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1Cardiovascular Research Group, School of Medicine, University of Manchester, Manchester, United Kingdom; 2Kagawa Prefectural College of Health Sciences, Mure, Kagawa, Japan; 3Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom; and 4Department of Physiology, University of British Columbia, Vancouver, British Columbia, Canada
Submitted 7 December 2005 ; accepted in final form 27 September 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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62% with time constants of 1.5 and 26.9 min, whereas 83% of the M2 receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with 
-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and -arrestin 2 significantly increased IK,ACh desensitization and M2 receptor internalization during a 3-min application of agonist. Internalized M2 receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and -arrestin 2-dependent internalization of the M2 receptor into clathrin-coated vesicles could play a major role in IK,ACh desensitization.
acethylcholine-activated potassium current; acetylcholine; Kir3.1; Kir3.4; caveolin
20 s and minutes to hours, respectively (3, 30, 38). The fast phase is likely to be the result of a change in the channel itself (1). The slow phase developing over minutes may be the result of a change in the receptor because it is not observed whether the receptor is bypassed (38).
In a cell line [Chinese hamster ovary (CHO)] heterologously expressing the M2 receptor and the ACh-activated K+ channel (Kir3.1 and Kir3.4), expression of G protein-receptor kinase 2 (GRK2) (-adrenergic receptor kinase 1 or ARK1) and -arrestin 2 increases the slow desensitization developing over both minutes and hours, and this too points to the involvement of the receptor (28–31). In vitro, GRK2 is known to phosphorylate the agonist-bound M2 receptor (11, 14). In the heart, the M2 receptor is known to become phosphorylated in the presence of agonist, and this is presumably the result of GRK2 (15, 16). In the case of G protein-coupled receptors in general, arrestins are known to bind to receptor kinase-phosphorylated (and agonist bound) receptors to cause desensitization by 1) uncoupling the receptor from the G protein and 2) (in the case of the nonvisual arrestins, e.g., -arrestin 2) promoting internalization of the receptor by clathrin-coated pits (the nonvisual arrestins act as adaptor proteins and bind both the receptor and clathrin) (17, 23). In the case of the M2 receptor in particular, it is known that -arrestin and -arrestin 2 bind to the M2 receptor in a phosphorylation-dependent manner (21). In addition, there is evidence that they may cause M2 receptor uncoupling (21, 22). In the heart, during exposure to agonist, the M2 receptor is internalized (26, 32). However, it is not clear whether an arrestin is involved. In a cell line (HEK-tsA201), overexpression of -arrestin and -arrestin 2 causes internalization of the M2 receptor via a dynamin-dependent mechanism (likely to involve clathrin-coated pits); however, in the absence of arrestin overexpression, internalization appears to occur via a mechanism independent of arrestins or dynamin (21). Furthermore, in rat ventricular cells, Feron et al. (7) reported that the M2 receptor is internalized via caveolae (a clathrin-independent pathway), although in a cell line (HEK-293) and cat atrial cells Roseberry and Hosey (26) failed to see colocalization of the M2 receptor and caveolin in the presence of agonist.
The main aim of the present study was to investigate the role of internalization of M2 receptors in the slow desensitization of IK,ACh in heart cells. A secondary aim was to determine how the M2 receptor is internalized in heart cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When necessary, the cells were incubated in a solution containing 10 µM carbachol chloride (CCh; Sigma, Poole, UK) or ACh to activate the M2 receptor (in whole cell patch-clamp experiments, IK,ACh activated by 10 µM ACh or CCh was the same). When it was necessary to expose cells to agonist for a prolonged period, CCh (rather than ACh) was used because it is stable. A concentration of 10 µM was used because this amount is sufficient for maximal activation of IK,ACh (4).
Adult cells were used for all experiments apart from those involving transfection (because neonatal cells, unlike adult cells, survive for long periods under culture conditions). On the day after isolation, the neonatal atrial cells were transfected with either 1) GRK2 (pEF-GRK2), 2) -arrestin 2 (pCMV5--arrestin 2), 3) GRK2 and -arrestin 2, or 4) constitutively active mutant (CAM) -arrestin 2 (pCDNA3--arrestin 2 1–393-CAM) (29). All cells were also transfected with green fluorescent protein (pEGFP-N1; BD Biosciences, San Jose, CA) as a marker for successfully transfected cells. The FuGENE 6 transfection reagent (Roche Diagnostics, Lewes, UK) was used for transfection. On the day after transfection, neonatal atrial cells were used for experiments.
