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Am J Physiol Heart Circ Physiol 280: H2554-H2562, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 6, H2554-H2562, June 2001

Evidence of involvement of GIRK1/GIRK4 in long-term desensitization of cardiac muscarinic K+ channels

Zhigang Shui, Tomoko Takahashi Yamanushi, and Mark R. Boyett

School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cardiac M2 muscarinic receptor/G protein/K+ channel system was studied in neonatal rat atrial cells cultured with and without 10 µM carbachol (CCh) for 24 h. Channel activity in CCh-pretreated cells was substantially reduced as a result of long-term desensitization regardless of whether the channel was activated by ACh in cell-attached patches or GTP in inside-out patches. Channel activity in CCh-pretreated cells was also low when the receptor was bypassed and the G protein and channel were directly activated by [gamma -S]GTP or both the receptor and G protein were bypassed and the channel was directly activated by trypsin. Finally, in CCh-pretreated cells, the whole cell K+ current was low when the channel was activated via the independent adenosine receptor. This suggests that the channel is involved in long-term desensitization. However, in CCh-pretreated cells, although the receptor was internalized, there was no internalization of the channel. We suggest that the function of the muscarinic K+ channel declines in long-term desensitization of the cardiac M2 muscarinic receptor/G protein/K+ channel system.

heart; acetylcholine; muscarinic receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DESENSITIZATION OF THE CARDIAC muscarinic K+ channel to ACh is a complex process that plays an important role in maintaining cardiac sensitivity to vagal nerve stimulation. The muscarinic K+ channel is thought to be composed of two homologous channel subunits (GIRK1 and GIRK4, members of the family of inwardly rectifying K+ channels) (15) and is activated via the M2 muscarinic receptor. On binding of ACh to the receptor, GDP bound to a heterotrimeric G protein is exchanged for GTP. This results in the dissociation of the G protein into alpha - and beta gamma -subunits. The free beta gamma -subunits subsequently bind to and activate the channel (22). During a continuous application of ACh, the activity of the muscarinic K+ channel declines as a result of desensitization to ACh (e.g., Ref. 16). There are three phases of desensitization of the muscarinic K+ current that develop over ~20 s, several minutes, and up to 1-2 days; these phases are referred to here as the fast phase, intermediate phase, and long-term desensitization, respectively (3, 31). The fast phase of desensitization may involve dephosphorylation of the muscarinic K+ channel (12, 13, 24, 31) and a cytosolic protein and a G protein-independent pathway (10). Alternatively, it may be caused by the nucleotide exchange and hydrolysis cycle of the G protein (5). The intermediate phase of desensitization involves a G protein-coupled receptor kinase and perhaps a beta -arrestin (25-27). The receptor kinase is activated by the free beta gamma -subunits after binding of ACh to the receptor (9). During desensitization, the receptor becomes phosphorylated (17, 18): the activated receptor kinase phosphorylates the agonist-bound receptor on the third intracellular loop (21). This causes uncoupling of the receptor from the G protein and, thus, desensitization (19). Binding of a beta -arrestin to the phosphorylated receptor may cause further uncoupling and desensitization (19). Long-term desensitization involves internalization and degradation of the M2 muscarinic receptor perhaps via a beta -arrestin and clathrin-coated pits (1, 7, 19). This is facilitated by the phosphorylation of the receptor by the receptor kinase (28-30). It has also been suggested that long-term desensitization is homologous (i.e., not heterologous) and is, therefore, strictly a receptor phenomenon (3). This is based on the observation that, in guinea pig atrial cells pretreated with 10 µM carbachol (CCh; a stable muscarinic agonist) for 18-48 h, there is a large decrease in whole cell K+ current activated by ACh but not by phospholipid or adenosine (3).

