Regulation of heterologously expressed 5‐HT_1B receptors coupling to potassium channels in AtT‐20 cells

Abstract

Background and Purpose

5‐HT_1B receptors are widely expressed GPCRs and a target of triptans, the most commonly prescribed anti‐migraine drugs. There is very limited information about the acute, agonist‐induced regulation of 5‐HT_1B receptor signalling and so we sought to characterize this in a neuron‐like system.

Experimental Approach

Epitope‐tagged human 5‐HT_1B receptors were expressed in mouse AtT20 cells. 5‐HT_1B receptor signalling was assessed using whole‐cell patch‐clamp recordings of endogenous G protein‐gated inwardly rectified potassium (GIRK) channels, and receptor localization measured using immunofluorescence.

Key Results

5‐HT (EC₅₀ 65 nM) and sumatriptan (EC₅₀ 165 nM) activated GIRK channels in AtT20 cells expressing 5‐HT_1B receptors. Continuous application of both 5‐HT (EC₅₀ 120 nM) and sumatriptan (EC₅₀ 280 nM) produced profound desensitization of 5‐HT_1B receptor signalling within a few minutes. Complete recovery from desensitization was observed after 10 min. Both 5‐HT and sumatriptan induced significant heterologous desensitization of SRIF (somatostatin)‐activated GIRK currents, with the 5‐HT‐induced heterologous desensitization being blocked by the protein kinase inhibitor staurosporine. Both agonists induced modest 5‐HT_1B receptor internalization, with a time course much slower than receptor desensitization.

Conclusions and Implications

In AtT‐20 cells, 5‐HT_1B receptors undergo rapid and reversible desensitization at concentrations of agonist similar to those required to activate the receptor. Desensitization is incomplete, and the continued signalling of the receptor in the presence of the agonist may lead to cellular adaptations. Finally, 5‐HT_1B receptor activation causes significant heterologous desensitization, which may lead to a reduced effectiveness of unrelated drugs in vivo.

Abbreviations

GIRK

G protein‐coupled inwardly rectifying potassium channel

GRK

GPCR kinase

GSK‐3

glycogen synthase kinase‐3

I_Ca

voltage‐gated calcium channel current

SRIF

somatostatin (somatotropin release inhibiting factor)

Introduction

5‐HT_1B receptors (Alexander et al., 2017a) are expressed throughout the human body including the vasculature and the CNS. They are G_i/o protein‐coupled receptors, with prominent roles in mediating inhibition of neurotransmitter release and relaxation of cerebral blood vessels. These functions have led to 5‐HT_1B receptors being suggested as potential drug targets for anxiety, depression, aggressive‐like behaviour and migraine headaches (Sari, 2004).

The coupling of 5‐HT_1B receptors to cellular signalling pathways is well defined (McCorvy and Roth, 2015) but little is known about how receptor signalling is regulated in the continued presence of agonist. Acute regulation of GPCR signalling usually involves intracellular mechanisms which lead to desensitization of receptor signalling and often internalization of the receptor during sustained receptor activation. Phosphorylation of agonist‐occupied receptors by GPCR kinases (GRKs) and the subsequent binding of arrestin proteins has been implicated in the desensitization and internalization of several GPCRs, and it is generally assumed that similar mechanisms operate for many others (Kelly et al., 2008).

Desensitization of 5‐HT_1B receptor signalling has only been addressed in a few studies. Prolonged (>1 h) incubation with 5‐HT modestly reduces inhibition of AC by native 5‐HT_1B receptors in opossum kidney cells (Pleus and Bylund, 1992), with the loss of signalling at later time points (20 h) coincident with down‐regulation of receptor number. The mechanisms underlying acute loss of receptor signalling are not defined, although they did not apparently involve GRK2 (Lembo et al., 1999). By contrast, GRK2/3 was implicated in the rapid internalization of mouse 5‐HT_1B in fibroblasts, a process which was suggested to occur via a caveolin‐dependent pathway, distinct from the more commonly observed clathrin‐dependent endocytosis pathway for GPCR (Janoshazi et al., 2007). In keeping with potentially non‐canonical regulatory pathways for the 5‐HT_1B receptor, basal receptor activity and recycling after internalization was reported to depend on receptor association with and phosphorylation by glycogen synthase kinase‐3 (GSK‐3) (Chen et al., 2009). Importantly, there is a dearth of information about whether prolonged activation of 5‐HT_1B receptors only produces desensitization of its own signalling (homologous desensitization) or whether it can also affect the signalling of other receptors in the cell (heterologous desensitization).

The desensitization of GPCR signalling by agonists may limit the acute therapeutic actions of drugs as well as determining the degree to which non‐desensitized receptors continue to signal in the continued presence of agonist, a phenomenon which may lead to cellular adaptations to the drug (Kelly et al., 2008). The relationship between 5‐HT_1B receptor activation and agonist‐induced desensitization has not been well defined, so we created a model system where acute 5‐HT_1B receptor signalling and trafficking can be studied in real‐time in a neuronal‐like environment. We found that in mouse pituitary‐derived AtT20 cells, both 5‐HT and sumatriptan produced rapid but incomplete desensitization of 5‐HT_1B receptor activation of G protein‐gated inwardly rectifying potassium (GIRK; K_ir3.x) channels. This desensitization reversed rapidly, and it was not associated with a substantial loss of cell surface receptor. Activation of 5‐HT_1B receptors also produced a profound heterologous desensitization of GIRK channel activation by endogenous somatostatin SST receptors in the AtT20 cells (note SRIF will be used in this article instead of somatostatin). Although 5‐HT_1B receptors have not been shown to couple to GIRK channels in neurons, our results suggest that acute desensitization of the 5‐HT_1B receptor may have the potential to shape therapeutic responses to drugs acting on 5‐HT_1B receptors, as well as the responses of other GPCRs.