Electrophysiology. Experiments were carried out by the whole cell configuration of the patch-clamp technique at 35°C (adult atrial cells) or room temperature (22–25°C; neonatal atrial cells). Adult or neonatal atrial cells seeded onto glass coverslips, with and without CCh pretreatment, were placed in a recording chamber mounted on a Nikon Diaphot microscope; 470- to 490-nm light was used to excite the green fluorescent protein in successfully transfected cells. The green fluorescent light was passed through a 515-nm filter for observation. Cells with a middle level of green fluorescence were chosen for study. Extracellular solution contained (in mM) 140 KCl, 1.8 MgCl2, 5 EGTA, and 5 HEPES, pH 7.4 with KOH. Acetylcholine chloride (ACh, 10 µM; Sigma) or CCh was added to the extracellular solution when required. Pipette solution contained (in mM) 120 potassium aspartate, 20 KCl, 1 KH2PO4, 2.8 MgCl2 (1.8 free Mg2+), 5 EGTA, 0.1 Na3GTP, 3 Na2ATP, and 5 HEPES, pH 7.4 with KOH. Whole cell currents were recorded with an Axopatch-1D amplifier and acquired with pCLAMP software (Axon Instruments, Union City, CA). Currents were filtered at 2 kHz with an eight-pole Bessel filter and sampled every 1 ms.
Immunocytochemistry. Adult or neonatal atrial cells seeded onto glass coverslips, with and without CCh pretreatment, were washed with PBS. The cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, permeabilized with 0.1% Triton X-100 for 10 min, washed with PBS, and blocked with 10% normal donkey serum in PBS for 30 min.
Primary antibodies, rabbit anti-Kir3.1 polyclonal antibody (Alomone Labs, Jerusalem, Israel), rabbit anti-Kir3.4 antibody [
CIR-N2, amino acids 19–32; gift of G. B. Krapivinsky (13)], rabbit anti-G protein i-1,2,3 polyclonal antibody (Oncogene Research Products, San Diego, CA), rat anti-M2-muscarinic-receptor monoclonal antibody (Chemicon International, Temecula, CA), mouse anti-clathrin heavy-chain monoclonal antibody (Affinity BioReagents, Golden, CO), and mouse anti-caveolin-3 monoclonal antibody (BD Biosciences, San Jose, CA), were diluted with PBS containing 1.5% normal donkey serum and 1% BSA. The cells were incubated with primary antibody at 4°C overnight. Secondary antibodies, donkey anti-rabbit IgG (conjugated to FITC for Kir3.1, Kir3.4, and G protein staining), goat anti-rat IgG (conjugated to FITC for M2-receptor staining), donkey anti-mouse IgG [conjugated to tetramethylrhodamine isothiocyanate (TRITC) for clathrin staining], and rabbit anti-mouse IgG (conjugated to FITC or TRITC for caveolin-3 staining), were diluted in the same way as the primary antibodies. The secondary antibodies were from Chemicon International or Jackson ImmunoResearch Laboratories (West Grove, PA). Cells were incubated with secondary antibody at room temperature for 1 h. Before and after the incubation, cells were washed with PBS three times. Finally, cells were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA), sealed onto microscope slides with nail polish, and stored in the dark at 4°C.
Labeling in cells was visualized with the use of a laser scanning confocal microscope (TCS SP, Leica, Heidelberg, Germany) equipped with an argon laser (488 nm) for FITC labeling and a krypton laser (568 nm) for TRITC labeling. Images were recorded from the center of a cell. No labeling was detectable without either the primary or secondary antibody. Corel Photo-Paint (Ottawa, ON, Canada) was used to process images.
