Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2015 Jul 6;172(16):4066–4077. doi: 10.1111/bph.13197

Agonist- and antagonist-induced up-regulation of surface 5-HT3A receptors

Russell A Morton 1,2, Daniel T Baptista-Hon 3, Tim G Hales 2,3, David M Lovinger 1,2,✉
PMCID: PMC4543613  PMID: 25989383

Abstract

Background and Purpose

The 5-HT3 receptor is a member of the pentameric ligand-gated ion channel family and is pharmacologically targeted to treat irritable bowel syndrome and nausea/emesis. Furthermore, many antidepressants elevate extracellular concentrations of 5-HT. This study investigates the functional consequences of exposure of recombinant 5-HT3A receptors to agonists and antagonists.

Experimental Approach

We used HEK cells stably expressing recombinant 5-HT3A receptors and the ND7/23 (mouse neuroblastoma/dorsal root ganglion hybrid) cell line, which expresses endogenous 5-HT3 receptors. Surface expression of recombinant 5-HT3A receptors, modified to contain the bungarotoxin (BTX) binding sequence, was quantified using fluorescence microscopy to image BTX-conjugated fluorophores. Whole cell voltage-clamp electrophysiology was used to measure the density of current mediated by 5-HT3A receptors.

Key Results

5-HT3A receptors were up-regulated by the prolonged presence of agonists (5-HT and m-chlorophenylbiguanide) and antagonists (MDL-72222 and morphine). The up-regulation of 5-HT3A receptors by 5-HT and MDL-72222 was time- and concentration-dependent but was independent of newly translated receptors. The phenomenon was observed for recombinant rodent and human 5-HT3A receptors and for endogenous 5-HT3 receptors in neuronal ND7/23 cells.

Conclusions and Implications

Up-regulation of 5-HT3A receptors, following exposure to either agonists or antagonists suggests that this phenomenon may occur in response to different therapeutic agents. Medications that elevate 5-HT levels, such as the antidepressant inhibitors of 5-HT reuptake and antiemetic inhibitors of 5-HT3 receptor function, may both raise receptor expression. However, this will require further investigation in vivo.

Tables of Links

TARGETS
Ligand gated ion channels
5-HT3 receptors
GABAA receptors
nACh receptors
Open in a new tab
LIGANDS
5-HT
Citalopram
mCPBG, m-chlorophenylbiguanide
Open in a new tab

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).

Introduction

The 5-HT3 receptor is a member of the cysteine (Cys)-loop pentameric ligand-gated ion channel (pLGIC) family, which includes the nicotinic acetylcholine (nACh) receptors, GABAA receptors, glycine receptors and Zn2+-activated ion channels (Barnes et al., 2009). Five 5-HT3 subunit genes have been cloned, which encode subunits termed 5-HT3A, B, C, D and E (Maricq et al., 1991; Davies et al., 1999; Karnovsky et al., 2003; Niesler et al., 2003). The 5-HT3A subunit can form functional homomeric receptors, whereas the 5-HT3B subunit requires the presence of 5-HT3A subunits to form heteromeric receptors with altered channel properties compared with homomeric 5-HT3A receptors (Davies et al., 1999; Karnovsky et al., 2003; Niesler et al., 2003). The functional consequences of the other receptor combinations are less well established (Barnes et al., 2009).

5-HT3 receptors are highly expressed in the peripheral nervous system and in the gut where they regulate the vomiting reflex and gut motility (Glatzle et al., 2002; Raybould et al., 2003). Antagonists to 5-HT3A receptors are used clinically to treat irritable bowel syndrome as well as nausea and emesis following either chemotherapy or surgical anaesthesia (Machu, 2011). 5-HT3A receptors have also been implicated in visceral pain and inflammation processes (Costall and Naylor, 2004) and may participate in some of the maladaptive effects of chronic morphine analgesia, such as hyperalgesia, tolerance (Liang et al., 2011) and dependence (Chu et al., 2009). Morphine has both competitive and non-competitive antagonist actions on 5-HT3 receptors (Fan, 1995; Baptista-Hon et al., 2012).

5-HT3A receptors are expressed throughout the CNS and are localized both pre- and post-synaptically (Kilpatrick et al., 1987; van Hooft and Vijverberg, 2000; Morales and Wang, 2002). Mice lacking the 5-HT3A receptor show sex-dependent alterations in depression and anxiety-like behaviours (Bhatnagar et al., 2004), and 5-HT3 receptors have been implicated in alcohol intake in humans and rats (Li et al., 2001). 5-HT plays a major role in mood and most clinically used anti-depressants elevate extracellular levels of this amine. Although the anti-depressant effects are not mediated by 5-HT3 receptors (Millan et al., 2003), it remains unclear whether 5-HT3A receptors are modulated by prolonged exposure to agonist.

Receptor surface expression is a highly regulated process, and changes in surface expression can produce significant alterations in downstream signalling. Sustained activation of GPCRs often leads to the internalization of surface receptors, leading, in turn, to a down-regulation of surface receptors (Ferguson, 2001). However, prolonged exposure to agonist can increase the surface expression of nACh (see Govind et al., 2009) and GABAA receptors (Eshaq et al., 2010). It has been proposed that intracellular nicotine or GABA can act as a molecular chaperone within the endoplasmic reticulum aiding the assembly of subunits, leading to an increase in functional surface receptors (Lester et al., 2009; Eshaq et al., 2010). We have previously reported that 24 h of exposure to 100 μM 5-HT produced an increase in maximal binding of radioligands to 5-HT3A receptors expressed in HEK-293 cells (Sanghvi et al., 2009). However, it is unclear whether this elevated binding reflects an increase in functional cell surface receptors. In this study, we used fluorescence microscopy and whole-cell electrophysiology to investigate the functional cell surface expression of recombinant 5-HT3A receptors stably expressed in HEK-293 and receptors native to ND7/23 (mouse neuroblastoma/rat dorsal root ganglion hybrid) cells.

Methods

Cell cultures

HEK-293 cells and ND7/23 cells were purchased from American Type Culture Collection (Manassas, VA, USA). HEK-293 cells and ND7/23 cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% calf serum and 1% penicillin/streptomycin/glutamate incubated at 37°C and in a humid environment at 5% CO2.