Methods

Cell transfection and culture

AtT20 cells were stably transfected with plasmids containing cDNA for the human 5‐HT_1B receptor (UMR cDNA Resource Centre, Rolla, MO, USA) and were cultivated in DMEM supplemented with 100 U penicillin, 100 μg streptomycin, 10% FBS and 500 μg·mL⁻¹ G418 (Invitrogen or Invivogen, Melbourne, Australia). Electroporation (Amaxa GmbH, Germany) was used to transfect AtT20 cells with the purified cDNA. Geneticin (500 μg·mL⁻¹, Invitrogen) was used to select for clones expressing the 5‐HT_1B receptor, and six clones were picked and seeded in 100 mm dishes. The expression of 5‐HT_1B receptors was confirmed using qualitative PCR, and four clones apparently expressing high amounts of 5‐HT_1B receptor mRNA were chosen for further analysis. HEK 293 cells stably expressing Ca_v3.1 channels under control of a tetracycline repressor (Gilmore et al., 2012; HEK 293 FlpIn‐TRex; RRID:CVCL_U427) were grown as described above, with the exception of the substitution of hygromycin (80 μg·mL⁻¹) as selection antibiotic. Channel expression was induced by incubation with tetracycline (2 μg·mL⁻¹, overnight).

Characterization of AtT20 wild type cells and expression of human HA‐tagged 5‐HT_1B receptors in AtT20 cells

In our laboratory, wild type AtT20 cells do not respond to 5‐HT with activation of endogenous K channels or inhibition of current through voltage‐gated calcium channels (I _Ca) and levels of mRNA for 5‐HT receptors is reportedly low (Atwood et al., 2011). We confirmed the absence of 5‐HT_1B receptor mRNA using qualitative PCR with cDNA extracted from mouse trigeminal ganglia was used as a positive control for the expression 5‐HT receptor mRNA (not shown). Cells were initially screened for 5‐HT_1B responses using inhibition of I _Ca as an assay; while it was possible to detect modest, reversible modulation of I _Ca in transfected cells, the inhibition was too small to be used in experiments focussing on quantitative measures of receptor function (Borgland et al., 2003), so we measured activation of K channels instead (see Results section). One cell line with a substantial response to 5‐HT was used in this study.

Electrophysiology

Cells were grown in 35 mm plastic tissue culture plates at low density and used up to 3 days after initial plating, providing cells were not in contact. For all recordings, the whole‐cell configuration of the patch‐clamp technique was used. Dishes were continually perfused with HBS (mM): NaCl 150, KCl 2.5, CaCl₂ 1.8, MgCl₂ 1, HEPES 10, glucose 10, pH 7.3 (NaOH), 330 ± 5 mosmol·L⁻¹. Recordings were made with fire‐polished borosilicate pipettes with a resistance of about 2–5 MΩ. Recordings were made using an Axopatch 200 B amplifier (Axon Instruments, Foster City, CA, USA). The cell capacitance was between 8 and 20 pF, and series resistance was less than 15 MΩ. Drugs were applied via a series of flow pipes positioned above the cells at a flow rate of 1 mL·min⁻¹. All recordings were performed at 33°C, which was provided by heating the external solution via a Dual Automatic Temperature Controller TC‐344B (Warner Instruments, USA).

GIRK currents were recorded at 32–34°C. For recording of GIRK currents, the extracellular solution contained (mM): NaCl 35, KCl 130, CaCl2 1.5, HEPES 10, glucose 10, BSA 0.05%, pH 7.3 (NaOH), 330 ± 5 mosmol·L⁻¹. The intracellular solution contained (mM): KCl 130, HEPES 20, EGTA 10, CaCl₂ 2, MgATP 5, NaGTP 0.2, NaCl 5, pH 7.3 (NaOH), 285 ± 5 mosmol·L⁻¹. In one series of experiments, EGTA was replaced by 10 mM BAPTA; in another, BAPTA and EGTA were omitted entirely. For the step protocol, cells were voltage clamped at 0 mV. GIRK currents were evoked by steps of 200 ms length to −60 mV every 10 s and monitored for current stability before drugs were applied. In one set of experiments, cells were stepped between potentials of −100 and +20 mV to examine the current/voltage relationship of the drug‐activated currents (Supporting Information Figure S1). GIRK currents were sampled at 10 kHz and filtered at 5 kHz. The series resistance was compensated for by at least 80% in all experiments. Data were recorded using Axograph X software (Axograph Scientific, Australia) or Clampex 9.2 software (Molecular Devices, Sunnyvale, CA). For continuous recordings of GIRK currents, cells were voltage clamped at −60 mV. GIRK currents were sampled at 2 kHz and filtered at 1 kHz. Axograph X software (Axograph Scientific) was used to acquire and analyse data. Drugs were applied through a set of flow pipes positioned approximately 200 μM from the cell. In some experiments aimed at determining the potency of drug activation of 5‐HT_1B receptors, up to three ascending concentrations of drug were briefly superfused on a cell, with washes of at least 50 s between applications. Drug responses for determining concentration–response curves were normalized to those produced by 100 nM SRIF , which couples to GIRK channels via endogenous SST receptors in AtT20 cells (Günther et al., 2016). The prior application of lower drug concentrations did not affect the response to subsequent applications of drug; 10 μM 5‐HT alone activated K currents to 77 ± 5% of that produced by 100 nM SRIF, while 10 μM 5‐HT applied after two lower concentrations of 5‐HT activated K currents 73 ± 7% of SRIF, P = 0.65, n = 6.

In the experiments where protein kinase inhibitors were included in the recording pipette, the contents of the pipette solution were allowed to equilibrate with the intracellular environment of the cell for at least 15 min. Parallel control recordings where cells were perfused for 15 min with the solvent used to dissolve the inhibitors (DMSO) were made for comparison of drug effects.