For the experiments shown in 
Figs. 4 and 5, the labeling was quantified. In these cases, cells isolated from each heart were divided into two groups: a control group (cells not exposed to agonist) and an experimental group. For each heart, images were collected over a few days (experience shows that fluorescence intensity does not change over this time scale) with constant laser settings. The mean intensity of labeling from eight random points on the cell membrane in a cell was measured by the TCS analysis system (Leica). For each heart, the mean labeling of control cells was taken as 100%, and labeling of the experimental groups was expressed as a percentage of this. For each treatment, measurements were taken from up to 78 cells from at least two hearts.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The time course of desensitization during an exposure to CCh >4 min in duration could not be routinely measured with the method of Fig. 1 (stable recordings >4 min in duration are not routine), and a modification of the method was used. Atrial cells were exposed to 10 µM CCh (the conditioning dose). In the presence of CCh, a gigaseal was obtained, and the whole cell patch-clamp was established. CCh was then washed off for 10 s before a 30-s test dose of 10 µM ACh was applied. The advantage of this method is that the cells were subjected to whole cell recordings for approximately the same period of time regardless of the duration of the exposure to CCh. Figure 2A shows typical current traces after 1.8- to 114-min exposure to conditioning doses of CCh; currents at the end of the exposure to CCh and during the test dose of ACh are shown. Peak IK,ACh during the test dose of ACh (and also IK,ACh at the end of the test dose) declined as the duration of the conditioning dose of CCh was increased. In Fig. 2B, peak IK,ACh during the test dose of ACh (and IK,ACh at the end of the test dose) is plotted against the duration of the conditioning dose of CCh. In Fig. 2B, current is normalized for cell capacitance because peak IK,ACh during the conditioning dose of CCh was not recorded. Figure 2B shows that peak IK,ACh during the test dose of ACh and IK,ACh at the end of the test dose declined with time constants of 26.9 ± 10.8 and 31.6 ± 12.1 min (based on data from 30 cells), respectively. In summary, Figs. 1 and 2 suggest that IK,ACh declines during an exposure to 10 µM CCh in a double-exponential fashion with time constants of 1.5 and 26.9 min.
Figure 2C shows that the percent decrease of IK,ACh (primarily the result of fast desensitization) during the 30-s test dose of ACh is weakly correlated (R2 = 0.4) with the duration (0.5–120 min) of the conditioning dose of CCh, and it declines with a time constant of 1 min as the duration of the conditioning dose is increased.
Effects of expression of GRK2, -arrestin 2, and CAM -arrestin 2 on IK,ACh desensitization.
We have previously shown that, when the M2 receptor, Kir3.1, and Kir3.4 are heterologously expressed in CHO cells, desensitization of IK,ACh during a 3-min exposure to ACh is increased by coexpression of GRK2 and -arrestin 2 (29, 31). Figure 3 shows that overexpression of GRK2 and -arrestin 2 also affects desensitization of IK,ACh in atrial cells. Figure 3 shows typical traces of IK,ACh during 3-min exposures to 10 µM ACh of a control cell (i.e., an untransfected cell; Fig. 3A) and of cells transfected with GRK2 (Fig. 3B), -arrestin 2 (Fig. 3C), and GRK2 plus -arrestin 2 (Fig. 3D). When compared with the control cell (Fig. 3A), in the cells transfected with GRK2 (Fig. 3, B and D) and -arrestin 2 (Fig. 3, C and D), desensitization of IK,ACh was increased. Mean desensitization of IK,ACh (the difference between peak IK,ACh during exposure to ACh and IK,ACh at the end of the exposure expressed as a percentage of peak IK,ACh) for the different cell groups is plotted in Fig. 3F. This shows that transfection with GRK2 alone or -arrestin 2 alone significantly (P < 0.001) increased IK,ACh desensitization. Figure 3F also shows that the effect of transfection with GRK2 plus -arrestin 2 was greater than the effects of GRK2 or -arrestin 2 alone.
Our group (29) has previously shown that transfection of CHO cells (heterologously expressing the M2 receptor, Kir3.1, and Kir3.4) with CAM -arrestin 2 causes desensitization (i.e., a decrease) of IK,ACh even in the absence of agonist. Figure 3E shows IK,ACh during an exposure to ACh of an atrial cell transfected with CAM -arrestin 2; peak IK,ACh was decreased compared with peak IK,ACh in the control cell (Fig. 3A). Figure 3G shows peak IK,ACh in the different cell groups, and it shows that peak IK,ACh was unaffected by transfection with GRK2 and -arrestin 2 (also shown by Fig. 3, A–D), but it was significantly decreased (P < 0.05) by CAM -arrestin 2. CAM -arrestin 2 had little effect on desensitization of IK,ACh (Fig. 3F). Therefore, in atrial cells, CAM -arrestin 2 caused desensitization in the absence of agonist, as expected.