The mouse 5-HT3A subunit was tagged with a linker sequence and two bungarotoxin pharmatope-tags on the receptor C-terminus (5-HT3A/BBS) in pCDNA3.1. The human 5-HT3A subunit was tagged with the hemagglutinin (HA) epitope in pCDNA3.1 vector on the N-terminus. Mouse 5-HT3A/BBS and human 5-HT3A subunits were stably expressed in HEK-293 cells by transfection with polyethyleneimine (Sigma-Aldrich, St. Louis, MO, USA) (150 μM per 1 μg DNA) or lipofectamine (Invitrogen) respectively. Cells were incubated for 24–48 h at 37°C in 5% CO2 after transfection to allow for expression, yielding HEK-3A/BBS or HEK-h3A cells respectively. Approximately 1000 cells were seeded in a 10 cm dish and cultured in the presence of 400 or 1000 μg·mL−1 G418 (Gibco Invitrogen, Carlsbad, CA, USA) for HEK-3A/BBS and HEK-h3A respectively. Individual colonies from HEK-293 cells transfected with 5-HT3A/BBS were tested for expression using BTX/555 labelling (Sanghvi et al., 2009; Morton et al., 2011). For HEK-h3A cells, expression levels were determined under voltage clamp using the patch-clamp technique. In experiments investigating the effects of 5-HT pre-incubation, a saturating concentration of 5-HT (30 or 100 μM) was added from frozen stocks of 5-HT to the culture medium.

Microscopy

Surface 5-HT3A/BBS receptors were labelled with α-bungarotoxin (BTX) conjugated to Alexa fluorophores at 1 μg·mL−1 for 30 min at 4°C. Cell membranes were labelled with wheat germ agglutinin (WGA) conjugated to Alexa-555 at 10 μg·mL−1 for 30 min at 4°C (Invitrogen). Cells were fixed with 4% paraformaldehyde for 30 min at 25°C. Confocal images were collected using an inverted Zeiss LSM 510 (Zeiss USA, Thornwood, NY, USA). For green fluorescence, an argon laser was used with an excitation filter of 480/35 and an emission filter of 535/30. For red fluorescence, a HeNe laser was used with an excitation filter of 540/25 and an emission filter of 605/55. Images were acquired with an oil immersion Plan-Apochromat 63× 1.4 NA objective using LSM imager software. Laser intensity, gain and offset were set using a time zero or no agonist control and maintained throughout each experiment. The fluorescence intensities for the control conditions were comparable between replicate experiments.

Surface receptors were quantified, as previously described (Morton et al., 2011), by drawing a segmented line along the cell membrane using the WGA fluorescence as a marker in ImageJ software. The pixel intensities were averaged for the BTX/488 fluorescence along that line in three separate optical sections within each cell. The average fluorescence along that line was averaged from all three optical slices to estimate the surface expression of 5-HT3A/BBS receptors for an entire cell. All surface expression calculations were normalized to the time zero no agonist control for each individual experiment.

Electrophysiology

The whole-cell configuration of the patch-clamp technique was used to record currents mediated by 5-HT3A/BBS and 5-HT3A-HA receptors in HEK-293 cells, or 5-HT3 receptors native to ND7/23 cells. Cells were seeded at low densities in 35-mm-diameter culture dishes and visualized with a Nikon Eclipse TE300 microscope (Chiyoda, Tokyo, Japan) equipped with a Plan Fluor 20× objective with NA of 0.45. Cells were held at −60 mV and continuously superfused with extracellular solution (in mM): NaCl, 140 or 150; KCl, 2.5 or 2.8; MgCl2, 2 or 2.5; CaCl2, 1 or 2.5; glucose, 10; and HEPES, 10 (pH 7.4 with NaOH). Patch pipettes were pulled from thin wall filament-containing borosilicate capillary glass with a Sutter Flaming-Brown P-97 multi-stage puller. Patch pipettes were filled with intracellular solution containing either (in mM) CsCl, 150; BAPTA, 0.2; MgCl2, 1; Mg ATP, 3; GTP, 0.3; HEPES, 10 (pH 7.2 with CsOH) for HEK-3A/BBS; or CsCl, 140; MgCl2, 2; CaCl2, 0.1; EGTA, 1.1; and HEPES, 10 (pH 7.4 with CsOH) for HEK-h3A and ND7/23 cells. Cells were lifted clear of the dish bottom to facilitate rapid drug application. Unless otherwise stated, a maximally efficacious concentration of 5-HT was used to evoke currents. Agonist was applied locally by rapid perfusion exchange using a SF-77B fast step perfusion system (Warner Instruments, Hamden, CT, USA) that is able to achieve complete whole-cell solution exchange in ∼20 ms (Moykkynen et al., 2003). Cells were held for 2–5 min after establishing the whole-cell configuration and prior to recording to allow complete exchange of ions between the electrode and the cell interior.

Currents were amplified with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA), low-pass filtered at 1 kHz and digitized at 12.5 kHz using a Digidata 1322A interface (Molecular Devices). Data were collected, displayed and analysed using pCLAMP8.2 software. Experiments were performed at room temperature (20–24°C). No correction was made for the compensation for liquid junction potential, which had a calculated value of −5.4 mV. Pre-agonist baseline currents were leak-subtracted and peak amplitudes were measured between cursors placed at the baseline and at the end of the sweep. In all cases, mean current amplitudes are presented as current densities, by normalizing the peak amplitude to the cell capacitance.

Data analysis

All data points represent individual cells from at least three separate experiments and averaged data are presented as mean ± SEM. All statistical analyses were performed in GraphPad Prism 5.0 (San Diego, CA, USA) or SigmaPlot software (Systat Software Inc, London, UK). Statistical significance was determined by either a one-way anova with a post hoc Tukey's test or a Student t-test. P < 0.05 was considered statistically significant.

Materials

All compounds used in these experiments were supplied by Sigma-Aldrich.

Results

Prolonged agonist exposure enhances 5-HT3 receptor-mediated current density

Agonist-induced up-regulation of 5-HT3A receptors was investigated using multiple cell lines expressing both human (HEK-h3A) and mouse (HEK3A/BBS) isoforms of 5-HT3A receptors with pre-incubation with 5-HT at 100 or 30 μM respectively. The functional consequence of agonist pre-incubation was investigated by whole-cell voltage-clamp electrophysiology in HEK-293 cells stably expressing human 5-HT3A receptors (HEK-h3A) that were pre-incubated with a saturating concentration of 5-HT (100 μM) for 24 h (Figure 1A). There was a significant increase in maximal current density (pA/pF) evoked by rapid application of 5-HT (100 μM) to cells incubated in 5-HT compared with control cells. This phenomenon was also tested in rodent ND7/23 cells that endogenously express 5-HT3A receptors. Once again, 5-HT induced a significant up-regulation of the density of current mediated by 5-HT3 receptors (Figure 1B).

