Control of the Cardiac Muscarinic K+ Channel by beta -Arrestin 2*

Z. Shui, I. A. Khan, T. HagaDagger , J. L. Benovic§, and M. R. Boyett

From the School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom, the Dagger  Department of Neurochemistry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan, and the § Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, December 6, 2000, and in revised form, January 8, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES

Control of the cardiac muscarinic K+ current (iK,ACh) by beta -arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K+ channel, receptor kinase (GRK2), and beta -arrestin 2, desensitization of iK,ACh during a 3-min application of 10 µM ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta -arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of iK,ACh in cells transfected with beta -arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of iK,ACh on application and wash-off of ACh in cells transfected with beta -arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak iK,ACh) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta -arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 ± 0.05 to 0.1 ± 0.01 nA). We conclude that beta -arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K+ channel.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES

The cardiac muscarinic K+ current (iK,ACh)1 is responsible, at least in part, for the negative chronotropic, inotropic, and dromotropic effects of vagal stimulation on the heart (1-3). In the heart, ACh released from vagal nerves binds to the m2 muscarinic receptor (a G protein-coupled receptor) causing the dissociation of a trimeric Gi-protein into alpha  and beta gamma subunits and the free beta gamma subunits bind to and activate the muscarinic K+ channel (4). As in other G protein-coupled receptor systems, the free beta gamma subunits also bind to and activate receptor kinase and the activated receptor kinase binds to and phosphorylates the agonist-bound receptor (on the third intracellular loop in the case of the m2 muscarinic receptor) (5-7). The phosphorylation of G protein-coupled receptors, including the m2 muscarinic receptor, leads to receptor desensitization (5-7). The chronotropic, inotropic, and dromotropic effects of ACh on the heart fade in the presence of ACh (2, 8-10) and this is likely to be, in part at least, the result of a fade of iK,ACh as a result of desensitization (2, 11). In rat atrial cells and in a mammalian cell line (transfected with m2 muscarinic receptor, muscarinic K+ channel, and receptor kinase) we have previously obtained evidence that the phosphorylation of the receptor by receptor kinase is responsible for short-term desensitization of iK,ACh (12, 13).

Arrestins act in concert with receptor kinase to bring about desensitization. Receptor kinase-mediated phosphorylation of the receptor promotes the binding of an arrestin to the agonist bound receptor and this causes desensitization by (i) preventing receptor-G protein interaction and (ii) for the nonvisual arrestins (beta -arrestin, beta -arrestin 2) only, promoting internalization of the receptor via clathrin-coated pits (the nonvisual arrestins act as adaptor proteins and bind both the receptor and clathrin) (5). For the m2 muscarinic receptor specifically, there is some evidence that arrestins are involved in desensitization: beta -arrestin and beta -arrestin 2 bind to the m2 muscarinic receptor in a phosphorylation-dependent manner (14). There is evidence that beta -arrestin and beta -arrestin 2 may be involved in m2 muscarinic receptor uncoupling: deletion of a cluster of serine/threonine residues (phosphorylation sites) in the C-terminal part of the third intracellular loop of the m2 muscarinic receptor greatly reduced the binding of arrestins and in HEK293 cells abolished desensitization as a result of receptor-G protein uncoupling (measured as a reduction in the carbachol inhibition of a isoproterenol-stimulated increase in cAMP as a result of pretreatment with carbachol) (14, 15). The evidence that beta -arrestin and beta -arrestin 2 may also be involved in m2 muscarinic receptor internalization is less clear: Pals-Rylaarsdam et al. (14) showed that overexpression of beta -arrestin and beta -arrestin 2 in HEK-tsA201 cells resulted in an internalization of the m2 muscarinic receptor via a dynamin-dependent mechanism (arrestin-mediated internalization is known to occur via a dynamin- and clathrin-dependent mechanism). However, in the same study Pals-Rylaarsdam et al. (14) showed that internalization of the m2 muscarinic receptor in HEK-tsA201 cells in the absence of arrestin overexpression occurred via an unknown pathway that does not involve arrestins or dynamin. Furthermore, in rat ventricular cells, Feron et al. (16) reported evidence that the m2 muscarinic receptor is internalized via caveolae (clathrin-independent pathway), although we have observed co-localization of the m2 muscarinic receptor and clathrin after CCh pretreatment in the same cell type (17).

The aim of the present study was to study the possible role of arrestin in short-term desensitization of iK,ACh. beta -Arrestin 2 was chosen for study (both beta -arrestin and beta -arrestin 2 are ubiquitously expressed in tissues (18)).