Abundance of Kir3.1, Kir3.4, and Gi.
Desensitization of IK,ACh could be the result of a loss of the M2 receptor, the Gi protein, or the channel (a heterotetramer of Kir3.1 and Kir3.4). Figure 4 shows immunolabeling of Kir3.1 (Fig. 4A), Kir3.4 (Fig. 4B), and Gi (Fig. 4C) in atrial cells; control cells (Fig. 4, A–C, left) and cells exposed to 10 µM CCh for 2 h (Fig. 4, A–C, right) are shown. In control cells, the Kir3.1, Kir3.4, and Gi labeling was in (or close to) the cell membrane. In the cells exposed to CCh, the pattern of labeling was similar. The intensity of labeling in or close to the cell membrane was measured in groups of control cells and in cells exposed to CCh and is shown in Fig. 4, D–F. Exposure to CCh had no effect on Kir3.1 (Fig. 4D) and Gi (Fig. 4F) labeling, whereas it resulted in a small but significant decrease in Kir3.4 labeling (by 15.4 ± 4.7%; P < 0.05; Fig. 4E). This suggests that there could be a decrease in the function of the muscarinic K+ channel.
M2-receptor internalization.
In contrast to Gi and the K+ channel, there were large changes in the localization of the M2 receptor in atrial cells during an exposure to 10 µM CCh. Figure 5 shows typical examples of M2-receptor labeling in a control cell (no exposure to CCh; Fig. 5A) and a cell after a 20-min exposure to CCh (Fig. 5B). In the control cell (Fig. 5A), there was labeling of the M2 receptor in the cell membrane and around the nucleus. In contrast, in the CCh-treated cell (Fig. 5B), M2-receptor labeling in the cell membrane was greatly reduced (presumably as a result of internalization); there was still M2-receptor labeling in the cytosol. The intensity of M2-receptor labeling in the cell membrane was measured in groups of 33–64 cells and is plotted against the duration of the conditioning dose of CCh in Fig. 5D. Figure 5D shows that the intensity of M2-receptor labeling in the cell membrane during a 2-h exposure to CCh was reduced by 83% over a double exponential time course with time constants of 2.9 ± 1.7 and 51.8 ± 26.3 min. In comparison, the time constants for desensitization of IK,ACh under the same conditions were 1.5 ± 0.9 and 26.9 ± 10.8 min (see above).
In the same way that overexpression of GRK2 or -arrestin 2 increased agonist-dependent desensitization of IK,ACh (Fig. 3), it also increased agonist-dependent internalization of the M2 receptor. In response to a 3-min exposure to 10 µM CCh, M2-receptor labeling decreased by 42 ± 5% (n = 80) in control (i.e., untransfected) cells, whereas it decreased significantly more (P < 0.05) (by 64 ± 6%, n = 48, and 72 ± 11%, n = 20, respectively) in cells transfected with GRK2 or -arrestin 2. In the same way that expression of CAM -arrestin 2 caused agonist-independent desensitization of IK,ACh (Fig. 3), it also caused agonist-independent internalization of the M2 receptor: in the absence of agonist, in cells transfected with CAM -arrestin 2 (as compared with 81 control, i.e., untransfected, cells), M2-receptor labeling was reduced by 90 ± 4% (n = 31; P < 0.001).