A four parameter sigmoidal dose–response curve was fitted to the data using the following equation in GraphPad Prism 4 (www.graphpad.com):

$$\mathrm{Y}=\text{Bottom}+\left(\mathrm{Top}–\text{Bottom}\right)/(1+10^(\left({\text{logEC}}_{50}–\mathrm{X}\right)*\text{Hillslope}$$

Immunocytochemistry

Immunocytochemistry was used to measure receptor internalization. Cells were seeded in black clear‐bottom 96‐well plates at a density of 25 000 cells per well and grown in DMEM. Twenty‐four hours after seeding, medium was changed to Leibowitz L‐15 Medium supplemented with 0.01% BSA, penicillin/streptomycin (50 U/5 μg·mL⁻¹) and geneticin (all from Invitrogen). Approximately 20 h after changing to L‐15 Medium, cells were incubated with blocking solution comprising 10% normal goat serum in L‐15 Medium at 37°C. A 1 h incubation with primary antibody at 37°C (mouse anti‐HA, 1:1000, Covance, USA, catalogue # MMS‐101P‐1000, RRID: AB_291261) was followed by incubation of cells with the appropriate concentration of either 5‐HT or sumatriptan in L‐15 Medium for 0–40 min at 37°C. The assay was terminated by placing the plate on ice for 30 s. Subsequently, cells were incubated in Alexa 488‐conjugated goat anti‐mouse secondary antibody (1:500, Molecular Probes, Melbourne, Australia, catalogue # A‐11001, RRID AB_138404) at room temperature for 30 min. Cells were fixed with 4% formaldehyde for 10 min at room temperature. Cells were washed twice PBS and then incubated for 10 min with DAPI (200 μg·mL⁻¹). After extensive washing with PBS, 50 μL PBS were added to each well and fluorescence intensity was measured using a plate reader (FlexStation 3, Molecular Devices). In wild type AtT‐20 cells, the fluorescence of the cells in the presence of the anti‐HA antibody was negligible. In each experiment, every time point was run in quadruplicate and experiments were repeated five times. Data were normalized to the fluorescence intensity of vehicle‐treated cells.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Results are expressed as the mean ± SEM of at least 5–6 independent determinations, with equal group sizes. On occasions, two cells from a single dish were included in the analysis; however, the experiments performed on these cells were always different. All data are expressed as a percentage change in pre‐drug values, largely to account for the differences between the size of the K currents in the different cells or the intensity of immunofluorescence between experiments. The nature of the experiments, which are for the most part perfusing known concentrations of drug onto single cells, makes blinding impractical. Control data were tested for normality using the D'Agostino and Pearson normality test in PRISM and were normally distributed. Student's t‐test was used to determine statistical significance of means. Statistical analysis of immunocytochemical experiments was performed using one‐way ANOVA followed by Dunnett's correction for multiple comparisons. Statistics were performed using GraphPad Prism v4‐7, RRID:SCR_002798). Statistical significance was set at P < 0.05.

Materials

SRIF 1‐14 (somatostatin) was supplied by AUSPEP (Tullamarine, VIC, Australia). 5‐HT hydrochloride, Pertussis toxin (PTX), GR127935 hydrochloride, staurosporine, BAPTA and BSA were obtained from Sigma‐Aldrich (Castle Hill, Australia). PP2 was supplied by Enzo Life Sciences (Plymouth Meeting, PA, USA). Sumatriptan succinate (Imitrex, 12 mg·mL⁻¹, GlaxoSmithKline, Boronia, VIC, Australia) was obtained from a pharmacy. This 40 mM solution was diluted in water for a 10 mM stock solution. For experiments, the 10 mM sumatriptan stock solution was diluted as necessary in either recording solution or in L‐15 Medium.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a, 2017b).

Results

Defining 5‐HT_1B receptor activation

5‐HT did not inhibit I _Ca, activate potassium channels or change intracellular Ca concentration in wild type AtT20 cells (not shown). However, application of 10 μM 5‐HT (Figure 1A) or 10 μM sumatriptan to AtT20–5‐HT_1B cells voltage clamped at 0 mV and repetitively stepped to −60 mV led to activation of a substantial inward current. The 5‐HT‐activated current was largely blocked by superfusion of Ba²⁺ (1 mM, P < 0.05, Figure 1B). Pretreatment of AtT20 cells with PTX (200 ng·mL⁻¹, overnight), which blocks signalling through Gi/o‐type G proteins, essentially abolished the inward currents produced by 5‐HT (10 μM) and SRIF (100 nM, Figure 1C, D). Native SST receptors in AtT20 cells have previously been reported to couple to GIRK channels, and the properties of the 5‐HT_1B receptor‐activated current are consistent with this (Supporting Information Figure S1). Application of the 5‐HT_1B/D receptor antagonist GR127935 (100 nM) reduced the response to co‐applied 5‐HT (10 μM) or sumatriptan (10 μM) by approximately 90% (Figure 1E, F). These data are consistent with the activation of GIRK channels in AtT20 cells by heterologously expressed 5‐HT_1B receptors.

Figure 1.

Signalling of AtT20–5‐HT_1B cells is due to activation of GIRK channels by 5‐HT_1B receptors. (A) Typical current trace of GIRK channel inhibition by 1 mM BaCl₂ (Ba). (B) 1 mM Ba significantly reduces GIRK currents activated by 5‐HT. (C) Typical current trace of the effects of PTX on GIRK channel activation. (D) Treatment of AtT20 5‐HT_1BR cells with PTX prevents the activation of GIRK currents by 5‐HT (10 μM) and SRIF (100 nM). (E) Typical trace of the effects of GR127935 (100 nM) on GIRK channel activation by 5‐HT (10 μM). (F) Application of the 5‐HT_1B receptor antagonist GR127935 (100 nM) inhibits the activation of GIRK currents by 5‐HT (10 μM) and sumatriptan (10 μM), compared with the response before GR127935 application. Data in panels A–D are from cells voltage clamped at 0 mV and repetitively stepped to −60 mV. The dotted line in A and C represents zero current. Data in panel E and F are from cells continuously voltage clamped at −60 mV. Each data point represents the mean ± SEM of at least six cells. *P < 0.05, significant effect of Ba or PTX; unpaired t‐test.

