British Heart Foundation Laboratories and Department of Medicine, University College London, London WC1E 6JJ, United Kingdom
Submitted 2 December 2003 ; accepted in final form 8 March 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
G protein-coupled receptor; potassium channel; inward rectifier; kinetics
The receptor-mediated response displays characteristic activation, desensitization, and subsequent deactivation phases in response to agonist application and removal. It was noted in some of the earliest studies on the atrial channel that the current desensitizes with a two-component time course: the fast component has a time constant of a few seconds, whereas the slower process occurs over tens of seconds (7, 24). Fast current desensitization also occurs in neurons (4, 16, 35), occurring within seconds of the peak current response. The slow component of desensitization is likely to occur at the level of the receptor and involve phosphorylation by G protein-coupled receptor (GPCR) kinases, uncoupling from G proteins, and receptor internalization (24, 37, 40, 45). However, the molecular events that underlie the fast component of desensitization remain elusive and controversial. A number of theories have been proposedreceptor-dependent effects that are independent of the G protein (5), effects at the level of the channel (1), a mechanism that is accounted for by the intrinsic hydrolysis cycle of the G protein (24), and, more recently, depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by concurrent agonist activation of a Gq/11-coupled muscarinic receptor (10, 20).
We previously investigated processes accounting for activation and deactivation kinetics (2, 3), and we now focus on desensitization. In this study we systematically tested the various hypotheses suggested to account for this phenomenon. Our data show that desensitization is a fundamental property of all GPCRs and indicate that fast desensitization is a property of the G protein cycle.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophysiology.
Whole cell membrane currents were recorded with an Axopatch 200B amplifier (Axon Instruments). Patch pipettes were pulled from filamented borosilicate glass (Clark Electromedical) and had a resistance of 1.52.5 M when filled with pipette solution (see Materials and drugs). Before filling, the tips of patch pipettes were coated with a Parafilm-mineral oil suspension. Data were acquired and analyzed with a Digidata interface (1200B or 1322; Axon Instruments) and pCLAMP software (version 6.0 or 8.0; Axon Instruments). Cell capacitance was
15 pF, and series resistance (<10 M
) was at least 75% compensated with the amplifier circuitry. Recordings of membrane current were commenced after an equilibration period of
5 min. Immediately after patch rupture a current-voltage relationship was determined to establish that currents were inwardly rectifying. Thereafter cells were voltage-clamped at 60 mV, and agonist-induced currents were measured at this potential. For current-voltage relationships, records were filtered at 1 kHz and digitized at 5 kHz. For continual data acquisition where cells were voltage clamped at 60 mV, records were digitized at 100 Hz. Rapid drug application was performed as previously described (2, 3, 26) with a "sewer pipe" system (Rapid Solution Changer RSC-160; Bio-Logic). Agonist was applied for at least 20 s, and current responses followed a typical profile: after rapid activation of currents to a peak amplitude, current subsequently waned during the presence of agonistwe termed this "desensitization." After removal of agonist, currents returned to baselinewe termed this "deactivation." In this article we focus on desensitization, the magnitude of which was quantified by measuring the relative reduction from peak current at a series of different time points. This is illustrated in Fig. 1A. Channel activation characteristically exhibited an initial "lag" between drug application and onset of current, which was then followed by a subsequent rise to peak amplitude ("time to peak," TTP). Activation kinetics were therefore quantified as "lag + TTP," which is the time period between initial drug application and the peak current amplitude (2). Deactivation kinetics were generally well fitted by a single-exponential decay function and were quantitated by the time constant for decay (deactivation
) (3).
|
Data analysis. Membrane currents were measured at 60 mV, and all data are presented as means ± SE, where n indicates the number of cells recorded from. Data were analyzed for statistical significance with either Student's t-test or one-way repeated-measures ANOVA with Bonferroni correction as appropriate.
Materials and drugs.