Colocalization of M2 receptor, clathrin, and caveolin-3. In rat ventricular cells, the M2 receptor has been suggested to be internalized by caveolae (7), although in HeLa cells they are internalized into endosomes of the clathrin-dependent pathway (5). Figure 6, A–C, shows the localization of the M2 receptor and clathrin in a control atrial cell (not exposed to CCh; cell double immunolabeled). In the control cell, the majority of the M2-receptor labeling was in the cell membrane (Fig. 6A), whereas the clathrin labeling was in the form of spots (presumably corresponding to clathrin-coated vesicles) in the cytosol (Fig. 6B). In Fig. 6C, the images of M2-receptor labeling in green and clathrin labeling in red have been superimposed to highlight colocalization of the two proteins (indicated by yellow). In the control cell, there was some colocalization of M2 receptor and clathrin labeling around the nucleus, as shown by yellow spots in Fig. 6C. Figure 6, D–F, shows another series of images from a cell exposed to 10 µM CCh for 2 h. In this cell, the M2-receptor labeling in the cell membrane was reduced and labeling of the M2 receptor was observed in the cytosol in the form of small spots (Fig. 6D). In this cell, clathrin labeling was again in the form of spots in the cytosol (Fig. 6E). The yellow spots in Fig. 6F show that the prominent M2-receptor labeling in the cytosol colocalized with clathrin. Similar results were obtained from 24 atrial cells. This suggests that the M2 receptor is internalized into clathrin-coated vesicles rather than in caveolae. To confirm this, cells were double immunolabeled for the M2 receptor and caveolin-3. Figure 6, G–L, shows the labeling of the M2 receptor (in green) and caveolin-3 (in red) in a control cell not exposed to CCh (Fig. 6, G–I) and a cell exposed to 10 µM CCh for 2 h (Fig. 6, J–L). In the control cell, the majority of both M2-receptor labeling (Fig. 6G) and caveolin-3 labeling (Fig. 6H) was colocalized in the cell membrane, as shown by yellow in Fig. 6I. In the cell exposed to CCh, the majority of M2-receptor labeling was in the cytosol (Fig. 6J), whereas the majority of the caveolin-3 labeling (Fig. 6K) remained in the cell membrane. The intensity of caveolin-3 labeling in cells exposed to 10 µM CCh for 2 h was 97 ± 6% (n = 29) of that in 25 control cells (not exposed to CCh). In the cell exposed to CCh shown in Fig. 6, J–L, there was little colocalization of M2 receptor and caveolin-3 (Fig. 6L).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IK,ACh desensitization and M2-receptor internalization.
The slow desensitization of IK,ACh in heart could be the result of the internalization of the M2 receptor. During a 2-h exposure to 10 µM CCh, IK,ACh declined as a result of slow desensitization by 62% over a double-exponential time course with time constants of 1.5 ± 0.9 and 26.9 ± 10.8 min (Figs. 1 and 2). The first time constant compares well with previous measurements of the time constant of desensitization of IK,ACh, e.g., 2.5 min in guinea pig atrial cells (38) and 1.2 min in rabbit sinoatrial node cells (12), and the second time constant corresponds to a much slower process of desensitization of IK,ACh described by Bünemann et al. (3) and Shui et al. (30, 32). Internalization of the M2 receptor was equally as rapid: under similar conditions, M2-receptor labeling in the cell membrane declined by 83% over a double-exponential time course with time constants of 2.9 ± 1.7 and 51.8 ± 26.3 min (Fig. 5). M2-receptor internalization, therefore, is rapid. Similar rapid internalization has been observed in HEK-293 cells heterologously expressing the M2 receptor: during a 30-min exposure to 1 mM CCh, 75% of the receptor internalized with a time constant of 2.6 min (25). In a study of CHO cells heterologously expressing the M2 receptor, during a 70-min exposure to 10 µM CCh, 62% of the receptor internalized with a time constant of 12.9 min (35). The approximate correspondence in the time courses of IK,ACh desensitization and M2-receptor internalization supports the possibility that IK,ACh desensitization is the result of M2-receptor internalization, although it cannot be ruled out that it is the result of receptor uncoupling immediately preceding internalization.
In the present study, factors known to act on the M2 receptor (GRK2, -arrestin 2, and CAM -arrestin 2) affected IK,ACh in the expected fashion: overexpression of GRK2 and -arrestin 2 increased desensitization of IK,ACh, whereas CAM -arrestin 2 caused desensitization in the absence of agonist (Fig. 3). Overexpression of GRK2 and -arrestin 2 is expected to increase agonist-dependent desensitization by increasing agonist-dependent phosphorylation, uncoupling, and internalization of the M2 receptor, whereas CAM -arrestin 2 is expected to cause agonist-independent desensitization by causing agonist-independent phosphorylation, uncoupling, and internalization of the M2 receptor (see Introduction). Consistent with this, overexpression of GRK2 and -arrestin 2 increased agonist-dependent internalization of the M2 receptor (from 42 ± 5% to 64 ± 6% and 72 ± 11% during a 3-min application of 10 µM CCh), and expression of CAM -arrestin 2 caused an agonist-independent internalization of the M2 receptor of 90 ± 4%. We have observed qualitatively similar actions of GRK2, -arrestin 2, and CAM -arrestin 2 on IK,ACh desensitization in CHO cells heterologously expressing the M2 receptor, Kir3.1, and Kir3.4 (29, 31); because desensitization pathways could vary among different expression systems (21), it was important to establish that the same pathways operate in heart cells.