Initially, experiments examining 5‐HT_1B receptor activation were performed by repetitively stepping the membrane potential of AtT20 cells from 0 to −60 mV, with the amplitude of evoked current being measured. For this step protocol, current amplitude was averaged for the last 50 ms of the step to −60 mV. In order to obviate the potential for voltage changes to affect drug action (Ben‐Chaim et al., 2003; Sahlholm, 2011), we also conducted experiments at a fixed holding potential of −60 mV. In these experiments, the change in current resulting from drug application was quantified. For these recordings, the peak current was quantified by averaging the current for 5 ms either side of the peak identified in Axograph.

When 5‐HT was applied to cells repetitively stepped from 0 to −60 mV (Figure 2A), it produced an increase in current at −60 mV with a pEC₅₀ of 7.17 ± 0.09 (65 nM) with a maximum effect of 73 ± 7% of the current produced by 100 nM SRIF (Figure 2B). Sumatriptan increased the current with a pEC₅₀ of 6.81 ± 0.13 (155 nM) to a maximum of 90 ± 6% of 100 nM SRIF (Figure 2C). When 5‐HT or sumatriptan were applied to cells voltage clamped at −60 mV, they produced an inward current of pEC₅₀ of 7.91 ± 0.08 (12 nM) and 7.23 ± 0.06 (60 nM), respectively, with maximum effects of 97 ± 2% and 92 ± 8% of SRIF (100 nM) respectively (Figure 2B, C).

Figure 2.

Activation of GIRK currents by 5‐HT_1B receptor agonists is concentration dependent. (A) A typical time course showing the concentration dependence of GIRK current activation by sumatriptan. Each point represents the amplitude of the inward current elicited by a step from ‐0 mv to ‐40 mV. Drugs were applied for the duration of the bars. (B) Comparing concentration response curves of 5‐HT_1B receptor activation by 5‐HT in experiments stepping from 0 to −60 mV and in experiments of continuous recordings at −60 mV. (C) Concentration response curves for 5‐HT_1B receptor activation by sumatriptan in experiments stepping from 0 to −60 mV and in experiments of continuous recordings at −60 mV. Activation of 5‐HT_1B receptors is displayed as percentage of 5‐HT‐induced GIRK current to SRIF‐induced GIRK current. Each data point represents mean ± SEM of at least six cells.

Desensitization of GIRK channels by 5‐HT_1B receptor agonists

Desensitization of 5‐HT_1B receptor signalling was determined by application of 5‐HT or sumatriptan for various times followed by testing the response to a probe concentration of 5‐HT (100 nM) (Figures 3 and 5). The probe concentration of drug was superfused before and then immediately after the test concentration of 5‐HT or sumatriptan. We initially examined desensitization utilizing conditions of repetitive stepping from 0 to −60 mV, where 5‐HT_1B receptor activity was repetitively probed in method analogous to our experiments on desensitization of μ‐opioid receptor modulation of I _Ca in AtT20 cells (Borgland et al., 2003) (Figure 3). Both 5‐HT (Figure 3C) and sumatriptan (Figure 3D) produced a profound reduction in the subsequent response to a submaximally effective concentration of 5‐HT in a time‐dependent manner. 5‐HT (10 μM) induced desensitization with a t _1/2 of 105 s, (95% CI 85–140 s), while sumatriptan (10 μM) induced desensitization with a t _1/2 of 240 s, (95% CI 210–280 s) (Figure 3A, B). Maximum desensitization for 5‐HT (10 μM) was reached at about 7 min with a value of 83 ± 7%. Desensitization induced by sumatriptan (10 μM) reached a maximum at 15 min with a value of 91 ± 4%. For all further experiments, we chose a duration of drug application where desensitization had reached about 80% for the highest concentrations tested (10 μM for both 5‐HT and sumatriptan). This was 6 min for 5‐HT and 10 min for sumatriptan.

Figure 3.

5‐HT_1B receptor signalling desensitizes in a time‐dependent manner. To examine the time dependence of 5‐HT_1B receptor desensitization by exposure to 10 μM 5‐HT or 10 μM sumatriptan, the GIRK current activation by an approximate EC₅₀ concentration of 5‐HT (100nM, clear bars) was repeatedly measured throughout the time course of the experiment. To elicit GIRK currents, the membrane potential was stepped from 0 to −60 mV. (A) 5‐HT_1B receptor desensitization by 5‐HT is time‐dependent with t _1/2 of 105 s. (B) Sumatriptan desensitizes 5‐HT_1B receptors in a time‐dependent manner with t _1/2 of 240 s. (C) Typical time plot of 5‐HT_1B receptor desensitization by exposure to 10 μM 5‐HT (black bars). (D) Typical time plot of 5‐HT_1B receptor desensitization by exposure to 10 μM sumatriptan (Sum; black bars). Each data point in A and B represents mean ± SEM of at least six cells. The dotted line represents the response to the probe concentration of 5‐HT before the challenge concentration of 5‐HT or sumatriptan was applied.

Figure 5.

Time course of desensitization of 5‐HT_1B receptor signalling by 5‐HT and sumatriptan at −60 mV. (A) Typical trace of continuous recording of 5‐HT (100 nM)‐induced desensitization used for post hoc analysis. (B) Illustration of the time dependence of desensitization for 100 nM 5‐HT with data derived from post hoc analysis of continuous recording experiments. (C) Typical trace of continuous recording of 5‐HT (10 μM)‐induced desensitization used for post hoc analysis. (D) Illustration of the time dependence of desensitization for 10 μM 5‐HT with data derived from post hoc analysis of continuous recording experiments. (E) Typical trace of continuous recording of sumatriptan (10 μM)‐induced desensitization used for post hoc analysis. (F) Time courses of desensitization induced by 10 μM sumatriptan with data points derived from post hoc analysis of continuous recordings. Each data point represents mean ± SEM of at least six cells. See Supporting Information Figure S2 for a diagram of how post hoc values were derived.

