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• W2162273168 abstract "Control of the cardiac muscarinic K+ current (iK,ACh) by β-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K+ channel, receptor kinase (GRK2), and β-arrestin 2, desensitization of iK,AChduring 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 β-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of iK,ACh in cells transfected with β-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 β-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 β-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 β-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K+channel. Control of the cardiac muscarinic K+ current (iK,ACh) by β-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K+ channel, receptor kinase (GRK2), and β-arrestin 2, desensitization of iK,AChduring 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 β-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of iK,ACh in cells transfected with β-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 β-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 β-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 β-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K+channel. The cardiac muscarinic K+ current (iK,ACh) 1The abbreviations used are: iKACh, cardiac muscarinic K+ currentCHOChinese hamster ovaryGTPγSguanosine 5′-3-O-(thio)- triphosphateAchacetylcholine is responsible, at least in part, for the negative chronotropic, inotropic, and dromotropic effects of vagal stimulation on the heart (1Boyett M.R. Kodama I. Honjo H. Arai A. Suzuki R. Toyama J. Cardiovasc. Res. 1995; 29: 867-878Crossref PubMed Scopus (53) Google Scholar, 2Boyett M.R. Kirby M.S. Orchard C.H. Roberts A. J. Physiol. 1988; 404: 613-635Crossref PubMed Scopus (60) Google Scholar, 3Nishimura M. Habuchi Y. Hiromasa S. Watanabe Y. Am. J. Physiol. 1988; 255: H7-H14PubMed Google Scholar). 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 α and βγ subunits and the free βγ subunits bind to and activate the muscarinic K+ channel (4Reuveny E. Slesinger P.A. Inglese J. Morales J.M. Iniguez-Lluhi J.A. Lefkowitz R.J. Bourne H.R. Jan Y.N. Jan L.Y. Nature. 1994; 370: 143-146Crossref PubMed Scopus (409) Google Scholar). As in other G protein-coupled receptor systems, the free βγ 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) (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 6Haga T. Haga K. Kameyama K. J. Neurochem. 1994; 63: 400-412Crossref PubMed Scopus (81) Google Scholar, 7Kwatra M.M. Leung E. Maan A.C. McMahon K.K. Ptasienski J. Green R.D. Hosey M.M. J. Biol. Chem. 1987; 262: 16314-16321Abstract Full Text PDF PubMed Google Scholar). The phosphorylation of G protein-coupled receptors, including the m2 muscarinic receptor, leads to receptor desensitization (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 6Haga T. Haga K. Kameyama K. J. Neurochem. 1994; 63: 400-412Crossref PubMed Scopus (81) Google Scholar, 7Kwatra M.M. Leung E. Maan A.C. McMahon K.K. Ptasienski J. Green R.D. Hosey M.M. J. Biol. Chem. 1987; 262: 16314-16321Abstract Full Text PDF PubMed Google Scholar). The chronotropic, inotropic, and dromotropic effects of ACh on the heart fade in the presence of ACh (2Boyett M.R. Kirby M.S. Orchard C.H. Roberts A. J. Physiol. 1988; 404: 613-635Crossref PubMed Scopus (60) Google Scholar, 8Martin P. Am. J. Physiol. 1983; 245: H584-H591PubMed Google Scholar, 9Martin P. Levy M.N. Matsuda Y. Am. J. Physiol. 1982; 243: H219-H225PubMed Google Scholar, 10Boyett M.R. Roberts A. J. Physiol. 1987; 393: 171-194Crossref PubMed Scopus (24) Google Scholar) and this is likely to be, in part at least, the result of a fade of iK,ACh as a result of desensitization (2Boyett M.R. Kirby M.S. Orchard C.H. Roberts A. J. Physiol. 1988; 404: 613-635Crossref PubMed Scopus (60) Google Scholar, 11Honjo H. Kodama I. Zang W.-J. Boyett M.R. Am. J. Physiol. 1992; 263: H1779-H1789PubMed Google Scholar). 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 (12Shui Z. Boyett M.R. Zang W.-J. Haga T. Kameyama K. J. Physiol. 1995; 487: 359-366Crossref PubMed Scopus (38) Google Scholar, 13Shui Z. Khan I.A. Tsuga H. Haga T. Boyett M.R. J. Physiol. 