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Originally published In Press as doi:10.1074/jbc.M303584200 on June 5, 2003

J. Biol. Chem., Vol. 278, Issue 36, 34150-34157, September 5, 2003
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Modulation of 5-HT3 Receptor-mediated Response and Trafficking by Activation of Protein Kinase C*

Hui Sun, Xian-Qun Hu, Edgar M. Moradel, Forrest F. Weight and Li Zhang {ddagger}

From the Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892-8115

Received for publication, April 7, 2003 , and in revised form, May 30, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of neurotransmitter-gated membrane ion channels by protein kinase C (PKC) has been the subject of a number of studies. However, less is known about PKC modulation of the serotonin type 3 (5-HT3) receptor, a ligand-gated membrane ion channel that can mediate fast synaptic transmission in the central and peripheral nervous system. Here, we show that PKC potentiated 5-HT3 receptor-mediated current in Xenopus oocytes expressing 5-HT3A receptors and mouse N1E-115 neuroblastoma cells. In addition, using a specific antibody directed to the extracellular N-terminal domain of the 5-HT3A receptor, treatment with the PKC activator, 4{beta}-phorbol 12-myristate 13-acetate (PMA), significantly increased surface immunofluorescence. PKC also increased the amount of 5-HT3A receptor protein in the cell membrane without affecting the amount receptor protein in the total cell extract. The magnitude of PMA potentiation of 5-HT3A receptor-mediated responses is correlated with the magnitude of PMA enhancement of the receptor abundance in the cell surface membrane. PMA potentiation is unlikely to occur via direct phosphorylation of the 5-HT3A receptor protein since the potentiation was not affected by point mutation of each of the putative sites for PKC phosphorylation. However, preapplication of phalloidin, which stabilizes the actin polymerization, significantly inhibited PMA potentiation of 5-HT-activated responses in both N1E-115 cells and oocytes expressing 5-HT3A receptors. On the other hand, latrunculin-A, which destabilizes actin cytoskeleton, enhanced the PMA potentiation of 5-HT3A receptors. The observations suggest that PKC can modulate 5-HT3A receptor function and trafficking through an F-actin-dependent mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The serotonin type 3 (5-HT3)1 receptor is a member of a ligand-gated ion channel (LGIC) supergene family including {gamma}-aminobutyric acid type A (GABAA), glycine, and nicotinic acetylcholine receptors (1). Although molecular studies have identified two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B (1, 2), homomeric 5-HT3A receptors are thought to be the dominant functional form in the central nervous system (3). The 5-HT3A receptors are differentially distributed in a number of important brain areas including the hippocampus, nucleus of the solitary tract, nucleus accumbens, substantia nigra, and ventral tegmental area (4–6). In some of these brain regions, 5-HT3 receptors have been found to modulate the release of neurotransmitters such as dopamine and GABA (7). In addition, 5-HT3 receptors are thought to be involved in reward mechanisms of some drugs of abuse and have been proposed to be involved in central nervous system phenomena such as anxiety, psychosis, nociception (8), and cognitive function (9).

Modulation of ligand-gated ion channel function by protein kinases has been the focus of a number of previous studies. For example, such studies have shown that activation of PKC can modulate glycine, GABAA, and N-methyl-D-aspartate (NMDA) receptors in various types of neurons and in cell lines expressing these receptors (10–15). Regulation of some of these receptors by PKC is thought to be important for synaptic modulation and neuronal plasticity. PKC has been found to induce internalization of GABAA receptors (14, 16) and, on the other hand, to promote trafficking of NMDA receptors to the cell surface (15). The regulation of receptor trafficking by activation of PKC is thought, at least in part, to contribute to PKC-induced alteration in the function of these receptors. Although PKC has been shown to regulate the phosphorylation of GABAA and NMDA receptor proteins (10, 17), it appears unlikely that the functional modulation of these receptors by PKC results from direct phosphorylation of the receptor proteins (14, 18). Rather, PKC is thought to phosphorylate receptor-associated proteins, which modulate receptor trafficking through intracellular signaling pathways (15, 18). Recent studies indicate that the actin cytoskeleton may play an important role in synaptic modulation and plasticity by anchoring, clustering, and targeting several LGICs (19–23). Activation of PKC by phorbol esters is proposed to disorder the dynamics of the actin filament network by removing a barrier to vesicle trafficking and docking, thereby promoting exocytosis (24–26). Consistent with this hypothesis, potentiation of NMDA receptor-mediated responses by activation of PKC is dependent on dynamic cycling of actin polymerization/depolymerization (27, 28).