Solutions were as follows (concentrations in mM): pipette solution: 107 KCl, 1.2 MgCl2, 1 CaCl2, 10 EGTA, 5 HEPES, 2 MgATP, and 0.3 Na2GTP (KOH to pH 7.2, 140 mM total K+); bath solution: 140 KCl, 2.6 CaCl2, 1.2 MgCl2, and 5 HEPES (pH 7.4). Cell culture materials were obtained from GIBCO BRL and Invitrogen. All chemicals were purchased from Sigma or Calbiochem. Drugs were made up as concentrated stock solutions and kept at 20°C: 5'-(N-ethylcarboxamido)adenosine (NECA), adenosine (9-
-D-ribofuranosyladenine), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), baclofen hydrochloride, carbachol (carbamylcholine chloride), quinpirole, and ()-norepinephrine hydrate (all obtained from Sigma Aldrich, Poole, UK).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Desensitization of currents is observed with both homomeric and heteromeric Kir3.0 channels. It was suggested recently that GIRK4 homomeric channels do not desensitize, suggesting that the desensitization phenomenon reflects molecular processes at the channel level (1). We examined the potential role that different Kir3.0 channel subunits might have in the desensitization response by investigating desensitization in different channel formations. In addition to the "neuronal" Kir3.1+3.2A channel-expressing cell line, we have also established a stable HEK-293 cell line expressing the "cardiac" channel subunits Kir3.1+3.4 (hereafter referred to as HKIR3.1/3.4). We also made functional homomultimers of Kir3.1 and Kir3.4 by introducing point mutations (Kir3.1F137S and Kir3.4S143T) into the coding sequence (43) and transiently transfected these into HEK-293 cells. The M4 muscarinic receptor was transiently transfected into the HKIR3.1/3.4 cell line and into the cells expressing the homomeric channel subunits and was stimulated with 10 µM carbachol. Figure 2A shows representative recordings from each of these channel formations, and quantification of the desensitization data is summarized in Fig. 2B and in Table 1. It is clear that, although there may be some differences in the relative degree of desensitization, the desensitization phenomenon itself is observed qualitatively for all subunits, Kir3.1, Kir3.2A, and Kir3.4, in both heteromeric and homomeric channel complexes.
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since it was first described in atrial myocytes, there has been considerable interest in the fast desensitization phenomenon of Kir3.0 currents in both native and cloned systems (10, 20, 24, 32, 37, 45). Most of the previous work by other investigators has focused on the M2 receptor and the native and cloned cardiac channel. However, in this study we have attempted to systematically dissect the mechanisms for the phenomenon of fast desensitization by using cloned atrial, neuronal, and novel homomeric Kir3.0 channels and several GPCRs. We first showed that a large number of pharmacologically distinct GPCRs, when activated at saturating agonist concentration, can all lead to fast desensitization of the current. Although the GPCRs may be pharmacologically distinct, it is clear from the data shown in Fig. 1 that they all lead to a quantitatively similar magnitude of current desensitization. It would appear that desensitization is a general phenomenon that is related generically to GPCR activation rather than having a specific pharmacological context. This is supported by observations in both cloned and native systems (4, 10); however, desensitization in response to opioid receptor stimulation in locus ceruleus neurons was more compatible with a slow desensitization process (10). In hippocampal neurons, one of us (25) has demonstrated that baclofen activation of endogenous GABAB receptors does lead to pronounced rapid desensitization similar to that observed in the current study. The dynamics and magnitude of the response were very similar. In addition, although we have looked at channel modulation via M4 in our heterologous system, our data are comparable to those obtained via M2 in atrial myocytes. Thus it is clear that desensitization is not an artifact of receptor and channel overexpression in the HEK-293 system. The phenomenon is qualitatively similar in native and heterologous systems.
Early studies in atrial myocytes found that desensitization only occurred at higher agonist concentrations. It was concluded that it was related to intrinsic properties of the G protein cycle (24), and this has been supported by studies on cloned channels (10). Here we show a similar agonist dependence in experiments using the A1 and GABAB heterodimeric receptorsdesensitization was less profound at lower agonist concentrationsand thus demonstrate that desensitization is a general feature of agonist concentration and GPCR activation. Furthermore, we showed that fast current desensitization is inhibited in our system by the inclusion of GDPS, which retards GDP/GTP exchange and consequently inhibits G protein cycling from inactive to active states. Paradoxically, we found that the rate of current deactivation is also enhanced by GDP
S; the reasons for this are unclear. In addition, we showed that where activation kinetics are modulated by changing the G protein in a ternary complex by constraining coupling to a specific G
subunit, this has a consequence similar to changes in agonist concentration on desensitization. Thus all our data support the hypothesis that desensitization is due to the rate of entry into the G protein cycle. It is an appealing hypothesis that desensitization reflects the transition from a nonequilibrium condition in which a large number of G protein
-subunits are simultaneously activated to the equilibrium condition in which they are actively cycling (i.e., binding and releasing G
). In the latter state, some G
will be sequestered in heterotrimers and thus inactive, resulting in smaller currents. In addition, the kinetics of deactivation are consistent with this process (3). It has been found that overexpression of regulators of G protein signaling (RGS proteins) enhances desensitization (10, 13). Our own studies (Benians A and Tinker A, unpublished observations) show a similar pattern, i.e., overexpression of RGS proteins increases desensitization and signaling via RGS-insensitive G
subunits leads to less (although this is not statistically significant). In a previous study (2), we found that G protein levels simply controlled the amplitude of current response but not the channel kinetics or the desensitization; this finding supports our general hypothesis.
One of the more recent and controversial proposals is that concurrent activation of a Gq/11 receptor leads to PIP2 depletion and thus current inhibition (9, 20, 32). A number of our observations tend to argue against such a mechanism being a broad one. First, GPCRs such as GABAB do not have a Gq/11-coupled counterpart, although it is conceivable that phospholipase C may be activated in these cell lines by G released from Gi/o heterotrimers (38). Thus we formally investigated this possibility by looking for activation of such pathways in our cell lines with Ca2+ imaging whereby the activation of phospholipase C and hydrolysis of PIP2 resulting in the generation of IP3 would lead to a rise in intracellular Ca2+ as it is released from intracellular stores. We found that neither baclofen nor NECA stimulation of the GABAB and A1 receptors mobilized intracellular Ca2+. This argues against PIP2 depletion as a general mechanism; instead, it may act to merely enhance desensitization. However, it is worth noting that even in the M4 line, where carbachol can stimulate an endogenous Gq/11-coupled muscarinic receptor, the degree of desensitization was no more prominent than with the other receptors. In our previous studies we have generally had to overexpress muscarinic M1 and M3 receptors in HEK-293 cells to observe channel regulation. Why haven't we seen regulation through the endogenously expressed receptor? Our pipette solution contains relatively little Ca2+ (
20 nM) that is heavily buffered, and it is known that phospholipase C activity is dependent on Ca2+ (38). Thus, to get significant enzymatic activity in the whole cell configuration, it seems likely that it is necessary to enhance signaling efficacy by increasing the levels of receptor expression.
A second possibility to account for desensitization is that a time-dependent decrease in external K+ concentration occurs with agonist application. Measurement of Erev before and after agonist application revealed little change. In addition, performing the experiments at more hyperpolarized potentials where the currents are larger (and accumulation should be more pronounced) led to a similar level of desensitization. Finally, Chuang et al. (10) only observed desensitization in inside-out macropatch recordings when switching from GDP to GTP. Thus our data do not suggest a central role for K+ depletion; however, we cannot exclude its importance in other situations or systems.
Here we also report an A1-mediated channel inhibition that is independent of G protein activation that is revealed when HKIR3.1/3.2/A1 cells are dialyzed with GTPS. This was a finding unique to the A1 receptorwe did not observe this with any other receptors tested. Other investigators have observed a similar phenomenon after adenoviral expression of the A1 receptor in atrial myocytes (5). This is likely to account for the more profound desensitization observed with the A1 receptor, and it may also underlie the transient current increase on agonist withdrawal. We suggest that it may represent a direct sequestration of G
subunits by the A1 receptor itself, but further studies are required to elucidate the underlying mechanism.
Recently, it was proposed that GIRK4 homomultimers do not desensitize and that desensitization reflects processes occurring at the level of the channel (1). It is worth noting that if channel activation by liberated free G is slow (relative to the kinetics of upstream events), recorded currents will not reflect the dynamics of the G protein cycle. If channel homomultimers in a given environment activate more slowly than heteromultimers then such an extrapolation cannot be made. To investigate this hypothesis we studied the desensitization of various hetero- and homomultimeric Kir3.0 channels in response to stimulation of M4 receptors. In contrast to Bender et al. (1), we found that desensitization was a general property of all GIRK channels regardless of their subunit composition.
Thus our data support the hypothesis that the G protein cycle, and in particular the rate of entry into it, is of central importance to a general mechanism of desensitization that can occur with any Gi/o-coupled GPCR at saturating agonist concentration. However, other processes such as PIP2 depletion and receptor-dependent G protein-independent processes may be able to attenuate or potentiate this response.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
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.
* J. L. Leaney and A. Benians contributed equally to this work.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Benians A, Leaney JL, Milligan G, and Tinker A. The dynamics of formation and action of the ternary complex revealed in living cells using a G-protein-gated K+ channel as a biosensor. J Biol Chem 278: 1085110858, 2003.
3. Benians A, Leaney JL, and Tinker A. Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K+ channels. Proc Natl Acad Sci USA 100: 62396244, 2003.
4. Blanchet C and Luscher C. Desensitization of µ-opioid receptor-evoked potassium currents: initiation at the receptor, expression at the effector. Proc Natl Acad Sci USA 99: 46744679, 2002.
5. Bosche LI, Wellner-Kienitz MC, Bender K, and Pott L. G protein-independent inhibition of GIRK current by adenosine in rat atrial myocytes overexpressing A1 receptors after adenovirus-mediated gene transfer. J Physiol 550: 707717, 2003.
6. Breitwieser GE and Szabo G. Uncoupling of cardiac muscarinic and beta-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature 317: 538540, 1985.[ISI][Medline]
7. Carmeliet E and Mubagwa K. Desensitization of the acetylcholine-induced increase of potassium conductance in rabbit cardiac Purkinje fibres. J Physiol 371: 239255, 1986.[Abstract]
8. Chan KW, Langan MN, Sui JL, Kozak JA, Pabon A, Ladias JA, and Logothetis DE. A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins. J Gen Physiol 107: 381397, 1996.[Abstract]
9. Cho H, Hwang JY, Kim D, Shin HS, Kim Y, Earm YE, and Ho WK. Acetylcholine-induced phosphatidylinositol 4,5-bisphosphate depletion does not cause short-term desensitization of G protein-gated inwardly rectifying K+ current in mouse atrial myocytes. J Biol Chem 277: 2774227747, 2002.
10. Chuang HH, Yu M, Jan YN, and Jan LY. Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K+ channels. Proc Natl Acad Sci USA 95: 1172711732, 1998.
11. Corey S and Clapham DE. Identification of native atrial G-protein-regulated inwardly rectifying K+ (GIRK4) channel homomultimers. J Biol Chem 273: 2749927504, 1998.
12. Dascal N, Schreibmayer W, Lim NF, Wang W, Chavkin C, DiMagno L, Labarca C, Kieffer BL, Gaveriaux-Ruff C, Trollinger D, Lester HA, and Davidson N. Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc Natl Acad Sci USA 90: 1023510239, 1993.[Abstract]
13. Doupnik CA, Davidson N, Lester HA, and Kofuji P. RGS proteins reconstitute the rapid gating kinetics of G-activated inwardly rectifying K+ channels. Proc Natl Acad Sci USA 94: 1046110466, 1997.
14. Drici MD, Diochot S, Terrenoire C, Romey G, and Lazdunski M. The bee venom peptide tertiapin underlines the role of IKACh in acetylcholine-induced atrioventricular blocks. Br J Pharmacol 131: 569577, 2000.
15. Giblin JP, Leaney JL, and Tinker A. The molecular assembly of ATP-sensitive potassium channels: determinants on the pore forming subunit. J Biol Chem 274: 2265222659, 1999.
16. Harris GC and Williams JT. Transient homologous mu-opioid receptor desensitization in rat locus coeruleus neurons. J Neurosci 11: 25742581, 1991.[Abstract]
17. Inanobe A, Yoshimoto Y, Horio Y, Morishige KI, Hibino H, Matsumoto S, Tokunaga Y, Maeda T, Hata Y, Takai Y, and Kurachi Y. Characterization of G-protein-gated K+ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra. J Neurosci 19: 10061017, 1999.
18. Jelacic TM, Kennedy ME, Wickman K, and Clapham DE. Functional and biochemical evidence for G-protein-gated inwardly rectifying K+ (GIRK) channels composed of GIRK2 and GIRK3. J Biol Chem 275: 3621136216, 2000.
19. Jelacic TM, Sims SM, and Clapham DE. Functional expression and characterization of G-protein-gated inwardly rectifying K+ channels containing GIRK3. J Membr Biol 169: 123129, 1999.[CrossRef][ISI][Medline]
20. Kobrinsky E, Mirshahi T, Zhang H, Jin T, and Logothetis DE. Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nat Cell Biol 2: 507514, 2000.[CrossRef][ISI][Medline]
21. Krapivinsky G, Gordon EA, Wickman K, Velimirovic B, Krapivinsky L, and Clapham DE. The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K+-channel proteins. Nature 374: 135141, 1995.[CrossRef][ISI][Medline]
22. Kubo Y, Reuveny E, Slesinger PA, Jan YN, and Jan LY. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364: 802806, 1993.[CrossRef][ISI][Medline]
23. Kurachi Y, Nakajima T, and Sugimoto T. Acetylcholine activation of K+ channels in cell-free membrane of atrial cells. Am J Physiol Heart Circ Physiol 251: H681H684, 1986.
24. Kurachi Y, Nakajima T, and Sugimoto T. Short-term desensitization of muscarinic K+ channel current in isolated atrial myocytes and possible role of GTP-binding proteins. Pflügers Arch 410: 227233, 1987.[ISI][Medline]
25. Leaney JL. Contribution of Kir3.1, Kir3.2A, and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons. Eur J Neurosci 18: 21102118, 2003.[CrossRef][ISI][Medline]
26. Leaney JL, Dekker LV, and Tinker A. Regulation of a G protein-gated inwardly rectifying potassium channel by a Ca2+-independent protein kinase C. J Physiol 534: 367379, 2001.
27. Leaney JL, Milligan G, and Tinker A. The G protein subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly rectifying K+ channels. J Biol Chem 275: 921929, 2000.
28. Leaney JL and Tinker A. The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel. Proc Natl Acad Sci USA 97: 56515656, 2000.
29. Lesage F, Duprat F, Fink M, Guillemare E, Coppola T, Lazdunski M, and Hugnot JP. Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain. FEBS Lett 353: 3742, 1994.[CrossRef][ISI][Medline]
30. Logothetis DE, Kurachi Y, Galper J, Neer EJ, and Clapham DE. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325: 321326, 1987.[CrossRef][ISI][Medline]
31. Luscher C, Jan LY, Stoffel M, Malenka RC, and Nicoll RA. G protein-coupled inwardly rectifying K+ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron 19: 687695, 1997.[ISI][Medline]
32. Meyer T, Wellner-Kienitz MC, Biewald A, Bender K, Eickel A, and Pott L. Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes. J Biol Chem 276: 56505658, 2001.
33. Noma A and Trautwein W. Relaxation of the ACh-induced potassium current in the rabbit sinoatrial node cell. Pflügers Arch 377: 193200, 1978.[ISI][Medline]
34. North RA. Drug receptors and the inhibition of nerve cells. Br J Pharmacol 98: 1328, 1989.[ISI][Medline]
35. Otis TS, De Koninck Y, and Mody I. Characterization of synaptically elicited GABAB responses using patch-clamp recordings in rat hippocampal slices. J Physiol 463: 391407, 1993.[Abstract]
36. Pfaffinger PJ, Martin JM, Hunter DD, Nathanson NM, and Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature 317: 536538, 1985.[ISI][Medline]
37. Pitcher JA, Freedman NJ, and Lefkowitz RJ. G protein-coupled receptor kinases. Annu Rev Biochem 67: 653692, 1998.[CrossRef][ISI][Medline]
38. Rebecchi MJ and Pentyala SN. Structure, function, and control of phosphoinositide-specific phospholipase C. Physiol Rev 80: 12911335, 2000.
39. Reuveny E, Slesinger PA, Inglese J, Morales JM, Iniguez Lluhi JA, Lefkowitz RJ, Bourne HR, Jan YN, and Jan LY. Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits. Nature 370: 143146, 1994.[CrossRef][ISI][Medline]
40. Shui Z, Khan IA, Tsuga H, Haga T, and Boyett MR. Role of receptor kinase in short-term desensitization of cardiac muscarinic K+ channels expressed in Chinese hamster ovary cells. J Physiol 507: 325334, 1998.
41. Sodickson DL and Bean BP. Neurotransmitter activation of inwardly rectifying potassium currents in dissociated hippocampal CA3 neurons: interactions among multiple receptors. J Neurosci 18: 81538162, 1998.
42. Soejima M and Noma A. Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells. Pflügers Arch 400: 424431, 1984.[ISI][Medline]
43. Vivaudou M, Chan KW, Sui JL, Jan LY, Reuveny E, and Logothetis DE. Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants. J Biol Chem 272: 3155331560, 1997.
44. Wickman K, Nemec J, Gendler SJ, and Clapham DE. Abnormal heart rate regulation in GIRK4 knockout mice. Neuron 20: 103114, 1998.[ISI][Medline]
45. Yamada M, Inanobe A, and Kurachi Y. G protein regulation of potassium ion channels. Pharmacol Rev 50: 723757, 1998.