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES

Cell Culture and Transfection-- Chinese hamster ovary (CHO)-K1 cells were cultured and transiently transfected as described previously (13). Cells were cultured in Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml Fungizone at 37 °C in 95% air and 5% CO2 (all media and chemicals from Life Technologies Ltd., Paisley, United Kingdom). In all experiments, cells from one of two cell lines were transiently transfected with plasmid vectors for Kir3.1 (pEF-GIRK1) and Kir3.4 (pEF-CIR) to form the muscarinic K+ channel heteromultimer using the calcium phosphate method (13). One cell line was already stably transfected with plasmid vector for human m2 muscarinic receptor (pEF-Myc-hm2) and the second cell line was already stably transfected with plasmid vectors for human m2 muscarinic receptor (pEF-Myc-hm2) and the G protein-coupled receptor kinase, GRK2, (pEF-GRK2). GRK2 is known to be present in the heart (19) and it phosphorylates the m2 muscarinic receptor both in vitro and in vivo (20, 21). The cell lines will be referred to as clones 1 and 2, respectively. To test whether the use of a particular clone influenced the results obtained, experiments were repeated using both clones. The results obtained with the two clones were indistinguishable and they have been combined, although the clones used are identified in figure legends. Depending on the experiment, cells of clone 1 were transiently co-transfected with neither, either, or both GRK2 and wild-type beta -arrestin 2 (pCMV5-beta -arrestin 2). Depending on the experiment, cells of clone 2 were transiently co-transfected with neither or either wild-type beta -arrestin 2 or constitutively active mutant beta -arrestin 2 (pCDNA3-beta -arrestin CAM). Finally, all cells were transiently co-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): Kir3.1, 400; Kir3.4, 400; GRK2, 400; beta -arrestin 2, 400; green fluorescent protein, 200. 10 ml of the transfecting solution was added to ~1-2 × 106 cells in a 100-mm diameter plastic tissue culture dish. The expression levels of the receptor and GRK2 in the stably transfected cells were measured and are given in Shui et al. (13). A few hours before electrophysiological experiments, 0.02% EDTA solution was used to remove the adherent cell layer from the dish. The cells were then centrifuged for 3 min at 100 × g and resuspended in fresh medium on fragments of glass coverslip.

Atrial Cell Isolation-- Adult rats were killed by stunning and cervical dislocation. Atrial cells were prepared as described previously (22).

Electrophysiology-- CHO cells were placed in a recording chamber mounted on a Nikon Diaphot microscope. 470-490-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. Experiments were carried out in the whole cell configuration of the patch clamp technique at room temperature (22-25 °C). Extracellular solution contained (in mM): KCl, 140; MgCl2, 1.8; EGTA, 5; HEPES, 5; pH 7.4. 10 µM ACh was added to the extracellular solution when required. Pipette solution contained (in mM): potassium aspartate, 120; KCl, 20; KH2PO4, 1; MgCl2, 2.8 (free Mg2+, 1.8); EGTA, 5; HEPES, 5; Na3GTP, 0.1; Na2ATP, 3; pH 7.4. Whole cell currents were recorded with an Axopatch-1D amplifier and acquired with pClamp software (Axon Instruments Inc., Foster City, CA). Currents were filtered at 2 kHz with an 8-pole Bessel filter and sampled every 1 ms. Decline in currents was fitted with a single exponential function with a least squares method using SigmaPlot (Jandel Corp., San Rafael, CA). Statistical tests (one way analysis of variance) were carried out using SigmaStat (Jandel Corp.).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES

Effect of Expression of beta -Arrestin 2 on Desensitization of IK,ACh-- In the present study, as in our previous study (13), we have recorded iK,ACh in CHO cells transfected with the m2 muscarinic receptor and the muscarinic K+ channel (as well as other proteins). In the previous study, the cell-attached and inside-out configurations of the patch clamp technique were used and the basic properties of the channel (single channel conductance, current-voltage relationship, single channel open time, dependence on receptor and agonist) were the same as those of the muscarinic K+ channel in heart cells (13). In the present study, the whole cell configuration of the patch clamp technique was used; 10 µM ACh was applied using a rapid solution changer for 3 min to activate iK,ACh maximally and current was recorded at a holding potential of -60 mV in a bathing solution containing 140 mM K+. Fig. 1, A and C, shows examples of iK,ACh recorded from CHO cells during an application of ACh. Mean traces from >= 8 cells are shown. In both cases, iK,ACh was activated and deactivated on application and wash-off of ACh, respectively. During the 3-min application of ACh, there was a fade of iK,ACh as a result of short-term desensitization. In the present study, iK,ACh was recorded from rat atrial cells under conditions identical to those used for the recording of iK,ACh from CHO cells in order that the muscarinic K+ channel system reconstructed in CHO cells can be compared with the native system in heart. Fig. 6A shows a typical recording of iK,ACh in a rat atrial cell. iK,ACh in CHO cells was qualitatively similar to that in rat atrial cells. However, there were differences. The semi-logarithmic plots in Fig. 1, B and D, show that the fade of iK,ACh in CHO cells as a result of desensitization was monoexponential, whereas it can be seen from Fig. 6A that in rat atrial cells it is biexponential. It is known that in heart cells, short-term desensitization is comprised of two independent phases: a fast phase that develops over ~20 s and a slower phase that develops over several minutes (23). Both of these phases are evident in Fig. 6A. It has been shown that the fast phase is the result of a change in the channel, whereas the slower phase is the result of a change in the receptor (23, 24). The fast phase of desensitization may involve a dephosphorylation of the muscarinic K+ channel (23-26), and a cytosolic protein and a G protein-independent pathway (27). Alternatively, it may be caused by the nucleotide exchange and hydrolysis cycle of the G protein (28). Based on the time course of desensitization (Fig. 1), the fast phase of desensitization observed in heart cells is absent in CHO cells (perhaps the underlying cellular machinery is absent) and the desensitization of iK, ACh in CHO cells is equivalent to the slower phase in heart cells.



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Fig. 1.   Effect of expression of beta -arrestin 2 on desensitization of iK,ACh. A, iK,ACh during 3 min application of 10 µM ACh (shown by the bar) in CHO cells transfected with GRK2 only (clone 2). Mean current from 15 cells shown. B, semi-logarithmic plot of the fade of iK,ACh in A. The difference between the current during the application of ACh and the calculated asymptote is plotted on a logarithmic scale against time. The data are fitted with an exponential function with the time constant shown. C, iK,ACh in CHO cells transfected with GRK2 and beta -arrestin 2 (clone 2). Mean current from eight cells shown. D, semi-logarithmic plot of the fade of iK,ACh in C. E, mean + S.E. amplitude of desensitization of iK,ACh in rat atrial cells. The amplitude of desensitization in rat atrial cells was measured as 100 × [(iK,ACh(20 s) - iK,ACh(end))/iK,ACh(20 s)] and the amplitude of desensitization in CHO cells was measured as 100 × [(iK,ACh(peak) - iK,ACh(end))/iK,ACh(peak)], where iK,ACh(peak), iK,ACh(20 s), and iK,ACh(end) are the peak current amplitude at the start of the ACh application and the currents amplitudes 20 s after the start and at the end of the ACh application, respectively. n numbers are shown in parentheses. All cells were transfected with the m2 muscarinic receptor and muscarinic K+ channel.

Fig. 1, A and C, shows iK,ACh in CHO cells transfected with (in addition to the receptor and channel) GRK2 alone (Fig. 1A) or GRK2 and beta -arrestin 2 (Fig. 1C). As compared with cells transfected with GRK2 alone (Fig. 1A), in cells transfected with GRK2 and beta -arrestin 2 (Fig. 1C), activation and deactivation of iK,ACh was slowed (see below), the peak amplitude of iK,ACh was not significantly different (p = 0.8) and the fade of iK,ACh as a result of desensitization was greater. The amplitude of desensitization (see Fig. 1 legend for measurement) in four groups of CHO cells is shown in Fig. 1E: (i) CHO cells not transfected with either GRK2 or beta -arrestin 2, (ii) CHO cells transfected with beta -arrestin 2, (iii) CHO cells transfected with GRK2, and (iv) CHO cells transfected with GRK2 and beta -arrestin 2. Desensitization was least in cells not transfected with either GRK2 or beta -arrestin 2. Desensitization was significantly greater in cells transfected with GRK2 alone (p < 0.05), but not with beta -arrestin 2 alone (p = 0.3). Desensitization was greatest in CHO cells transfected with both GRK2 and beta -arrestin 2, in this cell group desensitization was significantly greater than that in other cell groups (p < 0.05 in each case). In summary, the results show that desensitization was increased by transfection with GRK2 and beta -arrestin 2, but not with beta -arrestin 2 alone. For comparison, Fig. 1E also shows the amplitude of the equivalent phase of desensitization in rat atrial cells (see Fig. 1 legend for measurement).