Figure 1.

Figure 1

Open in a new tab

Pre-incubation of HEK-h3A cells and ND7/23 cells with 5-HT for 24 h increases functional 5-HT3A receptors. (A) Representative traces from HEK-h3A cells (top panel) and current density measurements of control and 5-HT (100 μM) pre-incubation (bottom panel) (each symbol represents an individual cell n = 11). (B) Representative traces from ND7/23 cells (top panel) and current density measurements (bottom panel) following pre-incubation (100 μM) for 24 h. Individual data points represent individual cells from several experiments. Means with SEM are shown; significantly different as indicated; unpaired Student's t-test.

Prolonged agonist exposure increases surface 5-HT3A receptors in HEK cells

The surface expression of 5-HT3A receptors was investigated using a heterologous HEK-293 system that stably expresses the mouse 5-HT3A subunit tagged with the α-bungarotoxin binding sequence (BBS) on the extracellular C-terminus (HEK-3A/BBS). Our previous studies demonstrated that the addition of the BBS to 5-HT3A receptors does not alter receptor function even in the presence of bound BTX (Sanghvi et al., 2009; Morton et al., 2011). This experimental design allows us to label only the surface receptors with fluorescently conjugated BTX and measure surface expression using fluorescent microscopy. Following a 24 h pre-incubation with 5-HT (30 μM), the surface expression of 5-HT3A/BBS receptors was measured in HEK-3A/BBS cells. Surface receptors were visualized following pre-incubation with BTX/488 and the cell membrane with BTX/488 and WGA conjugated to Alexa 555 (WGA/555) respectively. Following fixation, cells were imaged using confocal microscopy. Representative images of cells with or without 24 h 5-HT pre-incubation are shown in Figure 2A. Surface receptor expression was determined by BTX/488 fluorescence, averaged along the cell surface, as previously described (Morton et al., 2011). Exposure to 5-HT for 24 h increased surface expression of 5-HT3A/BBS receptors by approximately 30% (Figure 2B). These data suggest that prolonged exposure to 5-HT can induce the up-regulation of surface 5-HT3A/BBS receptors.

Figure 2.

Figure 2

Open in a new tab

Pre-incubation of HEK-3A/BBS cells with 5-HT for 24 h increases surface 5-HT3A/BBS receptors. (A) Representative confocal images of HEK-3A/BBS cells with or without pre-incubation of 5-HT (30 μM) for 24 h. Green = BTX/Alexa488-labelled surface 5-HT3A receptors; red = WGA/Alexa555 labelling of plasma membrane; and grey = DIC image (scale bar = 10 μm). (B) Averaged BTX/488 pixel intensities along WGA/555-labelled cell membrane normalized to control cells. Data are presented as percentage of control (n = 30). Individual data points represent individual cells from several experiments. Means with SEM are shown; significantly different as indicated; unpaired Student's t-test.

Agonist-induced up-regulation in serum-free growth medium

Agonist-induced up-regulation was assessed in the absence of serum to eliminate any confounding effects of 5-HT in serum (Mothersill et al., 2010). We used a chemically defined medium CD-293 (Invitrogen), which lacks 5-HT, but maintains growth and survival of cells without serum [serum-free growth medium (SFGM)]. HEK-3A/BBS cells were incubated in SFGM for 24 h in the absence or presence of 5-HT (30 μM), and 5-HT3A surface expression was measured via fluorescence microscopy (Figure 3A and B). Incubation in SFGM alone significantly reduced the surface fluorescence of 5-HT3A/BBS receptors. However, similar to our previous data, pre-incubation with 5-HT in SFGM increased surface 5-HT3A receptors to a similar magnitude (Figure 3C). The functional consequences of SFGM with or without 30 μM 5-HT incubation were assessed with whole-cell voltage-clamp electrophysiology. Representative current traces elicited by a saturating concentration of 5-HT (30 μM) are shown in Figure 3D. Pre-incubation with 5-HT (30 μM) in SFGM significantly increased the current densities from HEK-3A/BBS cells (Figure 3D).

Figure 3.

Figure 3

Open in a new tab

Agonist-induced up-regulation of mouse 5-HT3A receptors in the absence of serum in HEK-3A/BBS cells. Representative images of HEK-3A/BBS cells in the absence (A) or presence (B) of 5-HT (30 μM) for 24 h. Green = BTX/Alexa488-labelled surface 5-HT3A receptors; red = WGA/Alexa555 labelling of plasma membrane; and grey = DIC image (scale bar = 10 μm). (C) Averaged surface fluorescence from HEK-3A/BBS cells with or without pre-incubation with 5-HT (30 μM) for 24 h. Data are presented as percentage of control (n = 30). (D) Representative traces (top panel) and current densities (bottom panel) elicited by 30 μM 5-HT following pre-incubation in SFGM with or without 5-HT (30 μM). Data are expressed as current densities (pA/pF) (n = 26). Means with SEM are shown; significantly different as indicated; unpaired Student's t-test.

Agonist-induced up-regulation is dependent on both time and concentration

HEK-3A/BBS cells were exposed to 0, 1, 3, 30 and 100 μM 5-HT for 24 h in SFGM to determine whether agonist-induced up-regulation was concentration-dependent. The concentrations used span the 5-HT3A concentration response relationship previously described (Schreiter et al., 2003). Indeed, higher concentrations of 5-HT induced a larger up-regulation (Figure 4A). There was no significant up-regulation when the cells were pre-incubated with 1 or 3 μM 5-HT. However, after pre-incubation with 30 and 100 μM 5-HT, significant up-regulation was observed. The magnitude of up-regulation was similar when treated with 30 and 100 μM 5-HT, concentrations that produce maximal receptor activation, suggesting that maximal up-regulation occurs at concentrations that produce maximal receptor occupancy.

Figure 4.