Of all the LGICs, modulation of 5-HT3 receptor function by PKC has received relatively little attention. Previous studies reported the application of phorbol esters, activators of PKC, can potentiate 5-HT-activated current in Xenopus oocytes expressing 5-HT3A receptors (29), modulate the desensitization of 5-HT-activated current in HEK-293 cells expressing 5-HT3A receptors (30), and regulate the probability of occurrence of certain conductance levels of 5-HT-activated single channel currents in mouse neuroblastoma, N1E-115 cells (31). In addition, a recent study suggests that a tyrosine kinase may be involved in PMA potentiation of 5-HT3A receptors expressed in Xenopus oocytes (32). Another recent study indicates that 5-HT3A receptors are colocalized and coclustered with F-actin in NG108–15 cells, hippocampal neurons and in cells transiently transfected with cloned 5-HT3A receptors (33), suggesting that F-actin might be involved in the regulation of 5-HT3A receptor targeting and clustering.

Nevertheless, the molecular and cellular mechanisms by which PKC modulates 5-HT3 receptor function have not been determined. To address this question, we have used various approaches in this study and found that PKC can modulate 5-HT3 receptor function and receptor trafficking in N1E-115 cells and in Xenopus oocytes expressing 5-HT3A receptors. We have also found that PKC modulation of 5-HT3A receptor function is likely to occur via an F-actin-dependent mechanism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—Point mutations of a cloned mouse 5-HT3A receptor were introduced using a QuikChange site-directed mutagenesis kit (Stratagene). The authenticity of the DNA fragments that flank the mutation site was confirmed by double-strand DNA sequencing using an ABI Prism 377 automatic DNA sequencer (Applied Biosystems).

Preparation of Complementary RNAs and Expression of Receptors— Complementary RNAs were synthesized in vitro from linearized template cDNAs with a mMACHINE RNA transcription kit from Ambion Inc. The quality and the sizes of synthesized complementary RNAs were confirmed by denatured RNA agarose gels. Mature female Xenopus laevis frogs were anesthetized by submersion in 0.2% 3-aminobenzoic acid ethyl ester (Sigma), and a group of oocytes was surgically excised. The oocytes were separated, and the follicular cell layer was removed by treatment with type I collagenase (Roche Applied Science) for 2 h at room temperature. Each oocyte was injected with a total of 20 ng of RNA in 20 nl of diethyl pyrocarbonate-treated water and was incubated at 19 °C in modified Barth's solution (MBS: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 2.0 mM CaCl2, 0.8 mM MgSO4, 10 mM HEPES, pH 7.4).

Recording from Xenopus Oocytes—After incubation for 2–5 days, the oocytes were studied at room temperature (20–22 °C) in a 90-µl chamber. The oocytes were superfused with MBS at a rate of ~6 ml/min. Agonists and chemical agents were diluted in the bathing solution and applied to the oocytes for a specified time using a solenoid valve-controlled superfusion system. The agonist and chemical agents were diluted either directly in bathing solution or dissolved in dimethyl sulfoxide (Me2SO) before the dilution. The final Me2SO concentration was less than 0.1%, which did not produce detectable effects on the 5-HT-activated current under our experimental condition. Membrane currents were recorded by two-electrode voltage-clamp at a holding potential of –70 mV using a Gene Clamp 500 amplifier (Axon Instruments, Inc.). The recording microelectrodes were filled with 3 M KCl and had electrical resistances of 0.5–3.0 megaohms. Data were routinely recorded on a chart recorder (Gould 2300S). Average values are expressed as mean ± S.E.

Recording from Neuroblastoma Cells—N1E-115 cells (American Type Culture Collection) were prepared according to a method described previously (31). Cells were continuously superfused with a solution containing 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH, ~340 mosmol with sucrose). Membrane currents were recorded in the whole-cell patch clamp configuration using an Axopatch 200B amplifier (Axon) at room temperature. Cells were held at –60 mV. Data were acquired using pCLAMP 8 software (Axon).

Western Blot of Membrane Surface Proteins—Immediately after pretreatment with PMA, Xenopus oocytes expressing 5-HT3A receptors were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. The oocytes were then washed in PBS and incubated with N-hydroxysuccinimide-SS-biotin (NHS-SS-biotin; Pierce) at a concentration of 1.5 mg/ml in PBS for 30 min at 4 °C under a nonpermeabilized condition, as described previously (15, 34). The oocytes were then washed extensively and homogenized, and the homogenate was centrifuged repeatedly at 1000 x g for 10 min at 4 °C until all yolk granules and melanosomes were pelleted. The final supernatant was incubated with 100 µl of neutravidin-linked beads (Pierce) by end-over-end rotation for 2 h at 4 °C. The beads were centrifuged and washed extensively to isolate bead-bound proteins. Labeled proteins were eluted from the beads by dithiothreitol-containing SDS-PAGE loading buffer and loaded onto 10% SDS/PAGE. After transfer onto a polyvinylidene difluoride membrane (Invitrogen), the surface and total proteins were blocked with PBS, pH 7.5, containing 0.1% Tween 20 (Sigma) and 5% nonfat powdered milk and then incubated for 1 h with a polyclonal antibody (pAb120, 1:1000) directed to the extracellular N-terminal domain of the 5-HT3A receptor (5). The proteins were washed, blotted with a 1:600 dilution of fluorescein-linked anti-rabbit Ig in PBS, and incubated with anti-fluorescein AP conjugate at a 1:2500 dilution in PBS for 1 h. The proteins detected by a Western blotting kit (Amersham Biosciences) were scanned using a Molecular Dynamics Storm Gel and Blot Imaging System with ImageQuant Image Analysis Software (Amersham Biosciences).