If desensitization of IK,ACh is largely the result of internalization of the M2 receptor, it follows that there are two phases of slow desensitization of IK,ACh because there are two phases of internalization of the M2 receptor. However, it is not known why there are two phases of internalization.
M2-receptor recycling.
In the present study, after a 20-min exposure to 10 µM CCh, recovery of M2-receptor density in the cell membrane occurred over 2 h, whereas, after a 2-h exposure, recovery required >24 h (Fig. 5). This is reasonable: in HEK-293 cells heterologously expressing the M2 receptor, recovery of M2 receptor density in the cell membrane after a 30-min exposure to 1 mM CCh required 4 h (25), whereas, in guinea pig atrial cells, recovery of IK,ACh after a 24- to 40-h exposure to 10 µM CCh required 72 h (3). In the present study, recovery of the M2 receptor was slower after the longer exposure to agonist; a similar behavior has been observed for the recovery of IK,ACh in guinea pig atrial cells (3, 38) and the recovery of the M2 receptor in HEK-293 cells (25). This behavior suggests that different pathways are involved after short and long exposures to agonist. For example, data from HEK-293 cells suggest that protein synthesis is more involved in recovery of the M2 receptor after a 30-min exposure to CCh than a 10-min exposure (25); perhaps simple recycling of the M2 receptor to the cell membrane occurs after a short exposure (because there is little or no degradation of receptor), but M2-receptor synthesis has to occur after a long exposure (because there has been significant degradation of receptor) (35). In the present study, after an exposure to agonist, although there was an increase in intracellular labeling of M2 receptor, the increase did not match the decrease in membrane labeling of M2 receptor (data not shown), and this suggests that the M2 receptor may be degraded after internalization. After prolonged exposure to agonist, recovery may be slow because gene transcription has to occur [during prolonged exposure to agonist, there is a decrease in M2-receptor mRNA (8, 10, 20, 39)].
Role of other sites in IK,ACh desensitization.
Although the results from the present study suggest that slow desensitization of IK,ACh is largely the result of the M2 receptor, other possible factors cannot be excluded. Our group (32) has previously shown that, after a 24-h exposure to 10 µM CCh, activity of the ACh-activated K+ channel in rat atrial cells is greatly reduced even if the M2 receptor is bypassed and the channel is activated directly by GTP
S (32). This suggests that, as well as the receptor, the G protein and/or the channel is altered during the desensitization process. In the present study, we observed a small but significant reduction in the density of the Kir3.4 channel subunit in the cell membrane after a 2-h exposure to 10 µM CCh (Fig. 4). In embryonic chick atria, there is a decrease in mRNA for Kir3.1 and Kir3.4 during exposure to CCh (at concentrations 
0.5 µM) (20, 33).
Pathway of M2-receptor internalization.
Upon agonist stimulation, many G protein-coupled receptors such as the 2-adrenergic receptor are internalized via a -arrestin- and clathrin-dependent mechanism. -Arrestins are clathrin-binding proteins that act as adaptor molecules to link G protein-coupled receptors to clathrin-coated endocytotic vesicles (9). However, the pathway involved in internalization of the M2 receptor is unclear. Feron et al. (7) reported that, in adult rat ventricular cells, exposure to 100 µM CCh for 15 min caused translocation of the M2 receptor into a membrane fraction containing caveolin-3; this suggests that the M2 receptor is internalized into caveolae rather than in clathrin-coated vesicles. However, in the present study of adult rat atrial cells, after 2-h exposure to 10 µM CCh, the internalized M2 receptor was colocalized with clathrin and not caveolin-3 (Fig. 6), suggesting that the M2 receptor is internalized into clathrin-coated vesicles. The difference between the two studies could be the result of the difference in cell type or duration of exposure to agonist (see below).