The semi-logarithmic plots in Fig. 1, B and D, show that in CHO cells transfected with GRK2 alone (Fig. 1B) and GRK2 and beta -arrestin 2 (Fig. 1D) the fade of iK,ACh as a result of desensitization occurred with similar time constants of 69.5 and 53.7 s, respectively (based on recordings from 15 and eight cells, respectively). The time constants of desensitization were also similar for CHO cells not transfected with either GRK2 or beta -arrestin 2 (54.5 s; based on recordings from nine cells) or CHO cells transfected with beta -arrestin 2 alone (40.9 s; based on recordings from eight cells). The time constant of the equivalent phase of desensitization in rat atrial cells was 144.0 s (based on recordings from 10 cells).

Effect of Expression of beta -Arrestin 2 on Activation and Deactivation of iK,ACh-- Close inspection of Fig. 1 shows that activation and deactivation of iK,ACh on application and wash-off of ACh was slowed in CHO cells transfected with GRK2 and beta -arrestin 2 as compared with activation and deactivation in CHO cells transfected with GRK2 alone. This is clearly shown by Figs. 2 and 3.



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Fig. 2.   Effect of expression of beta -arrestin 2 on activation of iK,ACh. A, activation of iK,ACh on application of 10 µM ACh in rat atrial cells (n = 5) and in CHO cells transfected with GRK2 only (clone 2; n = 15) or GRK2 and beta -arrestin 2 (clone 2; n = 8). The traces shown are mean currents (from number of cells above) and have been normalized to the peak current on application of ACh. The application of ACh is shown by the bar, and the start of application is indicated by the dashed line. B, mean + S.E. value of the time to half-peak iK,ACh on application of ACh in rat atrial cells and in CHO cells transfected without either GRK2 or beta -arrestin 2 (clone 1), with GRK2 only (clones 1 and 2), with beta -arrestin 2 only (clone 1) and with GRK2 and beta -arrestin 2 (clones 1 and 2). n numbers are shown in parentheses. All CHO cells were transfected with the m2 muscarinic receptor and muscarinic K+ channel.



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Fig. 3.   Effect of expression of beta -arrestin 2 on deactivation of iK,ACh. A, deactivation of iK, ACh on wash-off of 10 µM ACh in rat atrial cells (n = 5) and in CHO cells transfected with GRK2 only (clone 2; n = 15) or GRK2 and beta -arrestin 2 (clone 2; n = 8). The traces shown are mean currents (from number of cells above) and have been normalized to the current at the end of the application of ACh. B, mean + S.E. value of the time to half-deactivation of iK,ACh on wash-off of ACh in rat atrial cells and in CHO cells transfected without either GRK2 or beta -arrestin 2 (clone 1), with GRK2 only (clones 1 and 2), with beta -arrestin 2 only (clone 1) and with GRK2 and beta -arrestin 2 (clones 1 and 2). n numbers are shown in parentheses. All CHO cells were transfected with the m2 muscarinic receptor and muscarinic K+ channel.

Fig. 2A compares the activation of iK,ACh on application of ACh in CHO cells, transfected with GRK2 alone or GRK2 and beta -arrestin 2, and rat atrial cells. Mean traces from 5 to 15 cells are shown. Fig. 2B shows the mean time to half-peak iK,ACh on application of ACh. The time to half-peak iK,ACh in CHO cells transfected with GRK2 and beta -arrestin 2 was significantly longer than in CHO cells transfected with GRK2 alone (p < 0.001), whereas the time to half-peak iK,ACh in atrial cells was not significantly different from that in CHO cells transfected with GRK2 alone (p = 0.08). Fig. 2B also shows the mean time to half-peak iK,ACh in CHO cells not transfected with either GRK2 or beta -arrestin 2 and in CHO cells transfected with beta -arrestin 2 only, these data show that the effect of beta -arrestin 2 on activation was independent of the absence or presence of overexpressed GRK2 (they also show that overexpressed GRK2 had no effect on activation).