In this study, we obtained evidence that both the M2 muscarinic receptor and the channel are involved in long-term desensitization of the muscarinic K+ channel to CCh. A preliminary account of this work has been communicated to the Physiological Society (23).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. Experiments were carried out in neonatal rat atrial cells because of the ease with which these cells can be cultured. Neonatal rats (1-3 days old) were killed by cervical dislocation. Both right and left atria were removed with fine forceps under a microscope and placed in Hanks' balanced salt solution without Ca2+ (Life Technologies; Paisley, UK). The atrial tissue was then incubated for 10-min periods at 37°C in Hanks' medium containing 0.05% collagenase (type II, Worthington; Lakewood, NJ) and 0.03% trypsin (Sigma; Poole, UK) by weight. The mixture was filtered with nylon gauze, and dissociated cells were sedimented by centrifugation at 180 g for 2 min. The supernatant was removed, and the cells were resuspended in culture medium. This process was repeated five times. Normally, cells from the third or fourth 10-min period were the best. The cells were plated onto fragments of glass coverslip coated with carbon using a Bunsen burner flame and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% horse serum, 100 U/ml penicillin G, 100 µg/ml streptomycin sulphate, and 0.25 µg/ml Fungizone at 37°C in 95% air-5% CO2. All chemicals for cell culture were from Life Technologies and sterilized before use. To investigate long-term desensitization, the neonatal rat atrial cells were cultured with and without 10 µM CCh (Sigma) for 24 h under normal cell culture conditions. Cultured neonatal rat atrial cells on glass fragments were placed directly from the culture medium into the recording chamber with no other treatment.

Chinese hamster ovary cells (CHO-K1) were continuously cultured in Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 50 U/ml penicillin G, and 50 µg/ml streptomycin sulphate at 37°C in 95% air-5% CO2. With the use of Lipofectamine Plus reagent (Life Technologies), the cultured cells were transiently transfected with plasmid vectors for GIRK1 (pEF-GIRK1) and GIRK4 (pEF-CIR) together with the S65T point mutation of green fluorescent protein (GFP) (plasmid pGFP-S65T; Clontech) as a marker for successfully transfected cells. On the day before the DNA transfection, ~1-2 × 106 cells were plated onto dishes with a diameter of 60 mm and cultured in high-glucose DMEM with 10% fetal bovine serum, 50 U/ml penicillin G, 50 µg/ml streptomycin sulphate, and 0.1 mM nonessential amino acid (NEAA) under normal cell culture conditions. About 60-80% cells were confluent on the next day for transfection. On the day of transfection, Lipofectamine Plus reagent and plasmids suspended in DMEM with NEAA were mixed for 15 min to form precomplex DNA at room temperature. The precomplex DNA and diluted Lipofectamine Plus reagent with high-glucose DMEM plus 0.1 mM NEAA were mixed for 15 min at room temperature to make the DNA solution. Final concentrations of the plasmid vectors added during the transfection were 0.0012-0.0058 µg/µl. The DNA solution (517 µl) was added onto cells and mixed gently after the cell culture medium was replaced with 2 ml of high-glucose DMEM plus 0.1 mM NEAA. Cells were incubated with the DNA solution at 37°C until the next day, and 2 ml of high-glucose DMEM with 0.1 mM NEAA and 20% fetal bovine serum were added to each well after 3-5 h of incubation. On the day after transfection, cells were washed twice with 0.01 M of phosphate-buffered saline (PBS; pH 7.4) and incubated with PBS containing 50% trypsin at room temperature for 1 min. Cells were then resuspended in DMEM after incubation at 37°C for 2 min and seeded onto carbon-coated glass coverslips for electrophysiological experiments.