We determined the potency of 5‐HT and sumatriptan to desensitize 5‐HT_1B receptor signalling using continuous recording at −60 mV, with a probe concentration of 100 nM 5‐HT applied before and after the test concentration of agonist. Under these conditions, 5‐HT reduced the probe response with a pEC₅₀ of 7.62 ± 0.23 (24 nM, Figure 4A), while sumatriptan reduced the probe response with a pEC₅₀ of 6.44 ± 0.08 (360 nM, Figure 4B). Thus, activation of K channels occurred at lower concentrations of agonist than recruitment of receptor desensitization. The apparent magnitude of 5‐HT_1B desensitization was less when the voltage was held at −60 mV than when the voltage was stepped repetitively from 0 to −60 mV. 5‐HT (10 μM) produced a desensitization of 45 ± 5% of the 100 nM 5‐HT probe at −60 mV, compared with 81 ± 7% at 6 min in the stepping protocol (Figure 5C, D). Similarly, the desensitization produced by sumatriptan (10 μM) was 54 ± 3% at a constant membrane potential of −60 mV, compared with 81 ± 6% at 10 min during the step protocol. The difference in apparent extent of receptor desensitization in the two voltage protocols is likely to reflect the fact that the 100 nM 5‐HT probe produces a response closer to the maximum observed when applied at a constant holding potential of −60 mV when compared with the step protocol. Loss of a similar number of receptors through desensitization in each case is likely to lead to a greater reduction in the apparent effect of the probe in the situation where the probe concentration was further away from the concentration required for a maximal effect. The currents produced by two applications of the probe concentration of 5‐HT (100 nM) were similar when applied 6 or 10 min apart (Figure 4C, D), indicating that the brief exposure to the probe concentration did not itself produce desensitization of signalling.

Figure 4.

Concentration dependence of 5‐HT_1B receptor activation and desensitization at −60 mV. (A) Concentration response curves of 5‐HT_1B receptor activation and desensitization by 5‐HT using continuous recordings at −60 mV. (B) Concentration response curves of 5‐HT_1B receptor activation and desensitization by sumatriptan using continuous recordings at −60 mV. Activation of 5‐HT_1B receptors is displayed as percentage of 5‐HT‐induced GIRK current to SRIF (100 nM)‐induced GIRK current. Desensitization of 5‐HT_1B receptor signalling was measured as a change in the amplitude of the 100 nM 5‐HT probe currents before and after the desensitizing drug exposure and is expressed as a percentage of the maximum desensitization observed (100%). (C, D) Response to 100 nM 5‐HT does not change after 6 and 10 min application of washing solution (external solution without drug). Each data point represents mean ± SEM of at least six cells.

The time course of desensitization for supramaximal concentrations of agonist (10 μM) at constant holding potential of −60 mV (5‐HT, 60 s, 95% CI 46–91 s Figure 5C, D; sumatriptan 106 s, 95% CI 92–125 s, Figure 5E, Supporting Information Figure S2) was similar to the repetitive stepping protocol (Figure 3A, B, see above). We also compared the desensitization rate at lower concentrations of 5‐HT; at 100 nM, the estimated t _1/2 was 104 (95% CI 80–148 s) for the step protocol and 102 s (95% CI 73–171 s) for continuous recordings (Figure 5A, B). The similarity of the time course and extent of desensitization under the two distinct protocols suggest that the desensitization process itself is not voltage dependent, unlike the initial receptor‐channel coupling.

Desensitization induced by continuous application of 5‐HT or sumatriptan (10 μM each) to cells voltage clamped at −60 mV largely reversed within 10 min (Figure 6A, B). The time course of recovery was determined by testing the response to 5‐HT applied every 2 min after the end of the desensitizing concentration of 5‐HT or sumatriptan. The t _1/2 time (t _1/2) for recovery after 10 μM 5‐HT was 140 ± 27 and 120 ± 32 s for 10 μM sumatriptan (Figure 6C). The amount of recovery from desensitization was the same whether the probe concentration of drug was applied every 2 min post‐desensitization or just once 10 min post‐desensitization (Figure 6D with P = 0.69 5‐HT, P = 0.32 sumatriptan).

Figure 6.

5‐HT_1B receptor signalling after desensitization recovers within minutes. Continuous GIRK current recordings were conducted by holding the membrane potential at −60 mV. According to the results from desensitization time experiments, 5‐HT was applied for 6 min, whereas sumatriptan was applied for 10 min; 100 nM 5‐HT was used as probe concentration. (A, B) Representative current traces of 5‐HT_1B receptor desensitization and recovery by 10 μM 5‐HT (A) and 10 μM sumatriptan (B) probing at 10 min. (C) Full recovery from desensitization occurs within 10 min for both 5‐HT and sumatriptan testing the response to 100 nM 5‐HT every 2 min of wash. (D) Recovery after 10 min wash is similar between probing every 2 min and probing at 10 min. Each data point in C and D represents mean ± SEM of at least six cells.

Sustained agonist application causes heterologous desensitization

We also examined the effect of sustained application of the 5‐HT_1B receptor agonist on the signalling of native SST receptors in AtT20 cells. In these experiments, the probe drug was SRIF (100 nM), applied before and after application of either 5‐HT or sumatriptan. Application of 5‐HT (10 μM) for 6 min reduced the subsequent SRIF response by 44 ± 6% (Figure 7A, B). When 100 nM SRIF was applied twice separated by 6 min, the second response was 12 ± 6% less than the first (Figure 7B). A 10 min application of sumatriptan caused a 43 ± 4% decrease in the second SRIF response, compared with a 6 ± 3% decrease observed for the parallel control experiments (Figure 7C, D). Superfusion of the 5‐HT_1B receptor antagonist GR127935 (100 nM) together with 5‐HT (10 μM) completely abolished the heterologous desensitization (Figure 7E, F).