Figure 4

Open in a new tab

Agonist-induced up-regulation of 5-HT3A receptors is both concentration- and time-dependent. (A) HEK-3A/BBS cells were pre-incubated in SFGM containing varying concentrations of 5-HT. Surface mouse 5-HT3A receptors were quantified by BTX/488 fluorescence, normalized to the no 5-HT exposure condition (n = 20). (B) HEK-3A/BBS cells were pre-incubated in SFGM with or without 1 μM 5-HT for 48 h, and surface mouse 5-HT3A receptors were quantified by BTX/488 fluorescence. Data are normalized to control cells and presented as percentage of control (n = 20). (C) HEK-3A/BBS cells were pre-incubated with 5-HT (30 μM) for varying amounts of time and surface receptors were quantified by fluorescence. The last bar represents surface expression of 5-HT3A receptors in HEK-3A/BBS cells that were exposed to 5-HT for 4 h followed by 20 h in the absence of 5-HT (n = 18). Data are presented as percentage of control. (D) HEK-h3A cells were exposed to 5-HT (100 μM) for varying lengths of time and current densities were recorded (n = 10). HEK-3A/BBS (E) (n = 14) and ND7/23 (F) (n = 10) cells were exposed to 5-HT (30 or 100 μM, respectively) for 24 h and current densities were measured every 2 h following the removal of 5-HT. Statistical significance was determined using an anova with a Tukey's post hoc comparison or an unpaired Student's t-test.

Our findings suggest that the length of agonist exposure and concentration are important and led us to examine whether a lower concentration of agonist could induce up-regulation of 5-HT3A/BBS receptors after longer periods of time. HEK-3A/BBS cells were exposed to 1 μM 5-HT for 48 h in SFGM and surface receptors were quantified by fluorescence. The lower concentration of 5-HT induced a significant up-regulation of 5-HT3A/BBS receptors after 48 h albeit to a smaller magnitude compared with higher concentrations for shorter periods (Figure 4B).

The time scale of 5-HT-induced up-regulation was examined by pre-incubating HEK-3A/BBS cells in SFGM supplemented with 30 μM 5-HT for 0, 4, 8 and 24 h. Surface receptor expression was again quantified by fluorescence analysis and normalized to time zero. Similar to our previous findings, 4 h of pre-incubation with 5-HT did not result in an increase in surface 5-HT3A/BBS receptors (Figure 4C). However, 8 and 24 h of pre-incubation with 5-HT significantly up-regulated surface 5-HT3A/BBS receptors (Figure 4C). It is possible that the signalling process required to induce receptor up-regulation could occur in the first few hours, but the remaining 4–20 h may be required for synthesis of new receptors or trafficking of more receptors to the plasma membrane. To examine this possibility, we pre-incubated cells with 5-HT (30 μM) in SFGM for 4 h and then incubated in SFGM only for an additional 20 h before measuring surface 5-HT3A/BBS receptors. We were unable to detect any difference in surface receptor expression after 20 h following the 4 h exposure compared with the time zero time point (Figure 4C). The time course of up-regulation was also assessed by incubating HEK-h3A cells in 5-HT (100 μM) for varying durations and functional receptors were assessed with whole-cell electrophysiology. While there was a trend to enhanced current density at shorter exposure durations, the difference became significant (P < 0.05) only after 24 h (Figure 4D). These data suggest that optimal up-regulation requires the presence of a saturating concentration of agonist for at least 8 h.

We investigated whether surface receptor expression returned to baseline levels following removal of 5-HT from the culture media. In both HEK-3A/BBS and ND7/23 cells, pre-incubation with 5-HT, 30 and 100 μM, respectively, resulted in significant up-regulation (Figure 4E and F). Within 2 h of 5-HT washout, the current densities in both cell lines were reduced, and within 4 h, the current densities had returned to baseline levels (Figure 4E and F). These data suggest that the agonist-induced up-regulation is reversible.

Antagonist-induced up-regulation of 5-HT3/BBS receptors

A possible mechanism for the up-regulation of 5-HT3A receptors is that the persistent opening of the receptor channel leads to an influx of Ca2+ that could activate downstream signalling and transcriptional mechanisms, resulting in an increase in 5-HT3A receptor expression. If the up-regulation were activity-dependent, then a competitive antagonist would be expected to inhibit up-regulation. HEK-3A/BBS cells were pre-incubated in SFGM with 5-HT (30 μM) and MDL-72222 (1 μM), a 5-HT3-specific competitive antagonist, for 24 h before surface expression was assessed by fluorescence. Interestingly, MDL-72222 did not block the 5-HT-induced up-regulation (Figure 5A). In fact, 24 h of exposure to 1 μM MDL-72222 alone caused a smaller but significant increase in surface expression of 5-HT3A/BBS receptors (Figure 5A). By contrast, picrotoxin, a non-competitive antagonist of 5-HT3A receptors (Das et al., 2003), did not increase surface expression of 5-HT3A/BBS receptors after 24 h of exposure (Figure 5B), consistent with the hypothesis that up-regulation requires occupancy of the orthosteric binding site.

Figure 5.

Figure 5

Open in a new tab

Up-regulation of 5-HT3A receptors was not blocked by MDL-72222 and was induced by either MDL-72222 or morphine. Surface receptors were quantified by BTX/488 fluorescence and normalized to control cells in HEK-3A/BBS cells that were pre-incubated in SFGM for 24 h with 30 μM 5-HT (n = 20), 30 μM 5-HT + 1 μM MDL-72222 (n = 20), 1 μM MDL-72222 (n = 20) (A) or with 30 μM 5-HT, and 100 μM picrotoxin (Pic) (B) (n = 20). (C) Mouse 5-HT3A/BBS currents were elicited by 5-HT (30 μM) following pre-incubation with or without MDL-72222 (n = 21) and expressed as current densities. (D) Current densities from HEK-h3A cells pre-incubated with or without 10 μM morphine for 24 h (n = 9). HEK-3A/BBS cells were exposed to varying concentrations of MDL-72222 for 24 h and surface receptors were quantified by fluorescence. Representative images of HEK-3A/BBS cells in the presence or absence of MDL-72222 (1 μM) are shown in (E) and averaged data in (F). Means with SEM are shown; significantly different as indicated; anova with Tukey's post hoc comparison or unpaired Student's t-test.