Electrophysiology. Neonatal rat atrial cells with CCh pretreatment, after being placed in a chamber on the stage of a microscope, were washed with high-K+ solution for 10 min to reverse the faster phases of desensitization. High-K+ solution contained (in mM) 140 KCl, 1.8 MgCl2, 5 EGTA, and 5 HEPES; pH 7.4 (titrated with KOH). CHO cells with a middle level of green fluorescence when illuminated by 470- to 490-nm light were chosen for study. Experiments were carried out in the cell-attached and inside-out configurations of the patch-clamp technique with the use of Sylgard-coated pipettes with a resistance of ~6 MOmega and the whole cell configuration with the use of pipettes with a resistance of ~4 MOmega . In the cell-attached configuration, both the pipette and bath contained high-K+ solution; in the case of atrial cells (but not CHO cells), the pipette and bath solution contained 0.1 mM ACh chloride. In the inside-out configuration, the pipette and bath again both contained high K+ solution; in the case of atrial cells (but not CHO cells), the pipette solution contained 0.1 mM ACh; in the case of both atrial and CHO cells, the bath solution did not contain ACh, but 0.1 mM GTP, 0.1 mM [gamma -S]GTP, or 0.2 mg/ml trypsin was added to the bath solution to activate the muscarinic K+ channel. In whole cell patch-clamp experiments, the pipette contained intracellular solution, and the bath contained high-K+ solution (with or without 0.1 mM ACh or 1 mM adenosine). The intracellular solution contained (in mM) 120 potassium aspartate, 20 KCl, 1 KH2PO4, 5.5 MgCl2 (1.8 free Mg2+), 5 EGTA, 5 HEPES, 3 Na2ATP, and 0.1 Na3GTP; pH 7.4 (titrated with KOH). Currents were recorded with an Axopatch-1D amplifier and acquired with pCLAMP software (Axon Instruments; Foster City, CA). Single channel currents were filtered at 5 kHz with an eight-pole Bessel filter and sampled every 0.2 ms, and whole cell currents were filtered at 1 kHz and sampled every 2 ms. The mean product of the apparent number (N) of active channels in a patch and the open probability of a channel (NPo) was calculated for consecutive 200-ms episodes as the mean current during an episode divided by the unitary current. All recordings were made at room temperature and at a holding potential of -60 mV (inside with respect to outside). Statistical tests were carried out using SigmaStat (Jandel Scientific; San Rafael, CA).

Immunocytochemistry. Neonatal rat atrial cells on glass coverslips were incubated with and without 10 µM CCh for 24 h under normal culture conditions. After incubation, the cells were washed with 0.01 M of PBS (pH 7.4). The cells were fixed with 4% paraformaldehyde in PBS for 15 min and then washed with PBS three times. The cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed with PBS three times, and blocked with freshly prepared 10% normal donkey serum (NDS) in PBS for 30 min. Primary antibody, rat anti-M2 muscarinic ACh receptor monoclonal antibody (Chemicon International; Temecula, CA) or rabbit anti-GIRK1 polyclonal antibody (Alomone Labs; Jerusalem, Israel), was diluted 100-fold into PBS containing 1.5% NDS and 1% BSA. The cells were incubated with primary antibody in a humid box at 4°C overnight. Secondary antibody, goat anti-rat IgG (conjugated to FITC) (Chemicon International) or donkey anti-rabbit IgG (conjugated to FITC) (Chemicon International), was diluted 50-fold in the same way as the primary antibody. The cells were incubated with secondary antibody in a humid box at room temperature in the dark for 1 h; before and after the incubation, cells were washed with PBS three times. Finally, the glass coverslips were mounted and 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 (Leica TCS SP; Heidelberg, Germany) equipped with an argon laser, which allowed excitation at a 488-nm wavelength. Images were recorded from the center of a cell. No labeling was detectable without either the primary or secondary antibody. The mean intensity of the muscarinic receptor or GIRK1 labeling from eight random points on the cell membrane in a cell was measured using the Leica TCS analysis system. All statistical analysis was carried out using SigmaStat (Jandel Scientific).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of muscarinic K+ channel by GTP, [gamma -S]GTP, and trypsin in neonatal rat atrial cells cultured with and without CCh for 24 h. Figure 1 shows recordings of muscarinic K+ channel activity made from neonatal rat atrial cells with the cell-attached and inside-out configurations of the patch-clamp technique. Recordings were made in bath and pipette solutions containing symmetrical 140 mM K+ at a holding potential of -60 mV. ACh (10 µM) was included in the patch pipette. Figure 1, A and C, shows single channel currents from typical patches on cells cultured with and without 10 µM CCh for 24 h. The average NPo from eight patches on pretreated cells and seven patches on untreated cells are shown in Fig. 1, B and D.


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  		Fig. 1.   Muscarinic K+ channel currents in neonatal rat atrial cells cultured with and without 10 µM carbachol (CCh) for 24 h. A and C: single muscarinic K+ channel currents in typical patches from pretreated (A) and untreated cells (C). B and D: mean values of the product of the apparent number (N) of active channels in a patch and the open probability of a channel (NPo) of the muscarinic K+ channel from 8 patches on pretreated cells (B) and 7 patches on untreated cells (D). In the beginning, the patches were cell attached. Arrow, point at which patches were excised. ACh (10 µM) was present in the pipette throughout. ACh (10 µM) was present in the bath solution when the patch was cell attached at the start of the experiment (but not subsequently). GTP or [gamma -S]GTP was applied to the intracellular surface of the cell membrane, as shown by the horizontal bars.

Initially, the patches were in the cell-attached configuration, and the muscarinic K+ channel was activated in the normal way via ACh binding to the M2 muscarinic receptor and the consequent activation of the G protein (Fig. 1). The channel activity in cell-attached patches was high in the untreated cells but low in pretreated cells. This is a result of long-term desensitization and is similar to that reported by Bünemann et al. (3). The cell-attached patches were then excised to form inside-out patches. One minute later, GTP was applied for 30 s to activate the channel and check the recording condition. It is important to confirm that after formation of inside-out patches the recording condition is reliable. In some patches, GTP could not activate the channel, and, in other patches, other channels (such as the ATP-sensitive K+ channel or the inward rectifier K+ channel) were present. Only patches that contained only the muscarinic K+ channel were used for analysis. Finally, the channel was activated by a nonhydrolyzable analog of GTP ([gamma -S]GTP) after the washout of GTP for 1 min. [gamma -S]GTP is widely used to bypass the receptor and activate the G protein and, therefore, the channel directly (e.g., Ref. 2) (see DISCUSSION for further consideration of the site of action of [gamma -S]GTP). The channel activity in the pretreated cells was substantially reduced as a result of long-term desensitization regardless of whether the channel was activated by GTP or [gamma -S]GTP in the inside-out patches. This suggests that not only the muscarinic receptor is involved in long-term desensitization, and the G protein or more likely the channel is also involved.

To confirm this conclusion, another set of experiments with the same procedure as above was carried out using trypsin instead of [gamma -S]GTP. It is known that trypsin is able to activate the muscarinic K+ channel in the absence of muscarinic agonist in an irreversible manner (14). Figure 2, A and C, shows single channel currents from typical patches on cells with and without CCh pretreatment. The average NPo from five patches on pretreated cells and from five patches on untreated cells are shown in Fig. 2, B and D. Figure 2 shows that the channel activity in the pretreated cells was also low during application of trypsin.


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  		Fig. 2.   Activation of muscarinic K+ channel currents by trypsin in neonatal rat atrial cells cultured with and without 10 µM CCh for 24 h. A and C: single muscarinic K+ channel currents in typical patches from pretreated (A) and untreated cells (C). B and D: mean value of NPo of the muscarinic K+ channel from 5 patches on pretreated cells (B) and 5 patches on untreated cells (D). In the beginning, patches were cell attached. Arrow, point at which the patches were excised. ACh (10 µM) was present in the pipette throughout. ACh (10 µM) was present in the bath solution when the patch was cell attached at the start of the experiment (but not subsequently). GTP or trypsin was applied to the intracellular surface of the cell membrane, as shown by the horizontal bars.

Figure 3 summarizes the results above. The average NPo of the muscarinic K+ channel in the cells with and without CCh pretreatment during application of GTP, [gamma -S]GTP, and trypsin are shown in Fig. 3A. The channel activity in pretreated cells was significantly lower (P < 0.01) as a result of long-term desensitization regardless of whether the channel was activated by GTP, [gamma -S]GTP, or trypsin. The percent decrease in channel activity in pretreated cells was calculated in two ways: In the first case, the difference in mean channel activity between untreated and pretreated cells was expressed as a percentage of the mean channel activity in untreated cells; this resulted in decreases in channel activity after pretreatment of 85.7, 91.9, and 76.9% when the channel was activated by GTP, [gamma -S]GTP and trypsin, respectively. In the second case, pretreated and untreated cells from same heart were randomly paired (cells from three hearts used), a percent change was calculated for each pair, and from these values a mean ± SE was calculated. As shown in Fig. 3B, this resulted in decreases in channel activity after pretreatment of 86.2 ± 11.2, 89.6 ± 6.5, and 69.3 ± 27.3% when the channel was activated by GTP, [gamma -S]GTP and trypsin, respectively.


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  		Fig. 3.   Summary of the activation of the muscarinic K+ channel by GTP, [gamma -S]GTP, and trypsin in neonatal rat atrial cells cultured with and without 10 µM CCh for 24 h. A: mean (+SE) values of NPo of the muscarinic K+ channel during exposures to GTP, [gamma -S]GTP, and trypsin in neonatal rat atrial cells with and without CCh pretreatment. B: extent of long-term desensitization of the muscarinic K+ channel. The decrease in NPo of the muscarinic K+ channel (activated by one of the agonists) in pretreated cells is shown as a percentage of NPo from untreated cells. The means + SE are shown (see text for details). Numbers in parentheses are the number of cells used.

Effect of trypsin on the reconstituted channel in CHO cells. The finding that channel activity was also depressed when the channel was activated by trypsin confirms that long-term desensitization does not only involve the receptor. This conclusion rests on the assumption that trypsin is able to bypass the receptor. To test this assumption, the effect of trypsin on the reconstituted channel in CHO cells was studied. CHO cells were transiently transfected with plasmid vectors for the channel proteins GIRK1 and GIRK4 as well as GFP but not the M2 muscarinic receptor. The procedure of the experiment was the same as above, but no ACh was included in the pipette solution.

Figure 4A shows a current trace from a typical patch on a CHO cell transfected with GIRK1 and GIRK4. The channel activity was low when the patch was cell attached and during application of GTP when the patch was inside-out. This is to be expected because activation in either case requires the presence of both agonist and receptor (both absent). However, the channel was active during application of trypsin. Figure 4, B and C, shows typical examples of an open time histogram and amplitude distribution histogram of the channel during application of trypsin. Both the mean open time (1.17 ± 0.34 ms, n = 4) and single channel conductance (36.7 ± 5.3 pS, n = 4) were similar to those in atrial cells. This indicates that trypsin is able to activate the channel in the absence of agonist and the M2 muscarinic receptor. It was also checked that trypsin did not activate any channels in CHO cells not transfected with GIRK1 and GIRK4 (n = 4).


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  		Fig. 4.   Activation of the muscarinic K+ channel by trypsin in Chinese hamster ovary (CHO) cells transfected with GIRK1 and GIRK4 but not the M2 muscarinic receptor. A: single muscarinic K+ channel currents in a typical patch from a CHO cell transfected with GIRK1 and GIRK4. In the beginning, the patch was cell attached. Arrow, point at which the patch was excised. GTP and trypsin were applied to the intracellular surface of the cell membrane, as shown by the horizontal bars. No muscarinic agonist was present in the pipette. B: open time histogram during application of trypsin. The channel open time was measured at 50% of the unitary single channel current amplitude, and the distribution was fitted with a single exponential function with the time constant (tau ; mean open time) shown. C: amplitude histogram of the channel current during application of trypsin.

Localization of the M2 muscarinic receptor and GIRK1 in neonatal rat atrial cells with and without CCh pretreatment. Figure 5 shows the distribution of the M2 muscarinic receptor and the muscarinic K+ channel subunit GIRK1 in neonatal rat atrial cells with and without CCh pretreatment. Cells were labeled with anti-M2 muscarinic receptor or anti-GIRK1 antibody. Typical examples of M2 muscarinic receptor labeling in an untreated cell and in a pretreated cell are shown in Fig. 5, A and B. In the untreated cell, the M2 muscarinic receptor label was on the cell membrane, whereas in the pretreated cell a substantial portion of the M2 muscarinic receptor label was within the cytoplasm and little was on the cell membrane. Figure 5, C and D, shows typical examples of GIRK1 labeling in an untreated cell and in a pretreated cell. In both cases, the majority of GIRK label was on the cell membrane.


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  		Fig. 5.   Distributions of M2 muscarinic receptor and GIRK1 in neonatal rat atrial cells cultured with and without 10 µM CCh for 24 h. A: untreated cell labeled with anti-M2 muscarinic receptor antibody. B: pretreated cell labeled with anti-M2 muscarinic receptor antibody. C: untreated cell labeled with anti-GIRK1 antibody. D: pretreated cell labeled with anti-GIRK1 antibody. E: mean + SE intensities of labeling of the M2 muscarinic receptor and GIRK1 in cells from 3 cell isolations with and without (control) CCh pretreatment. Numbers in parentheses are the number of cells used.

The mean intensity of M2 muscarinic receptor and GIRK1 labeling is summarized in Fig. 5E. The results were obtained from three independent cell isolations and three labeling treatments. The mean intensity of labeling of the M2 muscarinic receptor in untreated cells was significantly higher (P < 0.01) than that in pretreated cells. There was no detectable difference in the GIRK1 labeling intensity between untreated and pretreated cells.

Activation of whole cell K+ currents by ACh and adenosine in neonatal rat atrial cells with and without CCh pretreatment. According to the experiments above, long-term desensitization to CCh should be heterologous and the response to any agonist for which the muscarinic K+ channel is the effector should be depressed. However, in guinea pig atrial cells, Bünemann et al. (3) suggested that long-term desensitization is homologous because the response to ACh was depressed and the response to adenosine (assumed to activate the muscarinic K+ channel as well) was not. To resolve this discrepancy, we investigated whole cell K+ current activated by ACh and adenosine in neonatal rat atrial cells with and without CCh pretreatment. Figure 6, A and B, shows whole cell currents from a pretreated cell and an untreated cell. Intracellular and extracellular solutions contained 140 mM K+. ATP (3 mM) and GTP (0.1 mM) were present in the intracellular solution. ACh or adenosine at saturating concentrations was applied for 30 s to activate K+ current. Figure 6C summarizes the results obtained. Compared with untreated cells, in pretreated cells, the whole cell K+ current induced by ACh or by adenosine was greatly decreased, by 91 and 62%, respectively. In pretreated cells, the whole cell K+ current activated by adenosine was significantly greater than that activated by ACh. The results are different from those from guinea pig atrial cells (3). A possible explanation is given in the DISCUSSION.


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  		Fig. 6.   Whole cell K+ currents activated by ACh and adenosine (Ado) in neonatal rat atrial cells cultured with and without 10 µM CCh for 24 h. A and B: whole cell K+ current activated by ACh or adenosine from a pretreated cell (A) and an untreated cell (B). ACh or adenosine was applied for 30 s, as shown by the horizontal bars. Arrows, zero current. C: mean + SE amplitudes of whole cell current during application (from 10 to 30 s during a 30-s application) of ACh or adenosine. Numbers in parentheses are the number of cells used.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, evidence has been obtained to show that, in neonatal rat atrial cells during long-term desensitization caused by pretreatment with CCh for 24 h, a large portion of the muscarinic receptor is internalized and the function of the muscarinic K+ channel declines.

Possible role of long-term desensitization of muscarinic K+ current. Previous studies have shown that block of vagal activity by atropine almost doubles the heart rate in healthy resting young men (11) and in conscious dogs (8). Therefore, vagal tone is responsible for ~50% decrease in the heart rate under physiological conditions. This shows that the heart is continuously in the presence of ACh, and this is likely to induce long-term desensitization of the muscarinic K+ channel. This is supported by evidence that, in guinea-pig atrial cells cultured over several days, there is an increase (half-time ~2 days) in the sensitivity of the muscarinic K+ current to ACh and in the density of the M2 muscarinic receptor (4). The increase could be the result of recovery from long-term desensitization in vivo. The normal function of long-term desensitization may be to maintain the sensitivity of the heart to vagal stimulation at an appropriate level.

Is long-term desensitization of the muscarinic K+ channel homologous? Homologous and heterologous desensitization are distinct processes. Homologous desensitization is agonist and receptor specific and causes a decrease in response to agonists acting via the one receptor only, whereas heterologous desensitization is agonist and receptor nonspecific and causes a reduction in response to other agonists acting via other receptors.

Bünemann et al. (3) studied muscarinic K+ current in adult guinea pig atrial cells cultured with and without 10 µM CCh for 18-48 h. Compared with untreated cells, in cells cultured with CCh, there was a large decrease in the amplitude of the whole cell K+ current activated by ACh but not by phospholipid or adenosine acting via other receptors. On the basis of the prerequisite that ACh, phospholipid, and adenosine activate the same channel via the same signaling pathway, they suggested that long-term desensitization is homologous and is a receptor phenomenon. In the present study of neonatal rat atrial cells, the internalization of the M2 muscarinic receptor is presumably a homologous desensitization process. However, even though [gamma -S]GTP, trypsin, and adenosine do not activate the muscarinic K+ channel via the M2 muscarinic receptor (instead they activate the channel via the G protein, the channel itself, or the A1 purinergic receptor; see below), pretreatment with CCh depressed the response to [gamma -S]GTP, trypsin, and adenosine as well as ACh. This proves that, in neonatal rat atrial cells, a component of long-term desensitization is heterologous. Therefore, in neonatal rat atrial cells, long-term desensitization may have both homologous and heterologous components. The difference between the present study and that of Bünemann et al. (3) could be a species difference or a difference between the neonate and adult.

Involvement of sites other than M2 muscarinic receptor in long-term desensitization. To understand the mechanisms underlying long-term desensitization of the muscarinic K+ current, the current was activated by [gamma -S]GTP or trypsin rather than ACh. [gamma -S]GTP, like GTP, is able to activate the G protein (by exchanging for GDP on the G protein and causing dissociation of the G protein into alpha - and beta gamma -subunits). However, in the presence of [gamma -S]GTP, unlike in the presence of GTP, the G protein cannot be deactivated (deactivation is the result of the hydrolysis of GTP by the free alpha -subunit and the reformation of the G protein trimer; [gamma -S]GTP, unlike GTP, cannot be hydrolyzed). Although the action of [gamma -S]GTP initially depends on the presence of ACh-bound receptor to facilitate GDP-[gamma -S]GTP exchange (ACh was present in the experiments of Fig. 1), its action is subsequently independent of ACh and the receptor. [gamma -S]GTP is widely used to bypass the receptor and activate the G protein and, therefore, the channel directly (2, 16, 24, 31). Kirsch and Brown (14) first showed that trypsin, which cleaves proteins at both arginine and lysine residues, irreversibly activated the muscarinic K+ channel (in neonatal rat atrial cells). Trypsin activated the channel in the absence of ACh (14). In the present study, it was confirmed that trypsin activated the muscarinic K+ channel (in adult rat atrial cells) in the absence of muscarinic agonist (data not shown). This suggests that trypsin activation of the muscarinic K+ channel does not occur via the receptor. In the present study, the trypsin activation of the muscarinic K+ channel (GIRK1/GIRK4) expressed in CHO cells not transfected with the M2 muscarinic receptor (Fig. 4) confirms this. Kirsch and Brown (14) showed that trypsin activated the channel in the absence of intracellular Mg2+ and nucleotides, and this makes it unlikely that trypsin is activating the channel by the G protein. It is, therefore, likely that trypsin activates the channel by acting on the channel itself or on an ancillary regulatory protein (14). Trypsin affects the ATP-sensitive K+ channel by proteolytic cleavage (6).

The present study has shown that, as a result of long-term desensitization, channel activity in pretreated cells is still low when the channel is activated by [gamma -S]GTP or trypsin. We conclude that a site other than the M2 muscarinic receptor is involved in long-term desensitization. Other likely sites are the G protein and the channel itself. No evidence in favor of the G protein has been obtained in the present study, although this does not rule out an involvement of the G protein. If trypsin activates the channel by acting on the channel itself, then long-term desensitization must involve a modification of the channel that is not reversed by trypsin. One possibility, an internalization of the channel (similar to the internalization of the receptor) during long-term desensitization, was ruled out in the present study (Fig. 5). The possible mechanism underlying such a decline in function of the channel is unknown. It is interesting that the channel has also been implicated in the fast phase of desensitization (see introduction).

Involvement of the M2 muscarinic receptor in long-term desensitization. It is known that long-term desensitization of G protein-coupled receptors involves internalization (or sequestration) and downregulation (or degradation) of the receptors. For the first time, this study has demonstrated an internalization of the M2 muscarinic receptor in the heart during long-term desensitization (Fig. 5). Phosphorylation of agonist-bound receptors on the third intracellular loop by activated receptor kinase may play a role in this process. It has been reported that the sequestration of receptors is greatly reduced in CHO cells transfected with a mutant M2 muscarinic receptor (m2LD) lacking the third intracellular loop (20, 29). The sequestration of the M2 muscarinic receptor could be enhanced by overexpression of the receptor kinase and could be reduced by expression of a mutant form of the receptor kinase (DN-GRK2) lacking kinase activity (30). We have previously shown that, after 24 h pretreatment with 10 µM CCh, long-term desensitization of muscarinic K+ current occurred in CHO cells transfected with the M2 muscarinic receptor and receptor kinase as well as the channel (GIRK1/GIRK4) (28). However, the long-term desensitization of the muscarinic K+ current was greatly reduced in CHO cells transfected 1) without the receptor kinase, 2) with a mutant receptor lacking phosphorylation sites rather than the wild-type receptor, or 3) with a mutant receptor kinase lacking kinase activity rather than the wild-type receptor kinase.

In the summary, postulated events during long-term desensitization of the muscarinic K+ current are as follows: ACh binds to the M2 receptor, activating not only the G protein and the muscarinic K+ channel but also receptor kinase. The activated receptor kinase binds to the agonist-bound receptor and phosphorylates the third intracellular loop of the receptor. This uncouples the receptor from the G protein. The phosphorylation of the receptor promotes the binding of a beta -arrestin to the receptor, which leads to receptor sequestration and downregulation. In addition to the decrease in function of the receptor during long-term desensitization, there is a decrease in the function of the muscarinic K+ channel, although we do not know the mechanism underlying the change in the muscarinic K+ channel itself during long-term desensitization.


    FOOTNOTES

Address for reprint requests and other correspondence: M. R. Boyett, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9JT, UK (E-mail: m.r.boyett{at}leeds.ac.uk).

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.

Received 22 September 2000; accepted in final form 12 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(6):H2554-H2562
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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