1998; 507: 325-334Crossref PubMed Scopus (19) Google Scholar). ACh, cardiac muscarinic K+ current Chinese hamster ovary guanosine 5′-3-O-(thio)- triphosphate acetylcholine 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 (β-arrestin, β-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) (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). For the m2 muscarinic receptor specifically, there is some evidence that arrestins are involved in desensitization: β-arrestin and β-arrestin 2 bind to the m2 muscarinic receptor in a phosphorylation-dependent manner (14Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). There is evidence that β-arrestin and β-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) (14Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 15Pals-Rylaarsdam R. Hosey M.M. J. Biol. Chem. 1997; 272: 14152-14158Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The evidence that β-arrestin and β-arrestin 2 may also be involved in m2 muscarinic receptorinternalization is less clear: Pals-Rylaarsdam et al. (14Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) showed that overexpression of β-arrestin and β-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. (14Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) 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. (16Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) 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 (17Boyett M.R. Shui Z. Khan I. Dobrzynski H. J. Physiol. 1999; 521: 23Google Scholar). The aim of the present study was to study the possible role of arrestin in short-term desensitization of iK,ACh. β-Arrestin 2 was chosen for study (both β-arrestin and β-arrestin 2 are ubiquitously expressed in tissues (18Krupnik J.G. Benovic J.L. Annu. Rev. Pharamacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (853) Google Scholar)). Chinese hamster ovary (CHO)-K1 cells were cultured and transiently transfected as described previously (13Shui Z. Khan I.A. Tsuga H. Haga T. Boyett M.R. J. Physiol. 1998; 507: 325-334Crossref PubMed Scopus (19) Google Scholar). 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 (13Shui Z. Khan I.A. Tsuga H. Haga T. Boyett M.R. J. Physiol. 1998; 507: 325-334Crossref PubMed Scopus (19) Google Scholar). 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 (19Lohse M.J. Trends Cardiovasc. Med. 1995; 5: 63-68Crossref PubMed Scopus (28) Google Scholar) and it phosphorylates the m2 muscarinic receptor both in vitro and in vivo (20Kameyama K. Haga K. Haga T. Kontani K. Katada T. Fukada Y. J. Biol. Chem. 1993; 268: 7753-7758Abstract Full Text PDF PubMed Google Scholar, 21Tsuga H. Kameyama K. Haga T. Kurose H. Nagao T. J. Biol. Chem. 1994; 269: 32522-32527Abstract Full Text PDF PubMed Google Scholar). 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 β-arrestin 2 (pCMV5-β-arrestin 2). Depending on the experiment, cells of clone 2 were transiently co-transfected with neither or either wild-type β-arrestin 2 or constitutively active mutant β-arrestin 2 (pCDNA3-β-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; β-arrestin 2, 400; green fluorescent protein, 200. 10 ml of the transfecting solution was added to ∼1–2 × 106cells 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. (13Shui Z. Khan I.A. Tsuga H. Haga T. Boyett M.R. J. Physiol. 1998; 507: 325-334Crossref PubMed Scopus (19) Google Scholar). 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. Adult rats were killed by stunning and cervical dislocation. Atrial cells were prepared as described previously (22Harrison S.M. McCall E. Boyett M.R. J. Physiol. 1992; 449: 517-550Crossref PubMed Scopus (107) Google Scholar). 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.). In the present study, as in our previous study (13Shui Z. Khan I.A. Tsuga H. Haga T. Boyett M.R. J. Physiol. 1998; 507: 325-334Crossref PubMed Scopus (19) Google Scholar), 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 (13Shui Z. Khan I.A. Tsuga H. Haga T. Boyett M.R. J. Physiol. 1998; 507: 325-334Crossref PubMed Scopus (19) Google Scholar). 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,AChrecorded 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. 6 A 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. 6 A 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 (23Zang W.-J., Yu, X.-J. Honjo H. Kirby M.S. Boyett M.R. J. Physiol. 1993; 464: 649-679Crossref PubMed Scopus (48) Google Scholar). Both of these phases are evident in Fig.6 A. 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 (23Zang W.-J., Yu, X.-J. Honjo H. Kirby M.S. Boyett M.R. J. Physiol. 1993; 464: 649-679Crossref PubMed Scopus (48) Google Scholar, 24Shui Z. Boyett M.R. Zang W.-J. J. Physiol. 1997; 505: 77-93Crossref PubMed Scopus (27) Google Scholar). The fast phase of desensitization may involve a dephosphorylation of the muscarinic K+ channel (23Zang W.-J., Yu, X.-J. Honjo H. Kirby M.S. Boyett M.R. J. Physiol. 1993; 464: 649-679Crossref PubMed Scopus (48) Google Scholar, 24Shui Z. Boyett M.R. Zang W.-J. J. Physiol. 1997; 505: 77-93Crossref PubMed Scopus (27) Google Scholar, 25Kim D. Circ. Res. 1993; 73: 89-97Crossref PubMed Scopus (38) Google Scholar, 26Kim D. J. Physiol. 1991; 437: 133-155Crossref PubMed Scopus (60) Google Scholar), and a cytosolic protein and a G protein-independent pathway (27Hong S.-G. Pleumsamran A. Kim D. Am. J. Physiol. 1996; 270: H526-H537PubMed Google Scholar). Alternatively, it may be caused by the nucleotide exchange and hydrolysis cycle of the G protein (28Chuang H.H., Yu, M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11727-11732Crossref PubMed Scopus (107) Google Scholar). 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.Figure 6Resensitization of iK,ACh in rat atrial cells. A and B, iK,AChduring 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 arrowswith 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 1, A and C, shows iK,ACh in CHO cells transfected with (in addition to the receptor and channel) GRK2 alone (Fig. 1 A) or GRK2 and β-arrestin 2 (Fig.1 C). As compared with cells transfected with GRK2 alone (Fig. 1 A), in cells transfected with GRK2 and β-arrestin 2 (Fig. 1 C), activation and deactivation of iK,AChwas 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. 1 E: (i) CHO cells not transfected with either GRK2 or β-arrestin 2, (ii) CHO cells transfected with β-arrestin 2, (iii) CHO cells transfected with GRK2, and (iv) CHO cells transfected with GRK2 and β-arrestin 2. Desensitization was least in cells not transfected with either GRK2 or β-arrestin 2. Desensitization was significantly greater in cells transfected with GRK2 alone (p < 0.05), but not with β-arrestin 2 alone (p = 0.3). Desensitization was greatest in CHO cells transfected with both GRK2 and β-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 β-arrestin 2, but not with β-arrestin 2 alone. For comparison, Fig.1 E 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. 1 B) and GRK2 and β-arrestin 2 (Fig. 1 D) 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 β-arrestin 2 (54.5 s; based on recordings from nine cells) or CHO cells transfected with β-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). Close inspection of Fig. 1shows that activation and deactivation of iK,ACh on application and wash-off of ACh was slowed in CHO cells transfected with GRK2 and β-arrestin 2 as compared with activation and deactivation in CHO cells transfected with GRK2 alone. This is clearly shown by Figs.2 and3.Figure 3Effect of expression of β-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 β-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 β-arrestin 2 (clone 1), with GRK2 only (clones 1 and 2), with β-arrestin 2 only (clone 1) and with GRK2 and β-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 2 A compares the activation of iK,ACh on application of ACh in CHO cells, transfected with GRK2 alone or GRK2 and β-arrestin 2, and rat atrial cells. Mean traces from 5 to 15 cells are shown. Fig. 2 B 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 β-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. 2 B also shows the mean time to half-peak iK,ACh in CHO cells not transfected with either GRK2 or β-arrestin 2 and in CHO cells transfected with β-arrestin 2 only, these data show that the effect of β-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 β-arrestin 2 on deactivation of iK,ACh in CHO cells is shown in Fig. 3. Fig.3 A compares the deactivation of iK,ACh on wash-off of ACh in CHO cells, transfected with GRK2 alone or GRK2 and β-arrestin 2, and rat atrial cells. Mean traces from 5 to 15 cells are shown. Fig. 3 B 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 β-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. 3 B also shows the mean time to half-deactivation of iK,ACh in CHO cells not transfected with either GRK2 or β-arrestin 2 and in CHO cells transfected with β-arrestin 2 only, these data show that the effect of β-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). 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 andC) 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 β-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 β-arrestin 2 were 3 and 10 min, respectively (Fig. 4, Band D). The time courses of iK,AChresensitization in CHO cells transfected with GRK2 alone (squares) or GRK2 and β-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 β-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 β-arrestin 2 was 34 and 232 s, respectively.Figure 5Summary of the effect of expression of β-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 β-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 β-arrestin 2). The CHO cells were also transfected with the m2 muscarinic receptor and muscarinic K+ channel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 6 shows resensitization of iK,ACh in rat atrial cells. A typical trace during a control application of ACh is shown in Fig. 6 A and superimposed traces of iK,ACh during test applications of ACh are shown in Fig. 6 B. 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. 6 C 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 β-arrestin 2 (Fig.5). Arrestins preferentially bind to" @default.
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• W2162273168 title "Control of the Cardiac Muscarinic K+ Channel by β-Arrestin 2" @default.
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