Roseberry and Hosey (26) showed that, in HEK-293 cells, the M2 receptor is also internalized by a pathway independent of caveolae; however, they also showed that the pathway is independent of arrestin proteins and clathrin (26). The same group had previously showed that, in HEK-tsA201 cells, whereas desensitization as measured by an adenylyl cyclase assay (perhaps the result of an uncoupling of the receptor) is dependent on arrestins, internalization of the M2 receptor does not (21). This work suggests that internalization of the M2 receptor does not occur via a -arrestin- and clathrin-dependent mechanism, i.e., clathrin-coated pits. However, in HEK-293 cells, treatment with hypertonic sucrose, which is widely reported to specifically inhibit endocytosis through clathrin-coated pits, completely inhibits internalization of the M2 receptor (26). In CHO cells, hypertonic sucrose also inhibits internalization of the M2 receptor (34).
In the present study of neonatal rat atrial cells (Fig. 3) as well as in our study of CHO cells (29), slow desensitization of IK,ACh (activated by the M2 receptor) over 3 min in response to 10 µM ACh was greatly enhanced by expression of -arrestin 2. This enhancement of IK,ACh desensitization could be due to an enhancement of internalization of the M2 receptor (but as stated above it could be due to an enhancement of receptor uncoupling). If correct, these results show that the -arrestin-dependent pathway for internalization is present in the heart. However, they do not show that the -arrestin-dependent pathway is the preferred pathway for internalization (when -arrestin 2 is not overexpressed). In the present study, in adult rat atrial cells not overexpressing -arrestin 2, colocalization of the M2 receptor and clathrin was observed after internalization (Fig. 6F), suggesting that clathrin-coated vesicles are involved in the preferred pathway in the heart.
The data concerning M2-receptor internalization are, therefore, conflicting. However, it may be possible to rationalize the apparently conflicting data. First, cell type could be an important determinant of the pathway for M2-receptor internalization because the arrestin-independent pathway seen in HEK-tsA201 cells is perhaps absent in COS-20 cells (21). Second, in HeLa cells, although in response to CCh the M2 receptor is initially internalized via a clathrin-independent/Arf6-associated pathway, it is quickly transferred to endosomes of the clathrin-dependent pathway (5). Finally, Werbonat et al. (37) argued that most investigators, including their own group (36), have concluded that M2-receptor internalization does not occur via clathrin-coated pits, primarily on the basis that a dominant-negative inhibitor of dynamin (K44A mutant dynamin) does not inhibit it. However, Werbonat et al. (37) argue that this dominant-negative inhibitor is inappropriate, and they show that more appropriate dominant-negative inhibitors of dynamin do inhibit internalization of the M2 receptor. Clearly, there is a need for a more detailed experimental analysis of clathrin-dependent vs. clathrin-independent internalization of the M2 receptor in rat atrial cells in the future.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* T. T. Yamanushi and Z. Shui contributed equally to this work. 
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REFERENCES |
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-Arrestin acts as a clathrin adaptor in endocytosis of the 2-adrenergic receptor. Nature 383: 447–450, 1996.[CrossRef][Medline] subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin. J Biol Chem 267: 2222–2227, 1992.-adrenergic receptor kinase. Biochemistry 28: 4543–4547, 1989.[CrossRef][Medline]-arrestins in receptor signalling and desensitization. J Biol Chem 273: 18677–18680, 1998.-arrestins in the regulation of G-protein-coupled receptors. Nat Rev Neurosci 2: 727–733, 2001.[CrossRef][ISI][Medline] subunits. Nature 370: 143–146, 1994.[CrossRef][Medline]-arrestin 2. J Biol Chem 276: 11691–11697, 2001.
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) and IK,ACh at the end of the test dose (
) plotted against duration of the conditioning dose. Current is normalized to peak IK,ACh during the conditioning dose. Values are means ± SE (n = 4–6 cells). Data are fitted with single-exponential functions with time constants of 1.5 ± 0.9 min (peak IK,ACh) and 0.59 ± 0.95 min (IK,ACh at the end of the test dose). All data are from adult atrial cells. IK,CCh, carbachol chloride (CCh)-activated K+ current.
) and IK,ACh at the end of the test dose (
) of 29.6 ± 10.8 min (peak IK,ACh) and 31.6 ± 12.1 min (IK,ACh at the end of the test dose). C: dependence of fast desensitization of IK,ACh on the conditioning dose. The decline of IK,ACh during the 30-s test dose of ACh (as a percentage of peak IK,ACh during the test dose of ACh) is a measure of fast desensitization and is plotted against the duration (on a logarithmic scale) of the conditioning dose. Data are fitted with a single-exponential function R2 = 0.4, and 