Figure 7.

Prolonged 5‐HT_1B receptor stimulation causes heterologous desensitization. Heterologous desensitization was measured as level of GIRK current activation by 100 nM SRIF before and after application of 5‐HT_1B receptor agonists. (A) Typical trace of 5‐HT‐induced heterologous desensitization. (B) Desensitization of 5‐HT_1B receptors by 5‐HT (10 μM) caused significant reduction of SRIF response. (C) Typical trace showing the effect of prolonged sumatriptan application on the SRIF response. (D) The application of 10 μM sumatriptan induced significant desensitization of GIRK current activation by SRIF. Using the 5‐HT_1B receptor, specific antagonist GR127935 confirmed that 5‐HT‐induced heterologous desensitization was due to 5‐HT_1B receptor activation. (E) Typical trace of continuous recording of GIRK currents using 10 μM 5‐HT in combination with 100 nM GR127935. (F) There is no difference between the heterologous desensitization after antagonist treatment and control. Values are mean ± SEM of at least six cells per experiment. *P < 0.05, significantly different as indicated; unpaired t‐test.

Involvement of protein kinases in rapid 5‐HT_1B receptor regulation

To begin to address the potential involvement of protein kinases in the rapid desensitization of the activation of GIRK channels by 5‐HT_1B receptor agonists, we conducted a series of experiments where the broad spectrum protein kinase inhibitor staurosporine was included in the patch pipette. In these experiments, agonists were applied at least 15 min after rupture of the membrane in order to allow intracellular perfusion with the kinase inhibitor. Cells were voltage clamped at −60 mV throughout. Inclusion of staurosporine (10 μM) did not significantly affect the degree of homologous desensitization produced by prolonged application of either agonist (5‐HT or sumatriptan; Figure 8A, B). Intriguingly, staurosporine inhibited the heterologous desensitization of the SRIF response produced by activation of 5‐HT_1B receptors with 5‐HT, but not with sumatriptan (Figure 8C). Staurosporine did not affect SRIF (100 nM) currents alone. Further, staurosporine in the pipette did not inhibit the recovery of desensitization of either 5‐HT (100 nM) or SRIF (100 nM) after 6 min of 5‐HT (10 μM). The Src kinase‐specific inhibitor PP2 (10 μM) did not inhibit 5‐HT‐induced and sumatriptan‐induced heterologous desensitization of 5‐HT_1B receptors (Supporting Information Figure S3).

Figure 8.

Effects of staurosporine on 5‐HT_1B receptor desensitization and recovery. The broad spectrum kinase inhibitor staurosporine (1 μM) was used to elicit the involvement of a kinase in 5‐HT_1B receptor desensitization and recovery. (A) Staurosporine does not affect 5‐HT (10 μM)‐induced desensitization nor recovery from desensitization. (B) Sumatriptan (10 μM)‐induced desensitization and recovery from desensitization is not affected by staurosporine. (C) Staurosporine inhibits 5‐HT (10 μM)‐induced heterologous desensitization but not sumatriptan (10 μM)‐induced heterologous desensitization. Values are mean ± SEM of at least six cells per experiment. *P < 0.05, significantly different as indicated; unpaired t‐test.

Desensitization of the inhibition of glutamate release by 5‐HT_1B receptor agonists at the Calyx of Held synapse is blocked by inclusion of BAPTA in the presynaptic recording pipette (Mizutani et al., 2006). In AtT20–5HT_1B cells, the amount of desensitization produced by 100 nM 5‐HT (6 min) was unaffected by inclusion of BAPTA in the recording solution. A subsequent application of 5‐HT (100 nM) was inhibited by 37 ± 10% when EGTA (10 mM) was included in the pipette, and 38 ± 6% when BAPTA (10 mM) was in the pipette (n = 6). To demonstrate that BAPTA was active in whole cell recordings, Ca_v3.1 currents were measured in HEK 293 cells expressing human channels (Gilmore et al., 2012). Currents were evoked by stepping the cells from −100 to −30 mV every 10 s while superfusing the standard K current recording buffer. In the absence of intracellular BAPTA, Ca_v3.1 currents after 10 min stepping were 50 ± 14% of the initial current; in the presence of BAPTA, they were 112 ± 12% of the initial current. Inclusion of BAPTA prevented the Ca‐dependent rundown of the Ca_v3.1 channels.

5‐HT_1B receptors internalize in a time‐dependent manner

Desensitization of GPCR is often accompanied by rapid loss of cell surface receptors. We measured loss of 5‐HT_1B receptors from the cell surface using an immunoassay. 5‐HT and sumatriptan (10 μM) produced a time‐dependent loss of cell surface immunoreactivity (Figure 9C, D). After 40 min, cell surface 5‐HT_1B receptors were 72 ± 1% and to 70 ± 2% of untreated cells for 5‐HT and sumatriptan respectively (Figure 9A, B). Loss of cell surface receptor did not strongly correlate with desensitization of receptor signalling. Functional desensitization of 5‐HT_1B receptor coupling to K channels is largely complete after 5 min for 5‐HT and 10 min for sumatriptan, yet cell surface immunoreactivity at these times was not different from untreated cells [98 ± 4% for 5‐HT (10 μM) and 90 ± 7% for sumatriptan (10 μM)] (Figure 9A, B).

Figure 9.

Time dependence of 5‐HT_1B receptor internalization. Change in immunoreactivity after treatment of AtT20–5‐HT_1B cells with 10 μM 5‐HT (A) and 10 μM sumatriptan (B) for up to 40 min. Experiments were repeated five times with quadruplicates for each time point. Immunoreactivity is normalized to control (no drug) and values are mean ± SEM. *P < 0.05, significantly different from control (no drug); one‐way ANOVA followed by Dunnett's correction for multiple comparisons. (C, D) Confocal images illustrating the effect of 10 μM 5‐HT on 5‐HT_1B receptor distribution in AtT20–5‐HT_1B cells with HA‐like immunoreactivity at the time point of zero treatment, that is, no drug (C), and after 30 min of treatment (D).

Discussion

In this study, we have shown that 5‐HT_1B receptor coupling to GIRK channels undergoes rapid desensitization in a model system and that prolonged agonist exposure leads to only modest receptor internalization. The desensitization of GIRK channel activation was heterologous, it recovered quickly and did not seem to be associated with significant loss of receptors from the cell surface.

Despite the observations that AtT20 cells express mRNA for several G protein‐coupled 5‐HT receptors (Atwood et al., 2011), we are confident that the responses we observed were mediated by the heterologously expressed 5‐HT_1B receptors. Most importantly, we were unable to detect any responses to 5‐HT in untransfected cells, and had endogenous 5‐HT1A receptors been expressed, we would have expected them to couple to activation of GIRK channels or inhibition of I _Ca (McCorvy and Roth, 2015). We also found that 5‐HT_1B receptor activation and heterologous desensitization of SRIF‐activated responses was inhibited by the selective 5‐HT_1B/D receptor antagonist GR127935 (Pauwels and Colpaert, 1995), confirming that we were measuring the effects of 5‐HT_1B receptor activation in our experiments. 5‐HT was more potent than sumatriptan in activating GIRK channels and inducing desensitization, this is consistent with the affinity of the two agonists for 5‐HT_1B receptors (Jin et al., 1992) and previous studies comparing receptor activation (Zgombick et al., 1993; Lesage et al., 1998; Kiel et al., 2000). The coupling to GIRK channels and I _Ca by heterologously expressed 5‐HT_1B receptors is consistent with previous work showing similar effects mediated by heterologously expressed cannabinoid CB₁, μ‐ and κ‐opioid and dopamine D₃ receptors (Mackie et al., 1995; Kuzhikandathil et al., 1998; Borgland et al., 2003; Celver et al., 2004; Clayton et al., 2009).

The desensitization of 5‐HT_1B receptor activation of GIRK channels was rapid, consistent with previous studies of heterologously expressed μ‐opioid and dopamine D₃ receptors, as well as endogenously expressed SST receptors (Kuzhikandathil et al., 1998; Borgland et al., 2003; Celver et al., 2004). Intriguingly, the reversal of desensitization was also rapid, being essentially complete within 10 min. Coupled with our findings that there was minimal 5‐HT_1B receptor loss from the plasma membrane at times of maximal desensitization of receptor function, the data suggest that mechanisms involving reversible modifications of either the 5‐HT_1B receptor or GIRK channels were responsible. Our present experiments cannot definitively distinguish between these possibilities, and indeed, both could operate in parallel. However, we did find that the broad spectrum protein kinase inhibitor staurosporine significantly reduced the heterologous desensitization of SRIF responses produced by 5‐HT (but not sumatriptan) without affecting homologous desensitization. This suggests that separate mechanisms may be involved in homologous and heterologous desensitization induced by 5‐HT, if not sumatriptan.

Although the maximum responses to 5‐HT and sumatriptan were similar in each assay, 5‐HT was more potent and induced desensitization more rapidly, even at concentrations of drug (100 nM 5‐HT, 10 μM sumatriptan) that produced equivalent levels of desensitization. More interestingly, for 5‐HT, the potency to induce desensitization was only twice that for channel activation, while the difference for sumatriptan was six‐fold. These differences may reflect a higher intrinsic activity for 5‐HT than sumatriptan at 5‐HT_1B receptors, as has been suggested in other systems (Zgombick et al., 1993; Lesage et al., 1998; Kiel et al., 2000), but they could also conceivably reflect different processes recruited for receptor desensitization by the two agonists, an idea given some weight by the differential sensitivity to staurosporine of heterologous desensitization produced by the two agonists. There is evidence for agonist‐dependent patterns of μ‐opioid receptor phosphorylation (Just et al., 2013) and also distinct mechanisms of agonist‐induced μ‐opioid receptor desensitization (Kelly et al., 2008). These will be worth exploring further for the 5‐HT_1B receptor.

For many receptors, it is thought that agonist binding induces increases in GPCR phosphorylation, which leads to the recruitment of processes that drive receptor internalization (Kelly et al., 2008). Agonist‐induced internalization of 5‐HT_1B receptors in fibroblasts can be reduced by antibodies to GRK2/3, providing indirect evidence for an agonist‐induced receptor phosphorylation (Janoshazi et al., 2007). While it is often assumed that such receptor phosphorylation commits receptors to being internalized, reversal of agonist‐induced phosphorylation of both μ‐opioid and SST₂ receptors at the plasma membrane has been demonstrated, indicating that dephosphorylation of 5‐HT_1B receptors could potentially explain the reversal of agonist‐induced desensitization observed (Pöll et al., 2011; Doll et al., 2012). Unfortunately, there is no direct evidence in any system that 5‐HT_1B receptor phosphorylation is changed by agonist binding, and no evidence that phosphorylation mediates desensitization. On the contrary, it has been suggested that GSK‐3 phosphorylation is essential for maintaining activity of 5‐HT_1B receptors (Chen et al., 2009), and so acute dephosphorylation of the receptor could conceivably lead to loss of activity. However, neither 5‐HT_1B receptor desensitization nor recovery from desensitization was affected by staurosporine, a broad spectrum protein kinase inhibitor that inhibits both GSK‐3a and GSK‐3b (Karaman et al., 2008).

Clayton and colleagues (2009) identified a pathway for heterologous desensitization in AtT20 cells whereby κ‐opioid receptor activation desensitizes GIRK channel activity via p38 MAPK‐ and src‐dependent phosphorylation of Tyr12 of the Kir 3.2 subunit (Clayton et al., 2009). Intracellular perfusion of the Src inhibitor PP2 failed to modulate heterologous desensitization by either 5‐HT or sumatriptan, suggesting this mechanism may not be prominent. Heterologous desensitization is not always observed in AtT20 cells, for example, acute activation of CB₁ receptors results in a homologous loss of coupling to K channels (Cawston et al., 2013).

While activation of 5‐HT_1B receptors has been extensively studied in native cells, with the exception of one study showing that high concentrations of 5‐HT applied for several minutes produces desensitization of 5‐HT_1B receptor‐mediated inhibition of excitatory postsynaptic currents in rat brainstem slices (Mizutani et al., 2006), nothing is known about the kinetics or mechanisms underlying agonist‐induced desensitization. The desensitization of 5‐HT_1B receptors in AtT20 cells and at rat brainstem presynaptic terminals are differentially sensitive to rapid Ca chelation with BAPTA, but nothing more is known about the underlying molecular mechanisms in either preparation. The lack of information about 5‐HT_1B desensitization in brain may be because of the predominant presynaptic location for 5‐HT_1B receptors (Sari, 2004). Situations where desensitization of GPCRs mediating inhibition of neurotransmitter release is observed are uncommon (Pennock et al., 2012; Lowe and Bailey, 2015), and the reasons underlying this only beginning to be uncovered (Pennock and Hentges, 2016). Our data showed that 5‐HT concentrations, which do not produce maximum activation of GIRK channels, produced significant receptor desensitization, suggesting a reasonably close relationship between 5‐HT_1B receptor occupancy and desensitization of signalling, and implying that receptor signalling is under quite tight control. The rapid recovery of signalling on removal of agonist indicates a dynamic regulation. It remains to be established whether this rapid regulation of 5‐HT_1B receptors is observed in presynaptic compartments.

Intriguingly, the desensitization produced by sumatriptan occurred at concentrations achieved during acute administration for relief of migraine headache [~240 nM, (Duquesnoy et al., 1998)], implying that at least some loss of receptor activity may occur quite rapidly during therapy. Whether this might contribute to the ineffectiveness of triptans in a significant proportion of migraineurs is an open question (Ferrari et al., 2001), but it would be valuable to know if other triptan drugs shared the propensity of sumatriptan to desensitize receptor signalling. Finally, the apparent dissociation of receptor internalization from initial receptor desensitization suggests that several mechanisms may be involved in regulating receptor activity over periods of minutes to hours. It cannot be assumed that the processes leading to short‐term desensitization are simply the start of those that lead to receptor internalization or down‐regulation, and it may be possible to manipulate these processes independently.

Intriguingly, both 5‐HT and sumatriptan were more potent at activating GIRK channels via 5‐HT_1B receptors when cells were voltage clamped continuously at −60 mV rather than when stepped repetitively from 0 to −60 mV. This could reflect voltage dependence of agonist binding or signal transduction through the 5‐HT_1B receptor or voltage dependence of the activation of GIRK channels. Gβγ‐subunit interactions with GIRK channels are not thought to be voltage dependent (Doupnik et al., 1995), but several GPCRs including muscarinic M₂ and dopamine D₂ receptors show voltage‐dependent properties (Ben‐Chaim et al., 2003; Dekel et al., 2012; Sahlholm et al., 2012). This voltage dependence is likely to be associated with conformation changes in the receptors that could occur at the agonist binding site, in the transduction pathways between the agonist binding site and G protein interaction domain (Sahlholm, 2011). Other potential mechanisms contributing to voltage‐dependent signalling could include modulation of regulator of G‐protein signalling proteins (Jaén and Doupnik, 2006; Chuang and Chuang, 2012) or the activation of specific Gα_i and Gα_o subunits (Zhang et al., 2004).

5‐HT_1B receptors are potential targets for a number of conditions including migraine, anxiety, depression and attention‐deficit hyperactivity disorder (Sari, 2004; Guimaraes et al., 2009). In the experiments described here, we have used a model system to characterize agonist‐induced regulation of the receptor but it remains to be seen how relevant these findings are to 5‐HT_1B receptors in human brain. However, elucidation of the mechanisms underlying rapid, membrane‐delimited desensitization and the presumptive receptor trafficking‐dependent regulation of 5‐HT_1B receptor signalling are likely to provide important information for understanding how 5‐HT regulates 5‐HT_1B receptor‐dependent events, as well defining desirable properties for new drugs targeting the receptor.

Author contributions

M.H. performed and analysed most of the experiments, C.B. performed and analysed some experiments, and M.H. and M.C. designed the experiments, analysed data and wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1 5HT and SRIF activate currents with similar voltage‐dependence. The net current (pA) activated by either 5‐HT (100 nM) or SRIF (100 nM) in AtT‐20‐5HT1B cells. Data represents the difference in current between control conditions and in the presence of drug at test potentials between −90 and + 20 mV. The data is reported as mean ± s.e.m. of 4–5 cells per point. EK was 0 mV in these conditions.

Figure S2 Diagram of post hoc analysis of desensitization rate of continuous recordings of 5‐HT1B activated currents. The agonist‐induced current was measured every 30s during continuous application, expressed as a proportion of initial current and then plotted as mean ± s.e.m. of at least 6 cells. Trace and plot are from Figure 5.

Figure S3 Inclusion of the phosphatase inhibitor PP2 does not affect heterologous desensitization of SRIF responses by application of 5‐HT or sumatriptan. SRIF (100 nM) current was measured before and after superfusion of 5‐HT (10 μM, 6 min) or sumatriptan (10 μM, 10 min) in the presence of PP2 (10 μM) or DMSO in the recording pipette. Responses are expressed as a percentage of the first SRIF application and are plotted as mean ± s.e.m. of 6 cells.

Heblinski, M. , Bladen, C. , and Connor, M. (2019) Regulation of heterologously expressed 5‐HT_1B receptors coupling to potassium channels in AtT‐20 cells. British Journal of Pharmacology, 176: 451–465. 10.1111/bph.14547.