Cell Surface Immunolabeling of Oocytes and N1E-115 Cells—Xenopus oocytes expressing 5-HT3A receptors were labeled with pAb120. In a pre-screening, oocytes exhibiting electrophysiological responses to 100 µM 5-HT of 3000–5000 nA were selected for this experiment. These oocytes were then incubated in 3 ml of MBS in the absence or presence of 300 nM PMA for 20 min. After PMA treatment, the oocytes were fixed in 4% paraformaldehyde in calcium and magnesium-free PBS for 10 min, rinsed twice in PBS, and placed in blocking buffer (5% donkey serum, 0.5% bovine serum albumin, PBS, 0.04% Triton X-100) for 30 min. After an incubation with pAb120 at 1:1000 dilution for 1 h, the cells were washed 3 times in PBS for 5 min and labeled with donkey-anti-rabbit conjugated to fluorescein isothiocyanate secondary antibody (Jackson ImmunoResearch Laboratories) as described previously (15). In a holding chamber, each oocyte was placed in a position with the equator perpendicular to the plane of imaging since most foreign receptors have been found to express within the animal pole of oocytes (14). Cross-sectional and tangential images of oocytes were viewed and photographed using a laser scanning microscope (LSM 5 Pascal, Zeiss), and the intensity was quantified by image analyzing software (Scion Image, Scion Co). N1E-115 cells were incubated in external recording solution in the absence and presence of PMA (300 nM) and immunolabeled using a method described previously (15).

Data Analysis—Statistical analysis of concentration-response data was performed with the use of the nonlinear curve-fitting program ALLFIT (50). Data were fitted to the equation using KaleidaGraph 3.5 (Synergy Software),

(Eq. 1)
where X and Y are concentration and response, respectively, Emax and Emin are the maximal and minimal responses, respectively, EC50 is the half-maximal concentration, and n is the slope factor (apparent Hill coefficient). Data were statistically compared by the paired t test or analysis of variance, as noted. Average values are expressed as the mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PKC Potentiates 5-HT-activated Current in Oocytes and N1E-115 Cells—Xenopus oocytes have been widely used as an expression system to study functional regulation of recombinant receptors by protein kinases and the underlying molecular mechanisms (11, 35–38). Although pretreatment with 100 nM 4{alpha}-PMA, an inactive form of PMA, for 10 min did not significantly alter the amplitude of currents activated by 5-HT (Fig. 1A) in an oocyte expressing 5-HT3A receptors, in the same cell, treatment with 10 nM PMA for 10 min increased the amplitude of inward current activated by 5-HT (Fig. 1B). The potentiation reached a maximum 10–20 min after the beginning of PMA application, lasted for 30–50 min, and was inhibited by the intracellular injection of a PKC inhibitory peptide 19–31 (PKCI) (Fig. 1C). The graph in Fig. 1D plots average current potentiation after treatment of the oocytes with PMA (solid circles), 4{alpha}-PMA (open circles), or PMA after the intracellular injection of PKCI (solid triangles). These observations are in accord with previous results from this (29) and other (32) laboratories showing that activation of PKC can potentiate 5-HT3A receptors expressed in Xenopus oocytes. To determine whether PKC can modulate 5-HT3 receptor-mediated responses in N1E-115 cells, we performed whole cell recording on these cells. The amplitudes of currents evoked by 2 µM 5-HT at 2-min intervals were nearly identical under our experimental conditions (data not shown). Loading cells with 1 µM PKM (the constitutively active fragment of PKC) through a micropipette for 4 and 6 min increased the amplitude of 5-HT-activated currents (Fig. 2A). In a separate experiment, loading cells with 300 nM PMA (Fig. 2B) for 4 and 6 min increased the amplitude of the 5-HT current, whereas this did not occur using 4{alpha}-PMA (Fig. 2C). These findings suggest that enhancement of 5-HT current by PMA in N1E-115 cells is mediated by the activation of PKC. The potentiation appeared to reach the maximal magnitude within 4–6 min after PKM or PMA application. The bar graphs in Fig. 2D plot the average effect of 4-{alpha} PMA, PMA, and PKM without or with inclusion of 1 µM PKCI in the pipette. Whereas 4{alpha}-PMA was ineffective, PMA or PKM significantly increased the amplitude of 5-HT-activated current. Moreover, inclusion of PKCI significantly reduced the magnitude of potentiation by PMA or PKM from 209 ± 46 and 219 ± 40% of control to 104 ± 1 and 105 ± 5% of control, respectively (p < 0.001, unpaired t test, n = 5). The application of PKCI alone did not significantly alter the amplitude of 5-HT-activated current (data not shown).