The antagonist modulation of functional receptors was also assessed with whole–cell voltage-clamp electrophysiology. HEK-3A/BBS cells were exposed for 24 h to MDL-72222 in SFGM. Using a Mann–Whitney t-test, we determined that MDL-72222 significantly increased the current density in HEK-3A/BBS cells, suggesting that the presence of MDL-72222 alone induces up-regulation of functional surface receptors (Figure 5C). We also tested whether morphine, another antagonist of 5-HT3A receptors, which has competitive and non-competitive actions (Fan, 1995; Baptista-Hon et al., 2012), could induce up-regulation of 5-HT3A receptors using the HEK-h3A cell line. Exposure of HEK-h3A cells to 10 μM morphine for 24 h also induced up-regulation of 5-HT3A receptors (Figure 5D).

Receptor occupancy per se may also play a role in the antagonist-induced up-regulation. To test this possibility, HEK-3A/BBS cells were exposed to 0.1, 0.3 or 1 μM MDL-72222 for 24 h and surface expression was assessed by fluorescence. As observed with receptor agonists, the antagonist-induced up-regulation was concentration-dependent (Figure 5E and F). Lower concentrations of MDL-72222 induced a smaller but significant up-regulation of 5-HT3A receptors.

Agonist- and antagonist-induced up-regulation is independent of protein translation

We tested if the agonist-induced up-regulation was due to translation of new receptors by blocking protein translation with cycloheximide (CHX). HEK-3A/BBS cells were pre-incubated in SFGM without or with agonist in the absence or presence of 7 μM CHX for 24 h. This concentration of CHX has been shown to block translation-dependent up-regulation of dopamine receptors in HEK cells (Filtz et al., 1994). Surface 5-HT3A/BBS receptors were analysed via fluorescence, and we found that CHX alone did not alter the surface expression of 5-HT3A/BBS receptors compared with control. Furthermore, the presence of CHX did not block the agonist-induced up-regulation (Figure 6A). These data suggest that the up-regulation of surface receptors induced by 5-HT is not due to newly translated receptors, but rather receptors previously translated and stored intracellularly.

Figure 6.

Figure 6

Open in a new tab

Up-regulation is independent of receptor translation. (A) HEK-3A/BBS cells were pre-incubated in SFGM alone or SFGM containing 5-HT (30 μM). The same conditions were also performed in the presence of 7 μM CHX. Surface 5-HT3A receptors were quantified by BTX/488 fluorescence normalized to control cells (n = 20). Means with SEM are shown; significantly different as indicated; unpaired Student's t-test. (B) Surface 5-HT3A/BBS receptors in HEK-3A/BBS cells were labelled with BTX/488, cells were fixed, permeabilized and intracellular receptors were labelled with BTX/555. Representative images of HEK-3A/BBS cells are shown (green = BTX/488-labelled surface receptors; red = BTX/555-labelled intracellular receptors). Scale bar = 10 μm.

The presence of intracellular 5-HT3A/BBS receptors was examined in HEK-3A/BBS cells by labelling surface 5-HT3A/BBS receptors with BTX/488, fixing and permeabilizing the membranes, then labelling all intracellular receptors with BTX/555. A representative image is shown in Figure 6B, indicating that there is a significant intracellular pool of 5-HT3A/BBS receptors. Together, these data suggest that agonist induces the assembly and/or trafficking of intracellular receptors to the cell surface.

Active transport of 5-HT is not necessary for up-regulation

It has been suggested that nicotine and GABA cause the up-regulation of nACh and GABAA receptors, respectively, by entering the cell and acting as molecular chaperones within the ER enhancing subunit assembly. Unlike nicotine, 5-HT and GABA cannot cross the cell membrane without transporters. GABAA receptor up-regulation may be induced by GABA being synthesized by the cell, or by being actively transported across its membrane (Eshaq et al., 2010). We examined 5-HT-induced up-regulation in the presence of a 5-HT transporter inhibitor. Citalopram (1 μM) had no effect on the 5-HT-induced up-regulation of 5-HT3A/BBS receptors (Figure 7). We also pre-incubated HEK-3A/BBS cells with m-chlorophenylbiguanide (mCPBG), a selective 5-HT3 receptor agonist. Due to the difference in chemical structure, it is unlikely to be transported by any of the monoamine transporters. Like 5-HT, mCPBG in SFGM resulted in the up-regulation of surface 5-HT3A/BBS receptors after 24 h (Figure 7), demonstrating that transport is not required for the increase in receptor expression.

Figure 7.

Figure 7

Open in a new tab

Active transport of 5-HT is not required for the up-regulation of 5-HT3A/BBS receptors. HEK-3A/BBS cells were pre-incubated for 24 h in SFGM alone (CTR) (n = 30), SFGM + 30 μM 5-HT + 1 μM citalopram (Cit) (n = 20) or 1 μM mCPBG (n = 35). Surface 5-HT3A/BBS receptors were quantified by BTX/488 fluorescence and normalized to control cells. Means with SEM are shown; significantly different as indicated; anova with Tukey's post hoc comparison.

Discussion

In this study, we have demonstrated that recombinant and endogenous 5-HT3A receptors were up-regulated in the prolonged presence of the endogenous agonist 5-HT, or the 5-HT3A receptor specific agonist mCPBG. The up-regulation of 5-HT3A receptors was dependent upon the concentration and duration of agonist exposure. Furthermore, antagonists (MDL-72222 and morphine) also induced a significant up-regulation of functional surface receptors. Up-regulation was independent of receptor translation but was most likely generated from intracellular pools of receptors. Furthermore, our findings suggest that the up-regulation of 5-HT3A receptors is not mediated by the molecular chaperone mechanism.

Prolonged exposure to agonists has been shown to increase the surface expression of other members of the Cys-loop pLGIC family. Nicotine, an agonist for nACh receptors, is well known to increase the surface expression of nACh receptors in cultured cells (Bencherif et al., 1995), rodents (Schwartz and Kellar, 1985; Wonnacott, 1990) and human smokers (Benwell et al., 1988; Perry et al., 1999; Govind et al., 2009). Recently, Eshaq et al. (2010) showed that the GABAA receptors expressed in HEK cells are also up-regulated in the presence of GABA. Our results demonstrate that recombinant 5-HT3A receptors are also up-regulated in the prolonged presence of agonists or antagonists. These findings are consistent with our previous observation that exposure to 5-HT (100 μM) for 24 h leads to approximately a threefold increase in total binding to cells stably expressing 5-HT3 receptors (Sanghvi et al., 2009). Furthermore, Eshaq et al. (2010) demonstrated that 5-HT increases surface levels of 5-HT3A receptors, GABA was unable to up-regulate 5-HT3 receptors and 5-HT was unable to up-regulate GABAA receptors, suggesting that this phenomenon requires a ligand that binds to the appropriate orthosteric site.

