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1 School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom; 2 Department of Occupational Diseases, National Institute of Industrial Health, Ministry of Labour, Kanagawa 214; and 3 Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Desensitization of the cardiac muscarinic K+ channel was studied in cultured neonatal rat atrial cells and in Chinese hamster ovary (CHO) cells transfected with muscarinic receptor (HM2), G protein-coupled inward rectifying K+ channels 1 and 4, and G protein-coupled receptor kinase 2. In atrial cells incubated in 10 µM carbachol for 24 h, channel activity in cell-attached patches was substantially reduced as a result of long-term desensitization. The long-term desensitization was also observed in CHO cells transfected with the wild-type receptor and receptor kinase (as well as the channel). However, long-term desensitization was greatly reduced or abolished if the cells were 1) not transfected with the receptor kinase, 2) transfected with a mutant receptor lacking phosphorylation sites (rather than the wild-type receptor), or 3) transfected with a mutant receptor kinase lacking kinase activity (rather than the wild-type receptor kinase). We suggest that long-term desensitization of the cardiac muscarinic receptor-K+ channel system to muscarinic agonist may involve phosphorylation of the receptor by receptor kinase.
heart; acetylcholine; potassium current
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACETYLCHOLINE (ACh)
is released by the parasympathetic nerves to the heart, and it exerts
negative chronotropic, dromotropic, and inotropic effects, principally
by activating the muscarinic K+ channel (3, 4, 28,
39). ACh binds to the M2 muscarinic receptor. This
results in the dissociation of a trimeric G protein into a 
-subunit
and 
-complex. The -complex activates the muscarinic
K+ channel (30). In the presence of ACh, the
chronotropic, dromotropic, and inotropic effects of ACh fade (3,
5, 23). This may principally be the result of an
"inactivation" of the muscarinic K+ channel as a result
of desensitization (3, 14). There are at least three
phases of desensitization of the muscarinic K+ channel: a
fast phase developing over ~20 s, one or more intermediate phases
developing over several minutes, and a slow phase developing over
24-48 h (6, 17, 40). The slow phase is referred to as
long-term desensitization in this study.
Fast desensitization is heterologous: adenosine and phospholipids, acting via A1 and phospholipid receptors, respectively, also activate the muscarinic K+ channel; adenosine and phospholipids like ACh elicit fast desensitization and thus reduce the response to ACh (6, 20). This is consistent with fast desensitization being a channel phenomenon (the channel, but not the receptor, is common to the ACh, adenosine, and phospholipid pathways). The fast phase of desensitization is thought to be the result of a dephosphorylation of the muscarinic K+ channel (13, 17, 18, 31, 40). Bender et al. (2) have recently shown that the fast phase of desensitization is a function of the G protein-coupled inward rectifying K+ channel 1 (GIRK1) subunit of the muscarinic K+ channel (heterotetramer of GIRK1 and GIRK4): the fast phase of desensitization is observed when GIRK1 and GIRK4 are expressed as a heterotetramer and when GIRK1 is expressed as a homotetramer, but not when GIRK4 is expressed as a homotetramer. This supports the view that the fast phase of desensitization is a channel phenomenon. As an alternative, it has been suggested that the fast phase of desensitization is caused by the nucleotide exchange and hydrolysis cycle of the G protein (8).
