci 1025: 590–594.
Kobayashi T, Washiyama K, Ikeda K (2004b). Inhibition of G protein-activated inwardly rectifying K+ channels by various antidepressant drugs. Neuropsychopharmacology 29: 1841–1851.
Kobayashi T, Washiyama K, Ikeda K (2004c). Effects of interferon-α on cloned opioid receptors expressed in Xenopus oocytes. Life Sci 76: 407–415.
Krapivinsky G, Gordon EA, Wickman K, Velimirovic B, Krapivinsky L, Clapham DE (1995). The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K+-channel proteins. Nature 374: 135–141.
Kubo Y, Baldwin TJ, Jan YN, Jan LY (1993a). Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127–133.
Kubo Y, Reuveny E, Slesinger PA, Jan YN, Jan LY (1993b). Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364: 802–806.
Kuzhikandathil EV, Oxford GS (2002). Classic D1 dopamine receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) directly inhibits G protein-coupled inwardly rectifying potassium channels. Mol Pharmacol 62: 119–126.
Lesage F, Guillemare E, Fink M, Duprat F, Heurteaux C, Fosset M et al (1995). Molecular properties of neuronal G-protein-activated inwardly rectifying K+ channels. J Biol Chem 270: 28660–28667.
Lewohl JM, Wilson WR, Mayfield RD, Brozowski SJ, Morrisett RA, Harris RA (1999). G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action. Nat Neurosci 2: 1084–1090.
Liao YJ, Jan YN, Jan LY (1996). Heteromultimerization of G-protein-gated inwardly rectifying K+ channel proteins GIRK1 and GIRK2 and their altered expression in weaver brain. J Neurosci 16: 7137–7150.
Lüscher C, Jan LY, Stoffel M, Malenka RC, Nicoll RA (1997). G protein-coupled inwardly rectifying K+ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron 19: 687–695.
Malinowska B, Napiórkowska-Pawlak D, Pawlak R, Buczko W, Göthert M (1999). Ifenprodil influences changes in mouse behaviour related to acute and chronic ethanol administration. Eur J Pharmacol 377: 13–19.
Marquis P, Lecasble M, Passa P (1998). Quality of life of patient with peripheral arterial obliterative disease treated with ifenprodil tartate. Results of an ARTEMIS study. Drugs 56 (Suppl 3): 37–48.
McCool BA, Lovinger DM (1995). Ifenprodil inhibition of 5-hydroxytryptamine3 receptor. Neuropharmacol 34: 621–629.
Mikolajczak P, Okulicz-Kozaryn I, Kaminska E, Szulc M, Dyr W, Kostowski W (2003). Lack of ifenprodil anxiolytic activity after its multiple treatment in chronically ethanol-treated rats. Alcohol Alcholism 38: 310–315.
Mizusawa H, Yamane M, Sakai K (1976). Effects of ifenprodil tartate on the autonomic, peripheral and central nervous system. Folia Pharmacol Jpn 72: 185–199.
Morgan AD, Carroll ME, Loth AK, Stoffel M, Wickman K (2003). Decreased cocaine self-administration in Kir3 potassium channel subunit knockout mice. Neuropsychopharmacology 28: 932–938.
Nakagawa H, Yamano S, Nikawa K, Matsumoto Y, Suga T (1975). Metabolic fate of dl-erythro-2-(4-benzylpiperidino)-1-(4-hydrophenyl)-1-propanol I. Absorption, distribution and excretion in the rat and mouse. Pharmacometrics 10: 283–291.
Napiórkowska-Pawlak D, Malinowska B, Pawlak R, Buczko W, Göthert M (2000). Attenuation of the acute amnesic effect of ethanol by ifenprodil: comparison with ondansetron and dizocilpine. Fundam Clin Pharmacol 14: 125–131.
Narita M, Soma M, Narita M, Mizoguchi H, Tseng LF, Suziki T (2000). Implications of the NR2B subunit-containing NMDA receptor localized in mouse limbic forebrain in ethanol dependence. Eur J Pharmacol 401: 191–195.
Nash JE, Fox SH, Henry B, Hill MP, Peggs D, McGuire S et al (2000). Antiparkinsonian actions of ifenprodil in MPTP-lesioned marmoset model of Parkinson's disease. Exp Neurol 165: 136–142.
North RA (1989). Drug receptors and the inhibition of nerve cells. Br J Pharmacol 98: 13–28.
Otomo E, Kodama R, Tazaki Y, Nukada T, Fujishima M (1976). Double blind study of FX-505 (ifenprodil) on cerebrovascular disease -Phase III study- FX-505 project for phase III study. Clin Eval 4: 419–458.
Reimann F, Ashcroft FM (1999). Inwardly rectifying potassium channels. Curr Opin Cell Biol 11: 503–508.
Reuveny E, Slesinger PA, Inglese J, Morales JM, Iniguez-Lluhi JA, Lefkowitz RJ et al (1994). Activation of the cloned muscarinic potassium channel by G protein βγ subunits. Nature 370: 143–146.
Signorini S, Liao YJ, Duncan SA, Jan LY, Stoffel M (1997). Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K+ channel GIRK2. Proc Natl Acad Sci USA 94: 923–927.
Stanfield PR, Nakajima S, Nakajima Y (2002). Constitutively active and G-protein coupled inward rectifier K+ channels: Kir2.0 and Kir3.0. Rev Physiol Biochem Pharmacol 145: 47–179.
Suzuki T, Kato H, Tsuda M, Suzuki H, Misawa M (1999). Effects of the non-competitive NMDA receptor antagonist ifenprodil on the morphine-induced place preference in mice. Life Sci 64: PL151–156.
Thurgur C, Church J (1998). The anticonvulsant actions of σ receptor ligands in the Mg2+-free model of epileptiform activity in rat hippocampal slices. Br J Pharmacol 124: 917–929.
Torrecilla M, Marker CL, Cintora SC, Stoffel M, Williams JT, Wickman K (2002). G-protein-gated inwardly rectifying potassium channels containing Kir3.2 and Kir3.3 subunits mediate the acute inhibitory effects of opioids on locus ceruleus neurons. J Neurosci 22: 4328–4334.
Wickman K, Nemec J, Gendler SJ, Clapham DE (1998). Abnormal heart rate regulation in GIRK4 knockout mice. Neuron 20: 103–114.
Wickman K, Seldin MF, Gendler SJ, Clapham DE (1997). Partial structure, chromosome localization, and expression of the mouse Girk4 gene. Genomics 40: 395–401.
Williams K (2001). Ifenprodil, a novel NMDA receptor antagonist: site and mechanism of action. Curr Drug Targets 2: 285–298.
Witkin JM, Acri JB (1995). Effects of ifenprodil on stimulatory, discriminative stimulus, and convulsant effects of cocaine. Behav Pharmacol 6: 245–253.
Yajima T, Urano T, Nakamura K, Matsuura A, Nakamura K (1987). Cerebral vasodilating and vasospasmolytic action of the cerebral circulation improver 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)-4-([4-(2-methoxyphenyl)-1-piperazinyl]methyl)isoquininoline in experimental animals. Arzneim Forsch 37: 379–383.
Young AR, Barry DI, MacKenzie ET, Robert J-P (1983). Cerebro-circulatory effects of so-called ‘vasodilators’ in the anaesthetised rat. Eur Neurol 22: 142–153.
Yourick DL, Repasi RT, Rittase WB, Stanten LD, Meyerhoff JL (1999). Ifenprodil and arcaine alter amygdala-kinding development. Eur J Pharmacol 371: 147–152.
Zhou W, Arrabit C, Choe S, Slesinger PA (2001). Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels. Proc Natl Acad Sci USA 98: 6482–6287.
We thank Dr Kansaku Baba for his cooperation, and Tomio Ichikawa, Kazuo Kobayashi, and Kazuyo Sekikawa for their assistance. We also thank Dr Steven C Hebert for providing the Kir1.1 cDNA, and Dr Lily Y Jan for providing the Kir2.1 cDNA. This work was supported by research grants from the Ministry of Education, Science, Sports and Culture of Japan, and from the Ministry of Health, Labour and Welfare of Japan.
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Kobayashi, T., Washiyama, K. & Ikeda, K. Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Ifenprodil. Neuropsychopharmacol 31, 516–524 (2006). https://doi.org/10.1038/sj.npp.1300844
- GIRK channel
- Kir channel
- Xenopus oocyte
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