Maximal up-regulation of 5-HT3A/BBS receptors by 5-HT (30 or 100 μM) occurred within 24 h. A smaller up-regulation could be achieved in response to lower 5-HT concentrations over a longer time course. Similarly, the up-regulation of nACh and GABAA receptors requires saturating concentrations of agonist (Peng et al., 1994; Wang et al., 1998; Kuryatov et al., 2005; Vallejo et al., 2005; Eshaq et al., 2010). Up-regulation of nACh receptors by nicotine requires the presence of nicotine for at least 6–8 h (Wang et al., 1998; Kuryatov et al., 2005; Vallejo et al., 2005), similar to the time course presented here for 5-HT3A receptors. It remains unclear if the up-regulation of GABAA receptors is dependent upon agonist concentration. However, GABAA receptors were up-regulated with 1 h of GABA exposure followed by 5 h in the absence of agonist (Eshaq et al., 2010). This differs from the results with nACh and 5-HT3A receptors, which require the presence of agonist for several hours of exposure. This difference could be due to the proposed requirement for transport or synthesis of GABA intracellularly.

Up-regulation of 5-HT3A/BBS receptors by 5-HT was not blocked by MDL-72222, and MDL-72222 alone resulted in an increase in functional receptors. Furthermore, morphine was capable of inducing the up-regulation of functional 5-HT3A receptors. In contrast, the non-competitive antagonist picrotoxin did not induce up-regulation of 5-HT3A receptors. Morphine has competitive and non-competitive modes of inhibition at 5-HT3A receptors, (Baptista-Hon et al., 2012) and it is likely that both sites will be occupied in the absence of added 5-HT. As 5-HT and the competitive antagonist MDL-72222 both up-regulate functional receptors, it is likely that the ability of morphine to increase 5-HT3A receptor expression is also mediated through the orthosteric site. Recent data suggest that 5-HT3A receptors are involved in opioid-induced hyperalgesia, tolerance (Liang et al., 2011) and dependence (Chu et al., 2009). Morphine-induced up-regulation of functional 5-HT3A receptors may contribute to this effect. Antagonist-induced up-regulation has also been shown for GABAA receptors with a competitive antagonist (Eshaq et al., 2010). Mutations of the agonist-binding site of GABAA receptors completely blocked agonist-induced up-regulation (Eshaq et al., 2010). Competitive antagonists for nACh receptors are unable to induce up-regulation; however, non-competitive antagonists significantly up-regulate nACh receptors (Peng et al., 1994; Kuryatov et al., 2005). It could be that the non-competitive antagonists for nACh receptors are more effective than competitive antagonists at promoting subunit assembly in the endoplasmic reticulum.

It has been well established that agonist-induced up-regulation of both nACh receptors and GABAA receptors is independent of receptor translation (Peng et al., 1994; Wang et al., 1998; Kuryatov et al., 2005; Lester et al., 2009; Eshaq et al., 2010). Many hypotheses for the mechanism of the translation-independent up-regulation of nACh receptors have been put forward, including alterations in surface turnover (Peng et al., 1994) and the enhancement of subunit assembly by nicotine (chaperone effect) in the endoplasmic reticulum (Kuryatov et al., 2005; Lester et al., 2009). Furthermore, GABAA receptors are also thought to be up-regulated by a chaperone mechanism because up-regulation could be induced when cells synthesized GABA or expressed the GABA transporter (Eshaq et al., 2010). Our results are in agreement that the up-regulation of 5-HT3A receptors is independent of translation. However, our data also suggest that 5-HT3A receptor up-regulation is not due the transport of 5-HT across the cell membrane to act as an intracellular chaperone. Structural studies with the ACh-binding protein have shown that there are slight structural differences around the binding site between agonist bound and competitive antagonist-bound proteins (Shahsavar et al., 2012). Furthermore, there are distinct conformational changes in the extracellular domain of glycine receptors between activation and desensitization (Wang and Lynch, 2011). Our previous studies have shown that 5-HT3A receptors are constitutively internalized in HEK cells (Morton et al., 2011). It is possible that conformational changes due to agonist or antagonist binding in the 5-HT3A receptors could lead to changes in the rate of constitutive internalization. Our data indicated that the constitutive internalization of 5-HT3A receptors in HEK cells results in a near complete turnover of surface receptors in approximately 30 min at 37°C (Morton et al., 2011); therefore, even small alterations in receptor internalization could lead to significant changes in surface expression after 24 h. It remains unclear how any of these extracellular changes would affect the intracellular domains of the receptor leading to changes in internalization. However, the structure of the 5-HT3A receptor is now available and may give insight into any structural differences between the ligand bound and unbound states (Hassaine et al., 2014) and if such structural changes could alter receptor internalization.

In conclusion, we demonstrate here that pre-incubation with agonists or morphine will up-regulate 5-HT3A/BBS receptors. Furthermore, pre-incubation with agonist induced up-regulation of endogenous 5-HT3A receptors in a neuroblastoma/dorsal root ganglia hybrid cell line. Agonist/antagonist up-regulation could have implications for patients using 5-HT selective re-uptake inhibitors for depression, ondansetron for post-operative nausea and emesis, or patients using morphine for chronic pain. However, further studies are needed to confirm the up-regulation in neurons and other cell types.

Acknowledgments

The work of D. T. B.-H. and T.G. H. was supported by a grant from Tenovus Scotland. The work of R. A. M. and D. M. L. was supported by the Division of Intramural Clinical and Biological Research, NIAAA.