The effect of transfection of GRK2 and beta -arrestin 2 on deactivation of iK,ACh in CHO cells is shown in Fig. 3. Fig. 3A compares the deactivation of iK,ACh on wash-off of ACh in CHO cells, transfected with GRK2 alone or GRK2 and beta -arrestin 2, and rat atrial cells. Mean traces from 5 to 15 cells are shown. Fig. 3B shows the mean time to half-deactivation of iK,ACh on wash-off of ACh. The time to half-deactivation of iK,ACh in CHO cells transfected with GRK2 and beta -arrestin 2 was significantly longer than in CHO cells transfected with GRK2 alone (p < 0.001), whereas the time to half-deactivation of iK,ACh in atrial cells was significantly shorter than in CHO cells transfected with GRK2 alone (p < 0.001). Fig. 3B also shows the mean time to half-deactivation of iK,ACh in CHO cells not transfected with either GRK2 or beta -arrestin 2 and in CHO cells transfected with beta -arrestin 2 only, these data show that the effect of beta -arrestin 2 on deactivation was independent of the absence or presence of overexpressed GRK2 (they also show that overexpressed GRK2 had no effect on deactivation).

Effect of Expression of beta -Arrestin 2 on Resensitization of iK,ACh-- Resensitization of iK,ACh (i.e. the recovery of iK,ACh from desensitization) was studied by applying test applications of ACh at various test intervals after control applications of ACh. Fig. 4 shows typical traces of iK,ACh in response to control (Fig. 4, A and C) and test (Fig. 4, B and D) applications of ACh in CHO cells transfected with GRK2 alone (Fig. 4, A and B) or GRK2 and beta -arrestin 2 (Fig. 4, C and D). When the test application of ACh was applied soon after the control application, iK,ACh during the test application of ACh was smaller than the control as a result of insufficient time for recovery from the desensitization that developed during the control application of ACh. As the recovery interval between the two applications was increased, iK,ACh during the test application of ACh increased toward the control as a result of resensitization. The test intervals for full recovery of iK,ACh in CHO cells transfected with GRK2 alone or GRK2 and beta -arrestin 2 were 3 and 10 min, respectively (Fig. 4, B and D). The time courses of iK,ACh resensitization in CHO cells transfected with GRK2 alone (squares) or GRK2 and beta -arrestin 2 (circles) are shown in Fig. 5 from three or four cells, the peak amplitude of iK,ACh in response to the test application of ACh has been normalized to the peak amplitude of iK,ACh in response to the previous control application of ACh and plotted against the recovery interval between the two applications. Fig. 5 confirms that resensitization of iK,ACh was slower in CHO cells transfected with both GRK2 and beta -arrestin 2 than in CHO cells transfected with GRK2 alone. The time constant of resensitization in the CHO cells transfected with GRK2 alone or GRK2 and beta -arrestin 2 was 34 and 232 s, respectively.



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Fig. 4.   Effect of expression of beta -arrestin 2 on resensitization of iK,ACh. A and B, iK,ACh during 3 min control (A) and test (B) applications of 10 µM ACh (shown by bars) recorded from a CHO cell transfected with GRK2 only (clone 2). Four test responses are shown superimposed. The peak currents are indicated by the arrows with the intervals between control and test applications of ACh. C and D, iK,ACh recorded from a CHO cell transfected with GRK2 and beta -arrestin 2 shown in the same format as A and B (clone 2). The CHO cells were also transfected with the m2 muscarinic receptor and muscarinic K+ channel.



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Fig. 5.   Summary of the effect of expression of beta -arrestin 2 on resensitization of iK,ACh. Mean ± S.E. peak amplitude of iK,ACh during a test application of ACh (calculated as a percentage of the peak amplitude of the current during the previous control application) plotted against the test interval. Data for CHO cells transfected with GRK2 only (squares; n = 4 cells; clone 2) or GRK2 and beta -arrestin 2 (circles; n = 3 cells; clone 2) are shown. The data are fitted with single exponential functions with time constants of 34 s (GRK2) and 232 s (GRK2 and beta -arrestin 2). The CHO cells were also transfected with the m2 muscarinic receptor and muscarinic K+ channel.

Fig. 6 shows resensitization of iK,ACh in rat atrial cells. A typical trace during a control application of ACh is shown in Fig. 6A and superimposed traces of iK,ACh during test applications of ACh are shown in Fig. 6B. As discussed above, in rat atrial cells, unlike in CHO cells, there are two phases of desensitization of iK,ACh during a 3-min application of ACh. The two phases can be seen in Fig. 6, A and B. Resensitization of iK,ACh will be influenced by both the fast and slower phases, whereas in this study we are concerned with the slower phase only. For this reason, resensitization of iK,ACh was calculated in two ways: (i) peak iK,ACh during the test application of ACh was expressed as a percentage of peak iK,ACh during the previous control application of ACh and plotted against the recovery interval between the two applications; (ii) the amplitude of iK,ACh after the fast phase of desensitization (20 s after the start of the ACh application) was expressed as a percentage of the corresponding measurement from the previous control application of ACh and once again plotted against the recovery interval between the two applications. The time course of iK,ACh resensitization in rat atrial cells as determined by the two methods is shown in Fig. 6C and is roughly similar. The time constant of resensitization of iK,ACh in rat atrial cells was 64 and 75 s for methods i and ii, respectively. This is intermediate between the time constants of resensitization of iK,ACh in CHO cells with and without beta -arrestin 2 (Fig. 5).



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Fig. 6.   Resensitization of iK,ACh in rat atrial cells. A and B, iK,ACh during 3 min control (A) and test (B) applications of 10 µM ACh (shown by bar) recorded from a rat atrial cell. Five test responses are shown superimposed. The peak currents are indicated by the arrows with the intervals between control and test applications of ACh. C, mean ± S.E. (n = 3-5) amplitude of iK,ACh during a test application of ACh (calculated as a percentage of the amplitude of iK,ACh during the control application) plotted against the test interval. Both the peak amplitude of iK,ACh (squares) and the amplitude of iK,ACh after the fast phase of desensitization (20 s after the start of the ACh application) are shown. The data are fitted with single exponential functions with time constants of 64 s (peak amplitude) and 75 s (amplitude of current after the fast phase of desensitization).

Effect of Expression of a Constitutively Active beta -Arrestin 2 Mutant on iK,ACh-- Arrestins preferentially bind to the receptor once the receptor is agonist-bound and has been phosphorylated by receptor kinase. However, truncation of the C terminus of arrestins enhances binding to the dephosphorylated receptor, perhaps because the C terminus via an intramolecular interaction with the N terminus maintains arrestins in an inactive conformation (29, 30). With the phosphorylated but not dephosphorylated receptor, arrestins result in a high affinity agonist-binding state, which has been postulated to correlate with the desensitization process (31). The C-terminal truncation mutant, beta -arrestin 2-(1-393), is able to result in such a state even in the case of the dephosphorylated receptor (31). This suggests that beta -arrestin 2-(1-393) is constitutively active and binds regardless of the phosphorylation state of the receptor. Expression of such a constitutively active beta -arrestin 2 mutant might be expected to result in desensitization in the absence of the agonist, although this has never been tested. Fig. 7A shows iK,ACh recorded from CHO cells transfected with GRK2 only or GRK2 and the constitutively active beta -arrestin 2 mutant. Mean traces from >= 7 cells are shown in Fig. 7A. As predicted, the peak amplitude of iK,ACh was reduced upon transfection with the constitutively active beta -arrestin 2 mutant. Fig. 7B shows the peak amplitude of iK,ACh in CHO cells transfected with: (i) GRK2 and wild-type beta -arrestin 2, (ii) GRK2 alone, and (iii) GRK2 and the constitutively active beta -arrestin 2 mutant. The peak amplitudes of iK,ACh in CHO cells transfected with GRK2 alone or GRK2 and beta -arrestin 2 were similar. However, the peak amplitude of iK,ACh was significantly reduced in CHO cells transfected with GRK2 and the constitutively active beta -arrestin 2 mutant (versus CHO cells transfected with GRK2 and beta -arrestin 2, p < 0.001; versus CHO cells transfected with GRK2 alone, p < 0.001).



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Fig. 7.   Effect of expression of a constitutively active beta -arrestin 2 mutant on iK,ACh. A, iK,ACh during a 3-min application of 10 µM ACh (shown by bar) in CHO cells transfected with GRK2 alone (clone 2; n = 15) or GRK2 and the constitutively active beta -arrestin 2 mutant (clone 2; n = 7). Mean traces (from number of cells above) are shown. B, mean + S.E. value of the peak amplitude of iK,ACh in CHO cells transfected with GRK2 and beta -arrestin 2, with GRK2 only or GRK2 and the constitutively active beta -arrestin 2 mutant. n numbers are shown in parentheses.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES

In the present study, evidence has been obtained to show that beta -arrestin 2 is a multifunctional protein and it affects activation, desensitization, deactivation, and resensitization of iK,ACh: when beta -arrestin 2 was expressed in CHO cells, activation of iK,ACh on application of ACh was slowed, short-term desensitization of iK,ACh during the application of ACh was enhanced, deactivation of iK,ACh on wash-off of ACh was slowed, and resensitization of iK,ACh after the wash-off of ACh was slowed.

Short-term Desensitization of iK,ACh-- In the present study we have studied desensitization of iK,ACh during a 3-min application of ACh, in CHO cells there was a fade of iK,ACh with a time constant of 40.9-69.5 s depending on the components transfected (Fig. 1). This is equivalent to the short-term desensitization that develops over minutes in heart cells, e.g. rat atrial cells as used in the present study. Kobrinsky et al. (32) have recently studied desensitization of iK,ACh in neonatal rat atrial cells during a 2-min application of 100 µM ACh (same phase of desensitization investigated as in the present study). They have proposed a radical new hypothesis to explain the desensitization of iK,ACh in the heart (32). They have suggested that ACh, as well as activating iK,ACh, activates phospholipase C and this results in the hydrolysis of phosphatidylinositol bisphosphate and a consequent decrease in phosphatidylinositol bisphosphate binding to the muscarinic K+ channel and, thus, channel activity. In part this hypothesis was based on the observation that addition of the aminosteriod, U-73122, to inhibit phosphatidylinositol bisphosphate hydrolysis, abolished the desensitization of iK,ACh (32). According to this hypothesis the desensitization of iK,ACh is the result of a change in the channel rather than the receptor. However, Zang et al. (23) showed that in guinea pig atrial cells, if the muscarinic K+ channel was activated by GTPgamma S, which is known to bypass the receptor and activate the G protein and thus the channel directly, the phase of desensitization of iK,ACh that develops over minutes was abolished, this suggests that this phase of desensitization is a receptor, rather than channel, phenomenon. Furthermore, Shui et al. (12) showed that this phase of desensitization of iK,ACh was observed in whole cell, cell attached, and perforated outside-out recordings (in which the cytoplasm is retained) but was lost in inside-out and conventional outside-out recordings (in which the cytoplasm is lost). It was conjectured that receptor kinase was lost on loss of the cytoplasm and, consistent with this explanation, in inside-out recordings desensitization of iK,ACh was restored on adding exogenous GRK2 (12). This work suggested that, in heart, this phase of desensitization of iK,ACh is the result of phosphorylation of the receptor by receptor kinase (12). Subsequently Shui et al. (13) showed that in CHO cells transfected with the m2 muscarinic receptor, muscarinic K+ channel, and GRK2 short-term desensitization of iK,ACh was comparable to that in heart cells, but short-term desensitization was lost if the cells were not transfected with GRK2 or were transfected with a mutant m2 muscarinic receptor lacking the third intracellular loop containing the phosphorylation sites. The present study confirms that if CHO cells are not expressed with GRK2 (with or without beta -arrestin 2) then desensitization is reduced (Fig. 1E).

The present study has shown that transfection of CHO cells with both GRK2 and beta -arrestin 2 rather than GRK2 alone enhances desensitization of iK,ACh (Fig. 1E). This effect of beta -arrestin 2 is dependent on the presence of GRK2, if the cells were not transfected with GRK2, transfection with beta -arrestin 2 had no significant effect (Fig. 1E). The requirement for GRK2 is consistent with the known property of beta -arrestin 2 that it preferentially binds to receptor kinase-phosphorylated receptor. beta -Arrestin 2 is expected to enhance desensitization by uncoupling the receptor from the G protein and by promoting internalization of the receptor (see Introduction). The results of Kobrinsky et al. (32) are not necessarily in disagreement with our hypothesis that the desensitization is the result of a modification of the receptor rather than the channel, because U-73122 is known to inhibit m2 muscarinic receptor internalization (33). It is also possible that in native atrial cells beta -arrestin 2 acts in concert with other mechanisms, which act directly on the channel, to affect desensitization. Although we favor the hypothesis that beta -arrestin 2 affects desensitization by acting on the receptor, the possibility that it acts on another component in the pathway cannot be excluded. Recently, Appleyard et al. (34) have shown that, in Xenopus oocytes transfected with the rat kappa  opioid receptor and the muscarinic K+ channel, co-transfection with a receptor kinase (GRK3 or GRK5) and beta -arrestin 2 increased the desensitization of iK,ACh in the presence of the agonist, U69,593.

Resensitization of iK,ACh-- This study has shown that beta -arrestin 2 expression slows recovery from short-term desensitization (resensitization). Resensitization presumably involves unbinding of receptor kinase and the arrestin from the receptor and dephosphorylation of the receptor. There are at least two explanations for the slowing of resensitization with beta -arrestin 2 overexpression (for arguments in favor of overexpression see below): resensitization may be rate-limited by the dissociation of the arrestin and overexpression of beta -arrestin 2 might slow this dissociation. Another possibility is that the overexpression of beta -arrestin 2 pushes receptors to a more slowly resensitizing state: for example, in the absence of beta -arrestin 2 overexpression, desensitization-resensitization may involve receptor-G protein uncoupling-recoupling only (possibly fast processes), whereas with beta -arrestin 2 overexpression it may involve internalization-recycling to the membrane (possibly slower processes).

Activation and Deactivation of iK,ACh-- Overexpression with beta -arrestin 2 slowed both the activation and deactivation of iK,ACh on application and wash-off, respectively, of ACh (Figs. 2 and 3). Activation of iK,ACh involves ACh-receptor binding, G protein activation, and the subsequent activation of the channel by the G protein beta gamma subunits. It is known that binding of a beta -arrestin to the receptor inhibits receptor-G protein interaction (5). Although arrestins preferentially bind to the agonist-occupied receptor kinase-phosphorylated receptor, arrestins still bind but with reduced efficacy to the agonist-unoccupied, dephosphorylated receptor (29). Perhaps with beta -arrestin 2 overexpression, as in the present study, there is increased interaction of beta -arrestin 2 with the agonist-unoccupied, dephosphorylated receptor and this is manifested as impaired coupling with the G protein and a consequent slowing of the activation of iK,ACh on application of ACh. Alternatively, it is possible that beta -arrestin 2 has a direct effect on the muscarinic K+ channel to slow activation of iK,ACh.

On wash-off of ACh, deactivation of iK,ACh involves the splitting of GTP by the G protein alpha  subunit, unbinding of the G protein beta gamma subunits from the channel and reformation of the G protein trimer. It is possible that interaction of the G protein trimer with the receptor is involved in the deactivation process, perhaps by stabilizing the trimer. In this case, with beta -arrestin 2 overexpression as in the present study, there may be increased interaction of beta -arrestin 2 with the agonist-unoccupied, dephosphorylated receptor as argued above and this may impair coupling of the receptor with the G protein and this may slow deactivation of iK,ACh. Alternatively, it is possible that beta -arrestin 2 has a direct effect on the G protein or channel.

Comparison of iK,ACh in Atrial and CHO Cells-- While a comparison of iK,ACh in atrial and CHO cells may be useful, such a comparison must be considered cautiously, because of the absence/presence of additional regulatory components in the two cell types as well as the effects of protein overexpression on the relative stoichiometries of the different components. Fig. 1E shows that the amplitude of desensitization in rat atrial cells was comparable to that in CHO cells transfected with GRK2 alone and less than that in CHO cells transfected with GRK2 and beta -arrestin 2. Comparison of Figs. 5 and 6 shows that in rat atrial cells the speed of resensitization (time constant, tau , 64 or 75 s depending on the method of measurement) was intermediate between that in CHO cells in the absence and presence of beta -arrestin 2 (tau , 34 and 232 s, respectively). It is possible that transfection of CHO cells with beta -arrestin 2 resulted in overexpression of arrestin, i.e. a level of beta -arrestin 2 higher than normal, especially as CHO cells express endogenous arrestin (35). In the present study, activation and deactivation of iK,ACh in CHO cells transfected with or without beta -arrestin 2 were slower than in rat atrial cells (Figs. 2 and 3). Activation and deactivation of iK,ACh in heterologous expression systems are accelerated upon transfection with RGS proteins, activators of the GTPase activity of the G protein alpha  subunit (36), and it is possible that this difference between rat atrial cells and CHO cells is the result of a lack of RGS protein in CHO cells.


    SUMMARY
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES

Evidence from this study as well as our previous studies (12, 13, 23) suggests that the short-term desensitization of iK,ACh both in cardiac cells and in the reconstituted system in CHO cells is the result of a change in the receptor and, on the basis of the present study, it is concluded that beta -arrestin 2 has the potential to be involved in this desensitization process. In addition, it is concluded that beta -arrestin 2 has the potential to be involved in other properties of the channel (activation, deactivation, and resensitization). However, whether beta -arrestin 2 is involved in regulation of the muscarinic K+ channel in the heart remains to be elucidated.


    FOOTNOTES

* 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.

To whom correspondence should be sent: School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. Tel.: 44-113-2334298; Fax: 44-113-2334224; E-mail: m.r.boyett@leeds.ac.uk.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M011007200


    ABBREVIATIONS

The abbreviations used are: iK, ACh, cardiac muscarinic K+ current; CHO, Chinese hamster ovary; GTPgamma S, guanosine 5'-3-O-(thio)- triphosphate; Ach, acetylcholine.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
SUMMARY
REFERENCES


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