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  		FIG. 1.
Effect of 4{alpha}-PMA and PMA on 5-HT-activated current in Xenopus oocytes expressing 5-HT3A receptors. A, records of current activated by 0.25 µM 5-HT before (0 min) and 5–30 min after beginning the application of 100 nM 4{alpha}-PMA for 10 min. B, records of current activated by 0.25 µM 5-HT before (0 min) and 5–30 min after beginning the application of 10 nM PMA for 10 min. C, records of current activated by 0.25 µM 5-HT before (0 min) and 5–30 min after beginning the application of 10 nM PMA for 10 min in a cell previously injected with 10 µM PKCI. The solid bar above each record indicates the time of 5-HT application. D, graph plotting time-course of average percentage potentiation of 5-HT-activated current after application of PMA (•), 4{alpha}-PMA ({circ}), or PKCI plus PMA ({blacktriangleup}). The solid bar indicates the time of the application of PMA, 4{alpha}-PMA, and PKCI plus PMA. Each data point represents average of 5–7 oocytes. The error bars not visible are smaller than the size of the symbols.

 


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  		FIG. 2.
Effect of PKM, PMA, and 4{alpha}-PMA on 5-HT-activated currents in N1E-115 cells. A, records of current activated by 2.0 µM 5-HT during intracellular loading of the cell with 10 µM PKM for 4–6 min. B, records of current activated by 2.0 µM 5-HT during intracellular loading of the cell with 300 nM PMA for 4–6 min. C, records of current activated by 2.0 µM 5-HT during intracellular loading of the cell with 300 nM 4{alpha}-PMA for 4–6 min. The bar above each record indicates the time of 5-HT application. D, bar graphs of average 5-HT-activated current by loading the cells for 6 min with 4{alpha}-PMA, PMA, and PKM and with PMA and PKM in the presence of PKCI. Bars represent mean ± S.E.

 

PMA Increases Surface Expression of 5-HT3 Receptors in Oocytes and N1E-115 Cells—To determine whether PKC activation can alter 5-HT3 receptor trafficking, we used two different approaches. First, we used confocal microscopy to examine the effect of PMA on receptor surface expression by immunostaining nonpermeabilized oocytes. The uninjected oocytes and oocytes expressing 5-HT3A receptors with or without PMA pretreatment were incubated with pAb120 antibody followed by a fluorescein-tagged secondary antibody. The fluorescent surface labeling was viewed by confocal microscopy. The uninjected oocytes exhibited little or no immunofluorescence with this antibody directed against to the extracellular N-terminal domain of the receptor (data not shown). However, PMA increased membrane surface immunolabeling of oocytes expressing 5-HT3A receptors (Fig. 3B) as compared with controls (Fig. 3A). We did not observe intracellular fluorescence under any conditions, suggesting that the enhanced fluorescence is confined to the external surface of the cell membrane. A similar result was observed in our experiments using N1E-115 cells; as shown in Fig. 3, C and D, treatment with PMA for 5 min increased immunolabeling of N1E-115 cells (Fig. 3D) as compared with the controls (Fig. 3C). Quantitation of these images shows that the immunofluorescence in the oocytes expressing 5-HT3A receptors significantly increased to 282 ± 40% of control (p < 0.01, n = 8) after PMA treatment for 20 min (Fig. 3E). The immunofluorescence in the N1E-115 cells increased to 154 ± 24% that of control (p < 0.05, n = 8) after PMA treatment for 5 min (Fig. 3E). Next, we directly measured changes in surface expression of 5-HT3A receptors in oocytes using Western blots by labeling cell-surface proteins with sulfo-NHS-SS-biotin (39). The biotinylated surface proteins were separated from intracellular cell proteins by binding the biotinylated surface proteins to Neutravidin beads; these proteins were then eluted by treatment with dithiothreitol-containing buffer. Fig. 4A illustrates a representative Western blot of the surface expression of 5-HT3A receptors after treatment of the oocytes with 300 nM PMA for 20 min. PMA significantly increased the surface protein (Fig. 4A) with no change in the total protein (Fig. 4B) in oocytes expressing 5-HT3A receptors. Analysis of band densities (Fig. 4C) revealed an increase in surface expression of 5-HT3A receptors to 1.92 ± 0.14 times control (p < 0.01, n = 5), with no change in 5-HT3A receptor protein from the total cell (p > 0.05, n = 3). It should be noted that there is more immunoreactivity for the surface receptor than for the total receptor. This discrepancy might be due to the following. First, the starting material of the surface receptor protein produced by biotinylation experiments could be more concentrated than total receptor protein obtained from total cell lysate. Second, there might be a difference in the sample loading between the Western blot of the surface and the total receptor proteins.



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  		FIG. 3.
Increase in surface immunofluorescence of 5-HT3A receptors in oocytes and N1E-115 cells by PKC activation. Surface labeling of 5-HT3A receptors using a specific antibody directed against the N-terminal domain of the 5-HT3A receptors in nonpermeabilized oocytes expressing 5-HT3A receptors and in N1E-115 cells. A, cross-sectional confocal image of oocyte expressing 5-HT3A receptors before the application of PMA (Control). B, cross-sectional confocal image of oocyte expressing 5-HT3A receptors 5 min after the beginning of 300 nM PMA application. Note that PMA increased 5-HT3A receptor immunofluorescence. C, cross-sectional confocal image of N1E-115 cells before PMA application. D, cross-sectional confocal image of N1E-115 cells 5 min after the beginning of 300 nM PMA application. Note that PMA increased 5-HT3A receptor immunofluorescence. E, bar graphs of average surface 5-HT3A receptor immunofluorescence before (Control) and after the beginning of PMA application for Xenopus oocytes (left) and N1E-115 cells (right). Bar graphs plot surface fluorescence as the mean intensity per unit area. Bars represent mean ± S.E. from six or more cells.

 


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  		FIG. 4.
Increase in surface expression of 5-HT3A receptors in oocytes by PKC activation. A, representative Western blot of 5-HT3A surface receptor for uninjected oocytes (left), oocytes expressing 5-HT3A receptors (5-HT3AR) without PMA (middle), and oocytes expressing 5-HT3A receptors 20 min after the beginning of 300 nM PMA application (right). The surface protein was assessed by the biotin surface labeling method as described under "Experimental Procedures." B, a representative Western blot of total cell 5-HT3A receptor content for uninjected oocytes (left), oocytes expressing 5-HT3A receptors without PMA (middle), and oocytes expressing 5-HT3A receptors 20 min after the beginning of 300 nM PMA application (right). C, average 5-HT3A receptor band density for surface (left) and total cell (right) receptor. Bars represent the mean ± S.E. of gel band density from 3–5 experiments.

 

The Magnitude of PMA Enhancement of 5-HT3A Receptor Surface Expression Is Correlated with the Magnitude of PMA Potentiation of 5-HT3A Receptor-mediated Current—Next we compared the increase of receptor surface expression with the potentiation of 5-HT3A receptor-mediated current after treatment with PMA. The bar graphs in Fig. 5A plot the average potentiation of 5-HT-activated current by various concentrations of PMA from 10 to 1000 nM. The average potentiation induced by 10, 100, 300, and 1000 nM PMA was 152 ± 14% (p < 0.01, n = 11), 452 ± 24% (p < 0.001, n = 11), 582 ± 33% (p < 0.001, n = 14), and 492 ± 31% (p < 0.001, n = 7) that of control, respectively. Note that the potentiation was maximal at 300 nM PMA and that the potentiation by 1000 nM was significantly less than that by 300 nM (p < 0.01, unpaired t test, n = 7–14). Surface expression of the receptor assessed by Western blot analysis exhibited a pattern similar to that of the PMA potentiation of 5-HT3A receptor-mediated responses (Fig. 5B). The average normalized band density in Western blots after treatment with 10, 100, 300, and 1000 nM PMA was 110 ± 4% (n = 3), 152 ± 18% (n = 4), 192 ± 14% (n = 5), and 162 ± 12% (n = 5) that of control, respectively. These values are significantly different from control (analysis of variance, p < 0.05). In addition, the magnitude of PMA potentiation of 5-HT-activated current is correlated with the magnitude of PMA-induced increase in band density (Fig. 5C, R = 0.98, p < 0.001, n = 4).



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  		FIG. 5.
Correlation of PMA potentiation of 5-HT-activated current with PMA-induced increase in 5-HT3A receptor surface proteins. A, potentiation of 5-HT-activated current by 10–1000 nM PMA in oocytes expressing 5-HT3A receptors. Bar graphs plot the average percentage potentiation of current activated by 0.25 µM 5-HT 20 min after beginning PMA application. Bars represent the mean ± S.E. (n = 5–9). B, PMA-induced increase in 5-HT3A receptor abundance at the oocyte surface. Western blot gel bands of surface 5-HT3A receptor proteins (see "Experimental Procedures") 20 min after the application of 0, 100, 300, and 1000 nM PMA. Bar graphs plot the average band densities normalized as a percentage of control. Bars represent the mean ± S.E. (n = 3). C, correlation between the magnitude of PMA potentiation of 5-HT-activated current and the magnitude of PMA enhancement of surface 5-HT3A receptors (r = 0.98, p < 0.01, n = 4); the line is the best fit to a linear regression (Statistica).

 

PKC and PKA Sites in the Large Intracellular Loop (LIL) of the 5-HT3A Receptor Are Not Involved in the PMA Potentiation—It has been reported that protein kinase A (PKA) can phosphorylate the 5-HT3A receptor protein (40). The phosphorylation by PKA is abolished by a point mutation of a putative PKA phosphorylation site in the LIL of the receptor (40). To evaluate if PMA potentiation of the 5-HT3A receptor-mediated response is mediated by the putative PKC or PKA phosphorylation sites in the LIL of the receptor, we sequentially replaced all of the 11 serines (S) or threonines (T) in the LIL of the receptor with alanine (A). The sensitivity of these mutant receptors to both 5-HT and PMA potentiation was examined by two-electrode voltage-clamp in Xenopus oocytes previously injected with complementary RNAs of the receptors. Fig. 6A shows the EC50 values of the 5-HT concentration-response curves for the wild type (WT) and mutant 5-HT3A receptors. The bar graphs in Fig. 6B plot the average PMA potentiation of 5-HT-activated current at the EC5 concentration for each receptor. The potentiation by 10 nM PMA was 150 ± 14% for WT, 129 ± 21% for S274A, 151 ± 29% for S326A, 168 ± 27% for T372A, 126 ± 16% for T378A, 130 ± 14% for S382T/S384T, 153 ± 16% for T406A, 145 ± 13% for S412A, 170 ± 21% for S421A, and 146 ± 16% for S433A/S434A receptors. These values are not significantly different from the WT (analysis of variance, p > 0.1, n = 5–7). In view of sensitivity of S274A and T378A mutants to PMA potentiation, we also examined the sensitivity of these mutant receptors to high concentrations of PMA (300 nM) and found that the S274A or T378A mutation did not significantly alter the sensitivity of 5-HT3A receptor to 300 nM PMA (data not shown).



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  		FIG. 6.
Point-mutations of the putative PKC sites in the LIL of the 5-HT3A receptor do not significantly alter the receptor sensitivity to PMA. A, sequential replacement of each of 11 serine (S) and threonine (T) amino acids with alanine (A) in the LIL of the 5-HT3A receptor and the EC50 values of 5-HT concentration-response curves for the WT and mutant receptors in Xenopus ooyctes expressing the receptors. The EC50 values for 5-HT were obtained by fitting the 5-HT concentration-response curves to the Hill equation, as described under "Experimental Procedures." B, average PMA potentiation of the WT and mutant receptor-mediated responses. Bars represent the mean ± S.E. from 5–6 oocytes. The average potentiation for each of the mutant receptors by PMA was not significantly different from the PMA potentiation of the WT receptor (analysis of variance, p > 0.1). The current was activated by 5-HT at the EC5 concentration for each receptor.

 

Stabilizing Actin Cytoskeleton by Pretreatment with Phalloidin (PLD) Inhibits PMA Potentiation—In the light of recent studies reporting that the dynamics of actin cycling are essential for PKC modulation of NMDA receptor function (27, 28), we examined the effect of agents that disorder the dynamic movement of F-actin on the PKC potentiation of 5-HT3A receptor-mediated responses. The records in Fig. 7A show that treatment with 300 nM PMA greatly increased the amplitude of 5-HT-activated current in control oocytes expressing 5-HT3A receptors. Preincubation with 10 µM cytochalasin D (CCD), an agent that disrupts actin cytoskeleton, did not significantly alter the potentiation by 300 nM PMA (Fig. 7B). However, the intracellular injection of PLD (10 µM), which stabilizes F-actins, significantly reduced the magnitude of PMA potentiation of 5-HT-activated current. On average, injection of PLD reduced the PMA potentiation by 79 ± 5% (Fig. 7B, p < 0.01, n = 5). In addition, PLD did not affect either the EC50 value of the 5-HT concentration-response curve or the maximal amplitude of the 5-HT-activated current (data not shown). We observed similar results in a study of PMA potentiation of 5-HT-activated current in N1E-115 cells. Whereas 10 µM CCD did not significantly affect PMA potentiation, preincubation with PLD for 4 h abolished the PMA-induced increase in the amplitude of 5-HT-activated current (Fig. 7C). The percent potentiation by 300 nM PMA alone was 109 ± 8%, after CCD it was 106 ± 12% (p > 0.5, n = 4), and after PLD it was 3 ± 1.2% (p < 0.001, n = 4).



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  		FIG. 7.
PLD inhibits PMA potentiation of 5-HT-activated currents in oocytes and N1E-115 cells. A, records of potentiation of current activated by 0.25 µM 5-HT by 300 nM PMA before and after the application of CCD or PLD in Xenopus oocytes expressing 5-HT3A receptors. The solid bar above each record indicates the time of 5-HT application. CCD (10 µM) was preincubated for at least 4 h, and PLD (10 µM) was injected intracellularly for 2 h before testing the effect of PMA. B, bar graphs represent the average percentage potentiation of current activated by 0.25 µM 5-HT by 300 nM PMA before and after the bath application of CCD or intracellular injection of PLD. Each bar represents the mean ± S.E. from 5–6 oocytes. C, representative trace records of inward current activated by 2.0 µM 5-HT in the absence and presence of PMA after preincubation of 10 µM CCD or 10 µM PLD for 4 h in N1E-115 cells. D, bar graphs represent the average percentage potentiation of 5-HT-activated current by PMA after preincubation of CCD (10 µM) or PLD (10 µM) for 4 h. Each bar represents the mean ± S.E. of 4 cells.

 

Destabilizing Actin Cytoskeleton by Latrunculin-A (Lat-A) Enhanced the Potentiation of 5-HT3A Receptor-mediated Current by Low Concentrations of PMA—In view of reports that PMA by itself can destabilize actin dynamics (24–26), we wondered if pretreatment with Lat-A, a potent disrupter of actin cytoskeleton, can enhance PMA potentiation of 5-HT3A receptor-mediated responses. We found that preincubation with 1 µM Lat-A for 2 h greatly enhanced the potentiation of 5-HT3A receptor-mediated responses by 10 nM PMA (Fig. 8A). On the other hand, pretreatment with Lat-A for 2 h did not appear to affect the magnitude of PMA potentiation when 300 nM PMA was used (Fig. 8A). In cells preincubated with Lat A, the average magnitude of potentiation by 10 nM PMA increased by 370% (PMA alone versus PMA ± Lat A, 150 ± 25% versus 555 ± 39%; p < 0.01, n = 5), whereas Lat-A did not significantly affect the maximal potentiation by 300 nM PMA (Fig. 8B) (p > 0.05, unpaired t test, n = 5). Moreover, the magnitude of 10 nM PMA-induced potentiation after treatment with Lat-A was not significantly different from the maximal potentiation induced by 300 nM PMA alone (555 ± 39% versus 582 ± 33%; p > 0.1, unpaired t test, n = 5).



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  		FIG. 8.
Lat-A increases the sensitivity of 5-HT3A receptors to low concentration PMA. A, records of potentiation of current activated by 0.25 µM 5-HT by 10 and 300 nM PMA before and after application of 1 µM Lat-A for 2 h. The solid bar above each record indicates the time of 5-HT application. B, bar graphs plot the average potentiation of current activated by 0.25 µM 5-HT by 10 and 300 nM PMA with or without preincubation of 1 µM Lat-A (1 µM). Bars represent mean ± S.E. from 5–6 oocytes.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we confirmed previous reports that activation of PKC can enhance 5-HT-activated current in Xenopus oocytes expressing 5-HT3A receptors (29, 32). We also observed an ~2-fold potentiation of 5-HT-activated current in N1E-115 cells by either PKM or PMA. Moreover, the potentiation by either PKM or PMA is likely to be mediated by PKC since 4{alpha}-PMA did not affect 5-HT-activated current and the potentiation by PKM or PMA was inhibited by PKCI. Given the fact that N1E-115 cells are neuron-like cells containing both 5-HT3A and 5-HT3B subunits (41) that have been used for cloning and functional characterization of 5-HT3 receptors (42–44), our results also suggest that activation of PKC can modulate the function of native 5-HT3 receptors expressed in N1E-115 cells.

PKC has been found to modulate certain types of LGIC protein trafficking. However, such a study has not been reported for PKC modulation of 5-HT3 receptors. In the present study, we observed that pretreatment with PMA can enhance surface immunolabeling and surface expression of 5-HT3A receptors for both Xenopus oocytes and in N1E-115 cells. The increase in surface receptor expression by PKC activation is likely to occur via an increase in receptor trafficking rather than an increase of receptor protein synthesis since the quantity of receptor protein in the total cell extract remained unchanged after PMA treatment. In addition, consistent with previous reports in the studies of PKC regulation of NMDA and GABAA receptor trafficking (15, 45), our observation that the increase in surface expression of the 5-HT3A receptors developed rapidly, within a few minutes after the beginning of PMA application, in both Xenopus oocytes expressing 5-HT3A receptors and in N1E-115 cells also favors PMA modulation of receptor trafficking. An increase in receptor trafficking to the cell membrane may contribute, at least in part, to PKC potentiation of 5-HT-activated current because a correlation is observed between the magnitude of PKC-induced membrane receptor expression and the magnitude of PKC potentiation of 5-HT current.

In the study reported here, we found that the putative PKC phosphorylation sites in the LIL of the 5-HT3A receptor are unlikely to be involved in PMA potentiation of the receptor-mediated responses, because mutation of these sites did not significantly affect the sensitivity of the mutant receptor to PMA. These observations are consistent with a recent study in which point mutations of all of the known PKC/PKA sites in the LIL of the 5-HT3A receptor in a single clone did not alter the receptor sensitivity to PMA (32). These results are also consistent with observations in studies of PKC modulation of NMDA receptor channels in which PKC is thought to phosphorylate PKC-associated proteins rather than the receptor protein (15, 18).

Our results show that PMA potentiation of 5-HT-activated currents can be prevented when actin cytoskeleton is stabilized by treatment with PLD in both Xenopus oocytes expressing 5-HT3A receptors and N1E-115 cells. The observation that disruption of actin polymerization by Lat-A enhanced potentiation by low concentrations of PMA, whereas Lat-A did not alter maximal PMA potentiation, suggests that PMA modulation of 5-HT3A receptor function may involve F-actin. It has been well documented that the F-actin cytoskeleton can serve as a barrier to restrain the transport of vesicles to the cell membrane (25). It has also been reported that PKC isoforms can bind to actin after PMA treatment (46). As a result, the activation of PKC by phorbol esters could cause a rearrangement of actin filaments, which removes a negative clamp that prevents the exocytosis of proteins. In this regard, it seems likely that pretreatment with PMA may result in disassembly of actin cytoskeleton, thereby promoting transport of 5-HT3A receptors to the cell membrane. This hypothesis is supported by our observations that PLD inhibited PMA potentiation of 5-HT-activated responses and Lat-A increased the sensitivity of 5-HT3A receptors to PMA potentiation. This hypothesis is also consistent with a very recent study indicating that F-actin plays an important role in the regulation of 5-HT3A receptor targeting and clustering at cell membranes (33).

It should be noted that PMA may also modulate gating of the 5-HT3A receptor channel, given the observations that the potentiation of 5-HT-activated current by PMA is dependent on agonist concentration in Xenopus oocytes expressing 5-HT3A receptors (29) and PMA modulates subconductance states of 5-HT-activated single channel currents in N1E-115 cells (31). Such a scenario occurs in PKC modulation of NMDA receptor function in which PKC has been found to modulate gating of the receptor channels (15, 47). However, it is also thought that the alteration of NMDA receptor channel gating by PKC results at least in part from PKC modulation of the receptor trafficking such as insertion of new channels into cell surface membranes (15). It should also be pointed out that there are a number of other questions that remain to be determined. For instance, whether F-actin can either directly bind to the 5-HT3A receptor protein or whether it can interact with the receptor through intermediating proteins is not known. Moreover, the mechanisms by which PKC promotes an increase of 5-HT3A receptors in the cell membrane might also involve other mechanisms, such as a reduction in internalization of the receptor channels. In addition, a recent study suggests that tyrosine kinase may also be involved in regulation of 5-HT3A receptor function (32); in this regard, whether tyrosine kinase is involved in 5-HT3A receptor trafficking remains to be determined. These issues will need to be addressed in future studies.

For some LGICs, regulation of protein trafficking by PKC is thought to be critical for synaptic modulation and plasticity (48). Given the observation that 5-HT3A receptors are largely localized in the cytosol of central neurons (3), our observations that PKC can regulate 5-HT3A receptor function and trafficking through actin-dependent pathways raise the possibility that 5-HT3A receptors may be dynamically moved in and out of the cell membranes by PKC and other types of kinases. In light of observations reporting that neurotransmitter release can be regulated through an actin-dependent mechanism in the central nervous system (49) and that 5-HT3A receptors can modulate the release of dopamine and GABA in some important brain areas, it seems possible that enhancement of 5-HT3 receptor function and trafficking by PKC activation may play an important role in modulating the efficacy of serotonergic synaptic transmission, the release of neurotransmitters, and other 5-HT3 receptor-mediated phenomena.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, Park Bldg., Rm. 150, Bethesda, MD 20892-8115. Tel.: 301-443-1236; Fax: 301-480-6882; E-mail: lzhang{at}niaaa.nih.gov.

1 The abbreviations used are: 5-HT, serotonin; LGIC, ligand-gated ion channel; GABAA, {gamma}-aminobutyric acid (GABA) type A; PMA, 4{beta}-phorbol 12-myristate 13-acetate; PKC, protein kinase C; PKCI, PKC inhibitory peptide; PKA, protein kinase A; PKM, protein kinase C catalytic subunit; CCD, cytochalasin D; Lat-A, latrunculin-A; Me2SO, dimethyl sulfoxide; LIL, large intracellular loop; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; WT, wild type; PLD, phalloidin. Back


   ACKNOWLEDGMENTS
 
We thank Drs. David Julius and Sarah Lummis for providing us with mouse 5-HT3A receptor cDNA and a specific antibody against the 5-HT3A receptor, respectively, and Drs. Sarah Lummis and David Lovinger for comments on the manuscript.



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 DISCUSSION
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