Glossary

BTX

α-bungarotoxin

WGA

wheat germ agglutinin

CHX

cycloheximide

HEK-3A/BBS

HEK-293 cells stably expressing the mouse 5-HT3A subunit tagged with the α-bungarotoxin binding sequence (BBS) on the extracellular C-terminus

HEK-h3A

HEK-293 cells stably expressing human 5-HT3A receptors

mCPBG

m-Chlorophenylbiguanide

ND7/23

mouse neuroblastoma/rat dorsal root ganglion neuron hybrid

pLGICs

pentameric ligand-gated ion channels

SFGM

serum-free growth media

Author contributions

R. A. M. substantially contributed to the concept or design of the experiments, acquisition, analysis, interpretation of data and drafting of the manuscript. D. T. B.-H. substantially contributed to the acquisition, analysis, interpretation of data and drafting of the manuscript. T. G. H. substantially contributed to the concept or design of the experiments, interpretation of data and drafting of the manuscript. D. M. L. substantially contributed to the concept or design of the experiments, interpretation of data and drafting of the manuscript.

Conflict of interest

None.

References

  1. Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Catterall WA, et al. CGTP Collaborators. The Concise Guide to PHARMACOLOGY 2013/14: Ion Channels. Br J Pharmacol. 2013;170:1607–1651. doi: 10.1111/bph.12447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baptista-Hon DT, Deeb TZ, Othman NA, Sharp D, Hales TG. The 5-HT3B subunit affects high-potency inhibition of 5-HT3 receptors by morphine. Br J Pharmacol. 2012;165:693–704. doi: 10.1111/j.1476-5381.2011.01582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnes NM, Hales TG, Lummis SC, Peters JA. The 5-HT3 receptor – the relationship between structure and function. Neuropharmacology. 2009;56:273–284. doi: 10.1016/j.neuropharm.2008.08.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bencherif M, Fowler K, Lukas RJ, Lippiello PM. Mechanisms of up-regulation of neuronal nicotinic acetylcholine receptors in clonal cell lines and primary cultures of fetal rat brain. J Pharmacol Exp Ther. 1995;275:987–994. [PubMed] [Google Scholar]
  5. Benwell ME, Balfour DJ, Anderson JM. Evidence that tobacco smoking increases the density of (−)-[3H]nicotine binding sites in human brain. J Neurochem. 1988;50:1243–1247. doi: 10.1111/j.1471-4159.1988.tb10600.x. [DOI] [PubMed] [Google Scholar]
  6. Bhatnagar S, Nowak N, Babich L, Bok L. Deletion of the 5-HT3 receptor differentially affects behavior of males and females in the Porsolt forced swim and defensive withdrawal tests. Behav Brain Res. 2004;153:527–535. doi: 10.1016/j.bbr.2004.01.018. [DOI] [PubMed] [Google Scholar]
  7. Chu LF, Liang DY, Li X, Sahbaie P, D'Arcy N, Liao G, et al. From mouse to man: the 5-HT3 receptor modulates physical dependence on opioid narcotics. Pharmacogenet Genomics. 2009;19:193–205. doi: 10.1097/FPC.0b013e328322e73d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Costall B, Naylor RJ. 5-HT3 receptors. Curr Drug Targets CNS Neurol Disord. 2004;3:27–37. doi: 10.2174/1568007043482624. [DOI] [PubMed] [Google Scholar]
  9. Das P, Bell-Horner CL, Machu TK, Dillon GH. The GABA(A) receptor antagonist picrotoxin inhibits 5-hydroxytryptamine type 3A receptors. Neuropharmacology. 2003;44:431–438. doi: 10.1016/s0028-3908(03)00032-7. [DOI] [PubMed] [Google Scholar]
  10. Davies PA, Pistis M, Hanna MC, Peters JA, Lambert JJ, Hales TG, et al. The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature. 1999;397:359–363. doi: 10.1038/16941. [DOI] [PubMed] [Google Scholar]
  11. Eshaq RS, Stahl LD, Stone R, 2nd, Smith SS, Robinson LC, Leidenheimer NJ. GABA acts as a ligand chaperone in the early secretory pathway to promote cell surface expression of GABAA receptors. Brain Res. 2010;1346:1–13. doi: 10.1016/j.brainres.2010.05.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fan P. Nonopioid mechanism of morphine modulation of the activation of 5-hydroxytryptamine type 3 receptors. Mol Pharmacol. 1995;47:491–495. [PubMed] [Google Scholar]
  13. Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev. 2001;53:1–24. [PubMed] [Google Scholar]
  14. Filtz TM, Guan W, Artymyshyn RP, Facheco M, Ford C, Molinoff PB. Mechanisms of up-regulation of D2L dopamine receptors by agonists and antagonists in transfected HEK-293 cells. J Pharmacol Exp Ther. 1994;271:1574–1582. [PubMed] [Google Scholar]
  15. Glatzle J, Sternini C, Robin C, Zittel TT, Wong H, Reeve JR, Jr, et al. Expression of 5-HT3 receptors in the rat gastrointestinal tract. Gastroenterology. 2002;123:217–226. doi: 10.1053/gast.2002.34245. [DOI] [PubMed] [Google Scholar]
  16. Govind AP, Vezina P, Green WN. Nicotine-induced upregulation of nicotinic receptors: underlying mechanisms and relevance to nicotine addiction. Biochem Pharmacol. 2009;78:756–765. doi: 10.1016/j.bcp.2009.06.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hassaine G, Deluz C, Grasso L, Wyss R, Tol MB, Hovius R, et al. X-ray structure of the mouse serotonin 5-HT3 receptor. Nature. 2014;512:276–281. doi: 10.1038/nature13552. [DOI] [PubMed] [Google Scholar]
  18. van Hooft JA, Vijverberg HP. 5-HT(3) receptors and neurotransmitter release in the CNS: a nerve ending story? Trends Neurosci. 2000;23:605–610. doi: 10.1016/s0166-2236(00)01662-3. [DOI] [PubMed] [Google Scholar]
  19. Karnovsky AM, Gotow LF, McKinley DD, Piechan JL, Ruble CL, Mills CJ, et al. A cluster of novel serotonin receptor 3-like genes on human chromosome 3. Gene. 2003;319:137–148. doi: 10.1016/s0378-1119(03)00803-5. [DOI] [PubMed] [Google Scholar]
  20. Kilpatrick GJ, Jones BJ, Tyers MB. Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding. Nature. 1987;330:746–748. doi: 10.1038/330746a0. [DOI] [PubMed] [Google Scholar]
  21. Kuryatov A, Luo J, Cooper J, Lindstrom J. Nicotine acts as a pharmacological chaperone to up-regulate human alpha4beta2 acetylcholine receptors. Mol Pharmacol. 2005;68:1839–1851. doi: 10.1124/mol.105.012419. [DOI] [PubMed] [Google Scholar]
