Regulation of Human Kv1.4 Channel Activity by the Antidepressant Metergoline

Abstract

Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na⁺ channels in Xenopus laevis oocytes (Acta Pharmacol. Sin., 35, 2014, Lee et al.). In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. The IC₅₀ of peak currents of hKv1.4 currents was 3.6±0.6 µM. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity.

The ergot alkaloids are derived from ergot fungi. Metergoline (Fig. 1), an antagonist of serotonin receptors and agonist of dopamine receptors, is an ergot-derived drug that has been researched for use in a variety of clinical treatments, including seasonal affective disorder,¹⁾ prolactin hormone regulation,²⁾ premenstrual dysphoric disorder in women,³⁾ carbon dioxide-induced anxiety,⁴⁾ and in veterinary medicine as a pregnancy termination drug for dogs.⁵⁾ Metergoline and other ergot-derived drugs are used clinically in disorders associated with hyperprolactinemia, for inhibition of lactation and are used in the treatment of migraine headaches.⁶⁾ The pharmacological properties of metergoline act on the 5-hydroxytryptamine system. Metergoline acts as an antagonist on many of the serotonin (5-HT) receptor subtypes at a relatively low concentration⁷⁾; it also has dopamine agonist properties.⁸⁾ While metergoline is known to be an effective clinical treatment, little is known regarding the effects of metergoline on different types of receptors and ion channels. Voltage-gated K channels play important roles in a variety of physiological conditions, including regulation of neurotransmitter release, neuronal excitability, heart rate, muscle contraction, hormone secretion, epithelial electrolyte transport, cell volume, and cell proliferation in neuronal and non-neuronal cells.⁹⁾

Fig. 1. Chemical Structure of Metergoline

In the present study, we examined whether metergoline exerts inhibitory effects on peak currents of human Kv1.4 channel. We used the two-microelectrode voltage-clamp technique to investigate the effect of metergoline on human Kv1.4 currents in Xenopus laevis oocytes expressing wild-type Kv1.4 alpha subunits. The cRNAs encoding these channels were expressed in Xenopus laevis oocytes, a model system that has few endogenous ion channels and allows heterologous expression of ion channels for various biochemical studies.¹⁰⁾ Our results revealed that metergoline inhibited Kv1.4 channel currents in a concentration- and reversible manner. These results show that metergoline inhibits Kv1.4 channel currents and that Kv1.4 channel current inhibition could be one of the mechanisms by which metergoline affects the central nervous system.

MATERIALS AND METHODS

Materials

Metergoline (Tocris Bioscience, U.S.A.) was prepared by dissolving in dimethyl sulfoxide (DMSO) and diluted with bath solution before use. The final concentration of DMSO was less than 0.1% in all cases. The cDNA for human Kv1.4 channel (Gene bank accession number: NM_002233) was provided by Dr. Pongs (University of Hamburg, Germany). Other agent was purchased from Sigma (St. Louis, MO, U.S.A.).

Preparation of Xenopus laevis Oocytes and Microinjection

Xenopus laevis frogs were purchased from Xenopus I (MI, U.S.A.). Their care and handling were in accordance with the highest standards dictated by Chonnam National University institution guidelines. Oocyte preparation was performed according to the method previously reported.^10,11) The cRNAs of Kv1.4 channel (40 nL) were injected into each oocyte 1 d after isolation, using a 10 µL micro-dispenser (VWR Scientific, U.S.A.) fitted with polished glass pipette (20 nm in diameter).

in Vitro Transcription of Kv1.4 Channel cDNAs

The recombinant plasmids of Kv1.4 were linearized by appropriate restriction enzymes, and run off transcripts were prepared using the methylation cap analog mG(5′)3p(5′)G. The cRNAs were prepared by mMessage mMachine kit (Ambion, TX, U.S.A.) with RNA polymerase. The absence of degraded RNA was confirmed by electrophoresis followed by EtBr staining.

Data Recording

Oocyte recording was performed according to the method previously reported.^10,11) Data recordings were performed in ND96 bath solution (in mM: 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, and 5 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.5). All electrophysiological experiments were performed at room temperature using an Oocyte Clamp (OC-725C; Warner Instruments, CT, U.S.A.), and stimulation and data acquisition were controlled using a pClamp 10.2 (Axon Instruments, Union City, CA, U.S.A.).

Data Analysis

For concentration response curve describing the effect of metergoline on K⁺ currents, the peak currents at the different concentrations of metergoline were plotted. Origin software (Origin, MA, U.S.A.) was then used to fit the Hill equation plot, which is as follows: y/y_max=[X] and are n_H/([X]^n_H+[IC₅₀]ⁿ), where y is the peak at a treated concentration of metergoline, y_max is the maximal peak current, IC₅₀ is the concentration of metergoline that produces a half-maximum effect, [X] is the concentration of metergoline, and n_H is the regulation co-efficient. IC₅₀ values were obtained using the software. All values are presented as the mean±standard error of the mean (S.E.M.) The differences of the means of the control and treatment values were determined using an unpaired Student’s t-test. A value of p<0.01 was considered to be statistically significant.

RESULTS

Metergoline Inhibits Peak Current of Human Kv1.4 Channels

Using the two-electrode voltage-clamp technique, Kv1.4 channel currents were recorded from Xenopus laevis oocytes injected with cRNAs encoding human Kv1.4 channel proteins (Fig. 2). First, to assess the effect of metergoline on the current and voltage relationship, we figured IV curves with and without metergoline in the control. The current responses evoked by voltage steps (voltage pulses of 500 ms duration given in 10 mV increments and 10 s intervals from the holding potential of −80 mV) were used to construct the I–V curve. The Kv1.4 channel currents evoked by this voltage-clamp protocol were transient A-type K⁺ currents that rapidly inactivated, i.e., N-type inactivation¹²⁾ (Fig. 2, Con). In the absence of metergoline, human Kv1.4 channel currents were elicited by voltage pulses more positive than −40 mV, and current amplitude increased linearly with further depolarization (n=10). The presence of metergoline (10 µM) reduced peak current amplitude over the entire voltage range in which the Kv1.4 channel current was activated (Fig. 2, MG). Metergoline induced tonic inhibition was recovered to control current after washout. Therefore, the inhibitory effect of metergoline on Kv1.4 channel currents was reversible.

Fig. 2. Effects of Metergoline on Human Kv1.4 Channel Currents

A–C, Oocytes were injected with human Kv1.4 channel cRNAs and maintained for 2 to 4 d before Kv1.4 channel currents were recorded in ND96 using a two-electrode voltage clamp technique. Original K⁺ current traces are shown under control conditions, after bath superfusion of metergoline and washout. Voltage pulses of 500-ms duration were applied in 10-mV increments and at 10-s intervals from a holding potential of –80 mV. D, I–V relationships of human Kv1.4 channels in the absence or presence of 10 µM metergoline (MG). The current-voltage relationships of Kv1.4 channels were obtained using voltage steps between –80 and +60 mV taken in 10 mV increments. Each outward peak current was normalized to the current at +60 mV. Data are expressed as the mean±S.E.M. (* p<0.05 and ** p<0.01 compared to control, n=10).

Metergoline Exhibits Concentration-Dependent Inhibitory Effects on Human Kv1.4 Channels

Next, we examined concentration-dependent effects of metergoline on peak current of Kv1.4 channels. The inhibitory effect of metergoline on the peak current of human Kv1.4 channels was concentration-dependent (Figs. 3A, B). The currents were evoked by voltage stepping from a holding potential of −80 to +60 mV at 10-s intervals for a duration of 500 ms. The IC₅₀ value of peak currents was 3.6±0.6 µM (n=12; Fig. 3B). The Hill coefficients for peak currents were 1.0±0.1, suggesting an interaction between one metergoline molecule and human Kv1.4 channel. The V_max value of peak currents was 76.1±3.4%, in human Kv1.4 channels.

Fig. 3. Concentration–Response Curves and Delayed Recovery of Human Kv1.4 Channel Currents by Metergoline

A, Representative human Kv1.4 channel currents elicited in the absence and presence of various concentrations of metergoline. Superimposed current traces of Kv1.4 channel dynamics were obtained by applying depolarizing pulses from a holding potential of −80 to +60 mV every 10 s in the absence and presence of 1, 3, 10, 30 or 100 µM metergoline. B, Concentration–response curves for metergoline-induced inhibition of human Kv1.4 channels. Metergoline-induced inhibitions were measured at the peak of the depolarizing pulse of +60 mV. The currents were fit to the Hill equation as described in Materials and Methods. Data are expressed as the mean±S.E.M. (n=12). C, Recovery from inactivation was assessed in detail using the paired-pulse voltage-clamp protocol shown in the inset. After oocytes were depolarized for 50 ms (P1), fractional recovery during a subsequent test pulse (P2) was assessed after an intervening recovery interval. Channels recovered almost fully at this recovery interval in the absence of metergoline. In the presence of 10 µM metergoline, the test current measured during the second pulse (P2) was significantly reduced. Metergoline (●)-free channels recovered rapidly (<2 s), while complete recovery from inactivation was delayed for approximately 4 s in the presence of metergoline (○). Data represent the mean±S.E.M. (n=8/group).

The Inhibitory Effect of Metergoline on Kv1.4 Channels Shows Slow Recovery

Next, we examined whether the effects of metergoline on Kv1.4 channel currents were derived from delayed recovery of the channel inhibition. Current traces were recorded in the absence and presence of 10 µM metergoline, with a recovery time interval of 50 ms between pulses, and the results were analyzed as shown in Fig. 3C. After a 50-ms pre-pulse (P1), recovery from open channel block was assessed using a test pulse (P2) after increasing recovery intervals (Fig. 3C, insert). Our results revealed that Kv1.4 channels recovered rapidly in the absence of metergoline, likely due to the slow inactivation associated with Kv1.4 channels (1.6±0.6 s, closed circles). In contrast, metergoline-treated channels showed a delayed recovery from the channel inhibition (3.6±0.8 s). This slow recovery might be due to slow drug dissociation from the Kv1.4 channels.

DISCUSSION

Kv1.4, a shaker family potassium channel alpha subunit, is part of a family of voltage-gated potassium channels that mediate transient and rapidly inactivating A-type currents and N-type inactivation. Kv1.4 channels are mainly located on axons and at presynaptic terminals and function to modulate action potential waveforms and neurotransmitter release.¹³⁾ In cardiovascular system, Kv1.4 channels have critical roles in ventricular arrhythmias and atrioventricular blocking.^14,15) Thus, these channels are one of the targets of drugs for treatment of pathological conditions including human nervous system disease and cardiac diseases.

Metergoline is an ergot-derived drug that has been researched for use in a variety of clinical treatments.¹⁶⁾ In addition, accumulating evidence shows that metergoline has other beneficial effects, such as antidepressant activities,³⁾ regulation of the hormone prolactin,²⁾ anti-anxiety effects,^1,4) antifungal effects,⁵⁾ and neuroprotective actions.⁶⁾ For example, metergoline attenuated premenstrual dysphoric disorder symptoms by Roca et al., and other reports showed that depression suffered patients with seasonal affective disorder had dramatically decreased depressive symptoms after administration of metergoline.^1,3) In addition, CO₂-induced anxiety in volunteers was significantly increased by oral administration of metergoline.⁴⁾ However, very little is known about the therapeutic mechanisms of metergoline.

In the present study, we showed that the antidepressant metergoline could regulate the activity of Kv1.4 channels expressed in Xenopus laevis oocytes. Our major findings are as follows. Metergoline treatment produced tonic inhibition of Kv1.4 channel currents. Inhibition of Kv1.4 channel currents by metergoline was concentration-dependent and reversible (Figs. 2, 3). In addition, the inhibitory effects of metergoline on Kv1.4 channels also show slow recovery.

In conclusion, metergoline is a potent regulator of Kv1.4 channels in a concentration-dependent and reversible manner. These novel findings provide a possible molecular basis of the pharmacology of metergoline at the cellular and molecular levels.

Acknowledgment

This study was financially supported by Chonnam National University, 2014.

Conflict of Interest

The authors declare no conflict of interest.

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