Recently, the intermediate phase of desensitization has been proposed
to be the result of a depletion of phosphatidylinositol 4,5-bisphosphate (19), but this has been argued against
(24). Instead, it is possible that the intermediate phase
of desensitization is the result of the action of a receptor kinase
(34). Associated with G protein-coupled receptors, such as
the M2 muscarinic receptor, is a family of G
protein-coupled receptor kinases (GRKs) (9, 12). The
M2 muscarinic receptor is associated with a muscarinic receptor kinase but it is similar to or identical to the -adrenergic receptor kinase (12). After ACh is bound to the
M2 muscarinic receptor and after the dissociation of the G
protein, the -complex, in addition to activating the channel,
binds to and activates the receptor kinase in the presence of the
receptor (11). In vitro, the receptor kinase, once
activated, phosphorylates the agonist-bound M2 muscarinic
receptor (11). In vivo, the M2 muscarinic receptor in the heart is phosphorylated during exposure to an agonist
(21, 22), and the receptor kinase is presumably
responsible for this. The phosphorylation sites on the M2
muscarinic receptor are found on the third intracellular loop of the
receptor (9, 27, 29). Evidence for a role of receptor
kinase in the intermediate phase of desensitization comes from our
earlier studies (32-34), including a study of the
muscarinic receptor-K+ channel system reconstituted in a
cell line. The activated receptor kinase may bring about the
intermediate phase of desensitization in two ways: 1) by
simply binding to the receptor, and 2) by phosphorylating the receptor and facilitating the binding of a -arrestin (33, 34). Both the binding and the phosphorylation (and -arrestin binding) may cause desensitization by uncoupling the receptor from the
G protein (33, 34).
Long-term desensitization of the muscarinic K+ channel [induced by a 24- to 48-h exposure to the muscarinic agonist carbachol (CCh)] has been studied by Bünemann et al. (6) in cultured guinea pig atrial cells. During agonist washout, recovery from long-term desensitization occurs after a delay of several hours with a half time of ~20 h (6). Bünemann et al. (6) showed that long-term desensitization in cultured guinea pig atrial cells is homologous (not heterologous): exposure to a muscarinic agonist for 18-48 h greatly reduces the amplitude of the muscarinic K+ current activated by ACh, but not adenosine or phospholipid. In contrast, long-term desensitization in cultured rat atrial cells may have homologous and heterologous components (35). This suggests that long-term desensitization is in full or in part a receptor phenomenon, and Shui et al. (35) have shown that during long-term desensitization in cultured rat atrial cells the M2 muscarinic receptor is sequestered (internalized) from the outer cell membrane. Phosphorylation of the M2 muscarinic receptor by the activated receptor kinase may facilitate sequestration (and subsequent downregulation) of the receptor (9, 36). The aim of the present study was to determine whether receptor kinase plays a role in the long-term desensitization of the muscarinic K+ channel by using the muscarinic receptor-K+ channel system reconstituted in a cell line.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture.
Neonatal rat atrial cells were prepared as described previously
(35). Chinese hamster ovary (CHO-K1) cells were cultured and transiently transfected as described previously (34).
CHO cells were transiently transfected to introduce further DNA
plasmids to cell lines already stably transfected with plasmid vectors for wild-type human M2 muscarinic receptor HM2
(pEF-Myc-HM2) or a large deletion (LD) mutant of the
HM2 receptor M2LD (pEF-M2LD) and
possibly GRK2/ARK1 (pEF-GRK2). In the case of
M2LD, residues 233 to 380 encompassing most of the
third intracellular loop were deleted. In all cases, the cells were
transiently transfected with plasmid vectors for GIRK1 (pEF-GIRK1) and
GIRK4 (pEF-cardiac inward rectifier); GIRK1 and GIRK4 together form the
muscarinic K+ channel heteromultimer. In some
cases, the cells were transiently transfected with plasmid vector for
GRK2/ARK1 (pEF-GRK2) or its mutant DN-GRK2/GRK2-K220W
(pEF-GRK2-K220W). In the case of the mutant receptor kinase, a lysine
residue at position 220 within the catalytic domain of the protein was
substituted by a tryptophan residue. Finally, all cells were
transiently transfected with plasmid vector for the S65T point mutation
of green fluorescent protein (pGFP-S65T, Clontech) as a marker for
successfully transfected cells. The final concentrations of each of the
plasmid vectors added during transient transfections were as follows
(in ng/ml): 400 GIRK1, 400 GIRK4, 400 GRK2, 400 DN-GRK2, and 200 GFP.
Transfecting solution (10 ml) was added to ~1-2 × 106 cells in a 100-mm-diameter plastic tissue culture dish.
The expression levels of the wild-type receptor and receptor kinase in
the stably transfected cells are described in the study by Shui et al.
(34). GFP was used as a marker for successfully
transfected and expressing cells: cells expressing GFP fluoresced when
illuminated with 488-nm light. The assumption is that a cell
transfected with GFP had also been transfected with the other
components. To confirm that CHO cells expressing GFP were expressing at
least one of the channel proteins, immunofluorescence was used: cells
expressing GFP showed labeling by anti-GIRK1 (Alomone Laboratories;
Jerusalem, Israel). Three different CHO clones, i.e., stably
transfected CHO cell lines, were used (see Figs. 1-4 legends for
details). There was no discernible difference between clones: in the
three clones used, peak channel activity (cell-attached
patches; receptor kinase present) was similar (not shown). In the
present study, the experiments, with and without CCh pretreatment, were
carried out on different cells, because it is not possible to make
electrophysiological measurements on the same cells before and after
24-h CCh pretreatment. For this reason, we compared results from sets
of 7 to 18 cells with and without pretreatment. In all respects, the
cells were treated in the same way (apart from the difference in CCh
pretreatment). For each set of CHO cells, the cells were from two to
nine transfections. We observed no obvious variation between cells from
different transfections (but otherwise transfected and treated in the
same way). Control experiments on CHO cells (i.e., experiments in which the CHO cells were not pretreated with CCh) are taken from a previous study (34). In one case (see Fig. 2A), the CHO
cells were transfected with four components, whereas in the remainder
they were transfected with five. It could thus be argued that
transfection with a greater number of components may affect expression
levels. However, channel activity was least in the case in which the
cells were transfected with just four components, and intuitively this
is not what would be expected. In one case (Fig.
1), the CHO cells were stably transfected with two components, whereas in the remainder they were stably transfected with just one. However, similar results to those in Fig. 1
were obtained when cells stably transfected with just one component
were used instead; see RESULTS for details.
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Electrophysiology.
Faster desensitization processes were reversed by a 10-min washout of
CCh before patch clamp, after the cells were placed in the chamber that
was mounted on a Nikon Diaphot microscope. Experiments were carried out
in the cell-attached and whole cell configurations of
the patch-clamp technique using ~5 M
Sylgard-coated or ~4
M uncoated pipettes, respectively. Extracellular solution contained
(in mM) 140 KCl, 1.8 MgCl2, 5 EGTA, and 5 HEPES, pH 7.4. ACh (10 µM) was added to this solution when required. Intracellular solution contained (in mM) 120 potassium aspartate, 20 KCl, 1 KH2PO4, 2.8 MgCl2 (free
Mg2+, 1.8); 5 EGTA, 5 HEPES, 0.1 Na3GTP, and 3 Na2ATP, pH 7.4. Currents were recorded with an Axopatch-1D
amplifier and acquired with pCLAMP software (Axon Instruments; Union
City, CA). Patch 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 2 kHz and sampled every 1 ms. The product of the apparent
number of active channels in a patch and the channel open probability
(NPo) was calculated for consecutive 200-ms
episodes as the mean current during an episode divided by the unitary
current. A decline in channel activity was fitted with a single
exponential function with a least squares method using SigmaPlot (SPSS;
Chicago, IL). All recordings were made at room temperature and from a
holding potential of 
60 mV (inside with respect to outside).
Statistical tests were carried out using SigmaStat software (SPSS).
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To investigate long-term desensitization, cells were pretreated with 10 µM CCh (Sigma Chemical), a stable muscarinic agonist, for 24 h under normal culture conditions. In the case of cells not pretreated with CCh, the cells were not exposed to CCh and were used at the same time postisolation or posttransfection as those with CCh pretreatment.
Long-term desensitization.
Figure 1, A-C, shows recordings of muscarinic
K+ channel activity made from neonatal rat atrial cells
with the cell-attached configuration of the patch-clamp technique. ACh
(10 µM) was included in the patch pipette to activate the muscarinic
K+ channel. Recordings were made in bathing and pipette
solutions containing 140 mM K+ and at a holding potential
of 60 mV. Figure 1, A and B, shows multiple
single-channel currents; the currents are inward, but are shown as
upward deflections to allow comparison with Fig. 1C. The
single channel conductance and mean open time were typical of the
muscarinic K+ channel (not shown). Figure 1, A
and B, shows examples of channel activity recorded from
patches on control and test cells, respectively, over the first 3 min
after attachment of the pipette. Test cells (Fig. 1B) were
pretreated with 10 µM CCh (a stable muscarinic agonist) for 24 h. CCh was washed off for 10 min before the pipette was attached and
recorded to reverse short-term desensitization processes. Control cells
(Fig. 1A) were not pretreated with CCh. Figure 1A
shows that channel activity in the patch on the untreated cell was
high; there is often more than one channel in the patch (31,
32). In addition, in the patch on the untreated cell, there was
a fade in channel activity over the 3 min after the attachment of the
pipette, as previously described (32). This is short-term
desensitization (more specifically the intermediate phase of
desensitization) (32). In the patch on the pretreated cell, in contrast, channel activity was very low, and, furthermore, there was no decrease in activity over the 3 min after attachment of
the pipette (Fig. 1B). Figure 1C shows the
average NPo for 11 untreated and 12 pretreated
cells over the first 3 min after attachment of the pipette. Figure
1C confirms that channel activity was high in untreated
cells, but was low in cells pretreated with 10 µM CCh for 24 h.
The marked decline in channel activity after pretreatment cannot be
attributed to short-term desensitization because this would have been
reversed during the 10-min washout of CCh before recording
(40). Instead, it is attributed to long-term desensitization and is similar to that reported by Bünemann
et al. (6). Figure 1C also confirms that in
untreated cells channel activity declined as a result of the
intermediate phase of desensitization: it declined by 63 ± 11%
(n = 11). The decline was fitted with a single
exponential function with a time constant of 218 s. In contrast,
the average value of NPo remained low throughout
the 3 min for cells pretreated with CCh. In the cells pretreated with CCh, the decline in channel activity as a result of the intermediate phase of desensitization was largely absent.
Effect of absence of GRK2 on long-term desensitization. In Fig. 2, each trace shows the mean NPo from 7 to 18 patches during the first 3 min after attachment of the ACh-containing pipette. Data from untreated and CCh-pretreated cells are superimposed to show the effect of long-term desensitization. The results were obtained with four different transfection schemes, as described below. In all cases, the cells were transfected with the GIRK1 and GIRK4 channel subunits. In the first case, the cells were transfected with the wild-type receptor HM2 but not the receptor kinase GRK2 (Fig. 2A). In the control cells transfected with the wild-type receptor kinase GRK2, there was a marked decrease in channel activity after pretreatment with CCh (Fig. 1F), whereas in cells that were not transfected with the receptor kinase, there was only a small decrease in channel activity after CCh pretreatment (Fig. 2A). A possible implication of this finding is that receptor kinase is involved in long-term desensitization. However, there is an alternative explanation: in the absence of the receptor kinase (Fig. 2A), channel activity was already depressed (see below). Perhaps there was no scope for further desensitization. The results obtained with other transfection strategies (described below) are less ambiguous and support a role for the receptor kinase in long-term desensitization.
The absence of the receptor kinase GRK2 also affected the intermediate phase of desensitization, as we have reported before (34). Channel activity declined by 12 ± 12% (n = 8) in untreated cells and by 15 ± 12% (n = 12) in pretreated cells over the first 3 min after attachment of the pipette (Fig. 2A), whereas in the control cells (stably transfected with the wild-type receptor kinase GRK2), it declined by 75 ± 5% (Fig. 1F).Effect of transfection with mutant receptor lacking phosphorylation sites on long-term desensitization. To test whether phosphorylation of the receptor by the receptor kinase is involved in long-term desensitization, in the second case (Fig. 2B), the cells were transfected with the wild-type receptor kinase GRK2 and a deletion mutant of the receptor M2LD, which has residues P233 to S380 deleted. These residues are in the third intracellular loop, a relatively acidic region that is serine and threonine rich and is the region containing all but one of the known phosphorylation sites on the M2 muscarinic receptor (9, 27, 29). This deletion mutant, while having the same affinity and selectivity for the agonist and activating G protein in the same way, is not phosphorylated by the receptor kinase GRK2 while agonist bound (15). Without CCh pretreatment, peak channel activity in cells transfected with the mutant receptor M2LD was comparable to channel activity in cells transfected with the wild-type receptor HM2. Figure 2B shows that pretreatment of the cells transfected with the mutant receptor M2LD with CCh resulted in a decrease in channel activity, but the decrease was substantially smaller than that in the control cells transfected with the wild-type receptor HM2 (Fig. 1F). This shows that the third intracellular loop of the receptor is also involved in long-term desensitization.
Transfection with the mutant receptor M2LD also affected the intermediate phase of desensitization, as we have reported before (34). Channel activity declined by 20 ± 11% (n = 18) in untreated cells and by 24 ± 16% (n = 8) in pretreated cells over the first 3 min after attachment of the pipette (Fig. 2B), whereas in the control cells (transfected with the wild-type receptor HM2), it declined by 75 ± 5% (Fig. 1F).Effect of transfection with mutant receptor kinase lacking kinase activity on long-term desensitization. To test further whether phosphorylation of the receptor by the receptor kinase is involved in long-term desensitization, in the third case (Fig. 2C), cells were transfected with the wild-type receptor HM2 and a dominant negative (DN) mutant of the receptor kinase DN-GRK2 with the lysine residue at position 220 substituted with tryptophan. This mutant receptor kinase has no ability to phosphorylate agonist-bound M2 muscarinic receptor (37). Peak channel activity in cells transfected with the mutant receptor kinase DN-GRK2 was comparable to channel activity in cells transfected with the wild-type receptor kinase GRK2. Figure 2C shows that pretreatment of the cells transfected with the mutant receptor kinase DN-GRK2 with CCh resulted in little decrease in channel activity as a result of long-term desensitization. This is consistent with the hypothesis that long-term desensitization involves phosphorylation of the third intracellular loop of the receptor by the receptor kinase.
In cells transfected with the mutant receptor kinase DN-GRK2, the decline of channel activity during the intermediate phase of desensitization was still substantial (Fig. 2C), but it was reduced compared with that in the control cells transfected with the wild-type receptor kinase GRK2 (Fig. 1F), as we have reported before (34). Channel activity declined by 46 ± 7% (n = 17) in untreated cells and by 38 ± 9% (n = 7) in pretreated cells over the first 3 min after attachment of the pipette (Fig. 2C), whereas in the control cells (transfected with the wild-type receptor kinase GRK2), it declined by 75 ± 5% (Fig. 1F).Effect of transfection with both mutant receptor and mutant receptor kinase on long-term desensitization. In the fourth case (Fig. 2D), cells were transfected with both the mutant receptor M2LD and the mutant receptor kinase DN-GRK2. Peak channel activity was comparable to that in control cells transfected with wild-type forms (Fig. 1F). Figure 2D shows that channel activity was similar in both untreated and CCh pretreated cells, and, therefore, in cells transfected with both the mutant receptor M2LD and the mutant receptor kinase DN-GRK2, long-term desensitization was abolished.
In cells transfected with both mutants, the decline of channel activity during the intermediate phase of desensitization was almost completely abolished (Fig. 2D), as we have reported previously (32). Channel activity declined by 4 ± 9% (n = 12) in untreated cells and by 8 ± 12% (n = 8) in pretreated cells over the first 3 min after attachment of the pipette (Fig. 2D), whereas in the control cells (transfected with wild-type forms), it declined by 75 ± 5% (Fig. 1F).Summary. The results obtained are summarized in Fig. 3. Figure 3A shows average values (±SE) of NPo for untreated and CCh-pretreated cells. The peak value of NPo when an ACh-containing pipette was first attached to a cell is given. Figure 3A shows values for cultured neonatal rat atrial cells, control CHO cells (transfected with wild-type receptor and wild-type receptor kinase), and CHO cells prepared using the alternative transfection strategies. Figure 3B shows, for the various cell groups, the channel activity of CCh-pretreated cells as a percentage of the channel activity of untreated cells. Figure 3 confirms that the decrease in channel activity as a result of long-term desensitization was greater in rat atrial cells than in CHO cells transfected with the wild-type receptor HM2 and the wild-type receptor kinase GRK2 (compare bars 1 and 2). Figure 3 also confirms that, whereas transfection with both the mutant receptor M2LD and the mutant receptor kinase DN-GRK2 abolished the decrease in channel activity as a result of long-term desensitization (bar 6), transfection with only one of the mutants substantially reduced it but did not abolish it (bars 4 and 5).
Another effect of receptor kinase. Figure 3A shows that peak channel activity of untreated cells was similar in all groups, except for the CHO cells that were not transfected with receptor kinase (bar 3) (untreated cells: bar 3 significantly different from bars 5 and 6; P < 0.05; one-way analysis of variance). This is also evident by comparing the peak value of NPo in Fig. 2A in untreated cells with the peak value of NPo in Fig. 1F in untreated cells. One possible explanation is that the experiments in Figs. 1 and 2A were carried out on different clones: a cell line stably transfected with the receptor and receptor kinase and another cell line stably transfected with the receptor only (see Figs. 1 and 2 legends for further details of the transfections). To test this possibility, another series of experiments (not shown) was carried out with the cell line stably transfected with the receptor only (same as used for Fig. 2A): the cells were transiently transfected with the receptor kinase. The results obtained from six cells were similar to those in Fig. 1 from the cells stably transfected with both the receptor and receptor kinase: channel activity was high (and the decline in channel activity as a result of the intermediate phase of desensitization was similar to that in Fig. 1F). The unexpected observation was also checked by measurement of muscarinic K+ current in CHO cells with the whole cell patch-clamp technique. Figure 4A shows currents from untreated cells with and without the receptor kinase GRK2, and Fig. 4B shows the mean (±SE) peak amplitude of the current in the two groups of cells. The peak amplitude of the muscarinic K+ current was significantly smaller (P < 0.0005) in the cells without receptor kinase, and the decrease is consistent with the decrease in peak NPo in the cell-attached experiments (Fig. 3A). It is concluded that reduced channel activity observed in cells not stably transfected with the receptor kinase is due to the absence of receptor kinase rather than the use of a different clone or the technique used. The reason underlying this effect of receptor kinase is unknown.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The study of Bünemann et al. (6) was the first to show long-term desensitization of an ion channel gated by a G protein-coupled receptor, and this is the first study to investigate the underlying mechanisms.
In the present study, on exposure of cultured rat atrial cells to 10 µM CCh for 24 h, the peak activity of the muscarinic K+ channel (in response to 10 µM ACh) was depressed by 96.8% (Fig. 3). Before measurement of the muscarinic K+ channel activity after CCh pretreatment, CCh was washed off for 10 min. We have previously shown that 10 min is sufficient for full recovery from short-term desensitization (the fast and intermediate phases of desensitization) (40). When Bünemann et al. (6) exposed cultured guinea pig atrial cells to 10 µM CCh for 16-50 h, followed by a washout of CCh for at least 1 h, the activity of the muscarinic K+ channel (in response to 10 µM ACh) was depressed by a similar amount (~94%). In CHO cells transfected with the wild-type receptor HM2 and wild-type receptor kinase GRK2 (as well as the muscarinic K+ channel), channel activity (in response to 10 µM ACh) was substantially depressed (by 71.0%) after 24-h pretreatment with 10 µM CCh (followed by a 10-min washout of CCh) (Fig. 3). However, the decrease in channel activity was significantly less than that in the cultured rat atrial cells (decrease of 71.0 vs. 96.8%). The reason for this difference is not known, but it could be due to a difference in the expression level of one of the components involved in long-term desensitization.
Role of receptor kinase in long-term desensitization of muscarinic
K+ channel.
In the present study, long-term desensitization was greatly reduced
when the cells were not transfected with the receptor kinase GRK2 (Fig.
3). This suggests that receptor kinase may play a major role in
long-term desensitization. However, it is possible that the reduction
of long-term desensitization was a result of the low-channel activity
in the absence of the receptor kinase. Long-term desensitization of the
muscarinic K+ channel could be the result of a
sequestration and downregulation of receptor (see Introduction). Tsuga
et al. (36), using the same CHO cell line as in the
present study, showed sequestration and downregulation of the
transfected HM2 receptor after a 16-h exposure to 10 µM
CCh: if the cells were transfected with GRK2, [3H]quinuclidinyl benzilate ([3H]QNB)
binding (a measure of total number of HM2 receptors both in
the cell membrane and sequestered and, therefore, a measure of
downregulation) was reduced by ~68% after the CCh exposure. Furthermore, the downregulation was reduced in the absence of GRK2:
[3H]QNB binding was reduced by only ~47% after the CCh
exposure. In the present study, the comparable figures for muscarinic
K+ channel activity were 71.0% and 32.7%. Although the
percentage of reductions in muscarinic K+ channel activity
are qualitatively similar to the percentage of reductions in
[3H]QNB binding, they are not identical. However, the
relationship between receptor number and muscarinic K+
channel activity is unknown: for example, there is evidence
(6) that in the heart at least receptors are in excess,
and, therefore, the relationship between the two is unlikely to be
linear. In the present study, long-term desensitization was not
abolished when the cells were not transfected with the receptor kinase
GRK2 (Fig. 3): the long-term desensitization that occurred under these conditions may have been the result of endogenous receptor kinase present in CHO cells (25). GRK2, by virtue of its ability
to bind G-protein -complex, has previously been used to inhibit activation of the muscarinic K+ channel (30).
In the present study, no evidence of this action was observed (as
already discussed channel activity was greater in the presence of
GRK2). It is possible that much higher levels of GRK2 are required to
inhibit channel activity (an unphysiological phenomenon).
Role of receptor in long-term desensitization of muscarinic K+ channel. In the present study, long-term desensitization was dramatically reduced if the cells were transfected with M2LD, the mutant form of the HM2 receptor lacking the third intracellular loop, instead of the wild-type receptor HM2 (Fig. 3). This proves that the majority (if not all) of the long-term desensitization in CHO cells is a receptor phenomenon, i.e., is homologous. Because all but one of the known phosphorylation sites on the M2 muscarinic receptor are within the third intracellular loop (9, 27, 29), the reduction of long-term desensitization by the use of M2LD supports the hypothesis that long-term desensitization of the muscarinic K+ channel is the result of the phosphorylation of the third intracellular loop of the receptor by the receptor kinase. Some long-term desensitization remained when the mutant receptor was used (Fig. 3). Perhaps other regions of the receptor are also involved in long-term desensitization. Moro et al. (26) showed in a transfected cell line that expression of M2LD greatly reduced the sequestration of the M2 muscarinic receptor from 58% to 14% during a 2-h exposure to 1 mM CCh. In another study, Tsuga et al. (36), using the same CHO cell line as in the present study, showed that expression of M2LD reduced both sequestration (in response to exposures to CCh up to 1 h in duration) and downregulation (in response to exposures to CCh up to 16 h in duration) of the M2 muscarinic receptor.
Short-term desensitization of muscarinic
K+ channel.
During the first 3 min after the attachment of a pipette onto a rat
atrial cell or CHO cell, there was a decrease in the activity of the
muscarinic K+ channel as a result of the intermediate phase
of desensitization (Fig. 1). As shown in Fig. 2, the intermediate phase
of desensitization was changed by the procedures used in the present
study [see Shui et al. (34) for a discussion of these
changes]. In both the atrial and CHO cells pretreated with CCh, the
decline in channel activity as a result of the intermediate phase of
desensitization was largely absent. The reason for this is unknown;
however, a possible explanation is that, when the number of functional
receptors is greatly reduced, the concentration of free G protein
-complex is reduced, and this will reduce the concentration of
activated receptor kinases. Alternatively, after long-term
desensitization, the remaining receptors may be insensitive to phosphorylation.
Short- and long-term desensitization: working hypothesis.
Biochemical data (9, 36) and the data from this and our
previous studies (33, 34) from mammalian cell lines
suggest the following hypothesis: when ACh binds to the M2
receptor causing the dissociation of the G protein, the G protein
-complex, as well as activating the muscarinic K+
channel, binds to and activates the receptor kinase. The activated receptor kinase binds to the agonist-bound receptor; this may partly
uncouple the receptor from the G protein. The receptor-bound receptor
kinase then phosphorylates the third intracellular loop of the receptor
and further uncouples the receptor from the G protein. This may be
primarily the result of a facilitation of the binding of a -arrestin
to the receptor (transfection of CHO cells with arrestin 2 promotes the
intermediate phase of desensitization) (33). The
uncoupling of the receptor from the G protein may account for the
intermediate phase of desensitization of the muscarinic K+
channel. The phosphorylation of the third intracellular loop of the
receptor by the receptor kinase leads to receptor sequestration and
then downregulation, and we suggest that it is this that is principally
responsible for long-term desensitization of the muscarinic K+ channel. Because this hypothesis is based on work on
mammalian cell lines, it will be important to carry out experiments on
heart cells in the future (e.g., using dominant negative mutants) to test it.
-opioid receptor and the muscarinic K+ channel, cotransfection with a receptor kinase (GRK3 or
GRK5) and -arrestin 2 increases the short-term desensitization of
muscarinic K+ current in the presence of the agonist
U-69593.
Possible physiological importance of long-term desensitization. The vagal nerves innervating the cardiac pacemaker, the sinoatrial node, are tonically active. Katona et al. (16) showed that, in healthy resting conscious young men, a blockade of the M2 muscarinic receptor by atropine increases the heart rate by ~100%. A similar increase is observed in the conscious dog (10, 38). Therefore, the cells of the sinoatrial node are continuously exposed to sufficient ACh to decrease the heart rate by ~50%, and such an exposure is likely to induce long-term desensitization. Bünemann et al. (7) obtained some evidence of this: they isolated and then cultured guinea pig atrial cells, and over several days (half time, ~2 days) in culture they observed an increase in the sensitivity of the muscarinic K+ current to ACh, which they could account for by an almost sixfold increase in the density of the M2 muscarinic receptor. They argued that this was the result of recovery from long-term desensitization after the cells had been removed from the heart. The normal function of long-term desensitization may be to maintain the number of M2 muscarinic receptors at an appropriate level.
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FOOTNOTES |
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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.
May 2, 2002;10.1152/ajpheart.00515.2001
Received 21 June 2001; accepted in final form 25 April 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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; n = 11 in both cases) and
CCh-pretreated patches (
; C,
n = 12; F, n = 8) during the
first 3 min after attachment of the pipette. The data have been fitted
by single exponential functions; the time constant (
) for the data
from untreated cells is given. Transfection: stable transfection with
HM2 and G protein-coupled receptor kinase 2 (GRK2) and
transient transfection with G protein coupled inward-rectifying
K+ channel (GIRK)1, GIRK4, and green fluorescent protein
(GFP). F: untreated cells [data adapted from Shui et al.
(
). In all cases, cells were transfected with the
channel proteins GIRK1 and GIRK4. A: data from cells
transfected with wild-type receptor but not receptor kinase.
B: data from cells transfected with mutant receptor and
wild-type receptor kinase. C: data from cells transfected
with wild-type receptor and mutant receptor kinase. D: data
from cells transfected with mutant receptor and mutant receptor kinase.
Transfection: A, stable transfection with HM2
and transient transfection with GIRK1, GIRK4 and GFP; B,
stable transfection with M2 large deletion
(M2LD) and transient transfection with GRK2, GIRK1, GIRK4,
and GFP; C, stable transfection with HM2 and
transient transfection with dominant negative (DN)-GRK2, GIRK1, GIRK4
and GFP; D, stable transfection with M2LD and
transient transfection with DN-GRK2, GIRK1, GIRK4, and GFP. Data from
untreated cells were adapted from Shui et al. (