  22. Lester HA, Xiao C, Srinivasan R, Son CD, Miwa J, Pantoja R, et al. Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery. AAPS J. 2009;11:167–177. doi: 10.1208/s12248-009-9090-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li TK, Spanagel R, Colombo G, McBride WJ, Porrino LJ, Suzuki T, et al. Alcohol reinforcement and voluntary ethanol consumption. Alcohol Clin Exp Res. 2001;25:117S–126S. doi: 10.1097/00000374-200105051-00021. [DOI] [PubMed] [Google Scholar]
  24. Liang DY, Li X, Clark JD. 5-Hydroxytryptamine type 3 receptor modulates opioid-induced hyperalgesia and tolerance in mice. Anesthesiology. 2011;114:1180–1189. doi: 10.1097/ALN.0b013e31820efb19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Machu TK. Therapeutics of 5-HT3 receptor antagonists: current uses and future directions. Pharmacol Ther. 2011;130:338–347. doi: 10.1016/j.pharmthera.2011.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maricq AV, Peterson AS, Brake AJ, Myers RM, Julius D. Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel. Science. 1991;254:432–437. doi: 10.1126/science.1718042. [DOI] [PubMed] [Google Scholar]
  27. Millan MJ, Veiga S, Girardon S, Brocco M. Blockade of serotonin 5-HT1B and 5-HT2A receptors suppresses the induction of locomotor activity by 5-HT reuptake inhibitors, citalopram and fluvoxamine, in NMRI mice exposed to a novel environment: a comparison to other 5-HT receptor subtypes. Psychopharmacology (Berl) 2003;168:397–409. doi: 10.1007/s00213-003-1389-y. [DOI] [PubMed] [Google Scholar]
  28. Morales M, Wang SD. Differential composition of 5-hydroxytryptamine3 receptors synthesized in the rat CNS and peripheral nervous system. J Neurosci. 2002;22:6732–6741. doi: 10.1523/JNEUROSCI.22-15-06732.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Morton RA, Luo G, Davis MI, Hales TG, Lovinger DM. Fluorophore assisted light inactivation (FALI) of recombinant 5-HT(3)A receptor constitutive internalization and function. Mol Cell Neurosci. 2011;47:79–92. doi: 10.1016/j.mcn.2011.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mothersill C, Saroya R, Smith RW, Singh H, Seymour CB. Serum serotonin levels determine the magnitude and type of bystander effects in medium transfer experiments. Radiat Res. 2010;174:119–123. doi: 10.1667/RR2036.1. [DOI] [PubMed] [Google Scholar]
  31. Moykkynen T, Korpi ER, Lovinger DM. Ethanol inhibits alpha-amino-3-hydyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function in central nervous system neurons by stabilizing desensitization. J Pharmacol Exp Ther. 2003;306:546–555. doi: 10.1124/jpet.103.050666. [DOI] [PubMed] [Google Scholar]
  32. Niesler B, Frank B, Kapeller J, Rappold GA. Cloning, physical mapping and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E. Gene. 2003;310:101–111. doi: 10.1016/s0378-1119(03)00503-1. [DOI] [PubMed] [Google Scholar]
  33. Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP, et al. NC-IUPHAR. The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledge base of drug targets and their ligands. Nucl Acids Res. 2014;42(Database Issue):D1098–D1106. doi: 10.1093/nar/gkt1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Peng X, Gerzanich V, Anand R, Whiting PJ, Lindstrom J. Nicotine-induced increase in neuronal nicotinic receptors results from a decrease in the rate of receptor turnover. Mol Pharmacol. 1994;46:523–530. [PubMed] [Google Scholar]
  35. Perry DC, Davila-Garcia MI, Stockmeier CA, Kellar KJ. Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies. J Pharmacol Exp Ther. 1999;289:1545–1552. [PubMed] [Google Scholar]
  36. Raybould HE, Glatzle J, Robin C, Meyer JH, Phan T, Wong H, et al. Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying. Am J Physiol Gastrointest Liver Physiol. 2003;284:G367–G372. doi: 10.1152/ajpgi.00292.2001. [DOI] [PubMed] [Google Scholar]
  37. Sanghvi M, Hamouda AK, Davis MI, Morton RA, Srivastava S, Pandhare A, et al. Hydrophobic photolabeling studies identify the lipid-protein interface of the 5-HT3A receptor. Biochemistry. 2009;48:9278–9286. doi: 10.1021/bi901208j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schreiter C, Hovius R, Costioli M, Pick H, Kellenberger S, Schild L, et al. Characterization of the ligand-binding site of the serotonin 5-HT3 receptor: the role of glutamate residues 97, 224, and 235. J Biol Chem. 2003;278:22709–22716. doi: 10.1074/jbc.M301801200. [DOI] [PubMed] [Google Scholar]
  39. Schwartz RD, Kellar KJ. In vivo regulation of [3H]acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J Neurochem. 1985;45:427–433. doi: 10.1111/j.1471-4159.1985.tb04005.x. [DOI] [PubMed] [Google Scholar]
  40. Shahsavar A, Kastrup JS, Nielsen EO, Kristensen JL, Gajhede M, Balle T. Crystal structure of Lymnaea stagnalis AChBP complexed with the potent nAChR antagonist DHbetaE suggests a unique mode of antagonism. PLoS ONE. 2012;7:e40757. doi: 10.1371/journal.pone.0040757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vallejo YF, Buisson B, Bertrand D, Green WN. Chronic nicotine exposure upregulates nicotinic receptors by a novel mechanism. J Neurosci. 2005;25:5563–5572. doi: 10.1523/JNEUROSCI.5240-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang F, Nelson ME, Kuryatov A, Olale F, Cooper J, Keyser K, et al. Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells. J Biol Chem. 1998;273:28721–28732. doi: 10.1074/jbc.273.44.28721. [DOI] [PubMed] [Google Scholar]
  43. Wang Q, Lynch JW. Activation and desensitization induce distinct conformational changes at the extracellular-transmembrane domain interface of the glycine receptor. J Biol Chem. 2011;286:38814–38824. doi: 10.1074/jbc.M111.273631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wonnacott S. The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol Sci. 1990;11:216–219. doi: 10.1016/0165-6147(90)90242-z. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES