Rapid desensitization of G protein-gated inwardly rectifying K+ currents is determined by G protein cycle

Joanne L. Leaney,* Amy Benians,* Sean Brown, Muriel Nobles, David Kelly, and Andrew Tinker

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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of G protein-gated inwardly rectifying K+ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of Gi/o-coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several Gi/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A1, adrenergic {alpha}2A, dopamine D2S, M4 muscarinic, and GABAB1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A1 receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca2+ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific G{alpha} subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle.

G protein-coupled receptor; potassium channel; inward rectifier; kinetics


INWARDLY RECTIFYING K<SUP>+SUP> CHANNELS gated by G proteins (GIRK channels) were first characterized in atrial myocytes and are activated by acetylcholine binding to muscarinic M2 receptors (33). Stimulation of this current is responsible in part for slowing of the heart rate in response to vagal nerve stimulation (14, 44). It is now clear that analogous currents are present in central neurons and neuroendocrine cells. In central neurons GIRK currents are activated by a large variety of Gi/o-coupled receptors (34), including GABAB and adenosine A1, generating late inhibitory postsynaptic potentials (31, 41, 45). Activation of the current is membrane delimited (42), mimicked by nonhydrolyzable GTP analogs (6), and sensitive to pertussis toxin (PTX), implicating the inhibitory family of G proteins (Gi/o) (36). Activation of native and cloned G protein-gated K+ channels has been shown to involve a direct interaction with the G{beta}{gamma} dimer that is released from the activated G protein heterotrimer (30, 39, 45). The cloning of a subfamily of inwardly rectifying K+ channels (Kir3.0) revealed that the native channel is a heterotetrameric complex composed of Kir3.1 with Kir3.2, Kir3.3, or Kir3.4 subunits (8, 12, 17, 19, 21, 22, 29, 45). Recently it has become apparent that functional homotetrameric complexes of Kir3.0 may also occur in native cells (1, 11, 17) and that Kir3.2 and Kir3.3 may coassemble (18).

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 proposed—receptor-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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular biology, cell culture, and transfection. The methods for cell culture and transient transfection and the techniques for establishing stable cell lines have been described previously (15, 27). In addition to the stable lines described previously (2, 3), a line expressing Kir3.1 and Kir3.4 channel subunits was established. Kir3.1 and Kir3.4 were subcloned into the dual-promoter vector pBudCE4.1. Monoclonal cell lines were generated after transfection and growth under selective pressure (364 µg/ml Zeocin). Single colonies were picked, expanded, and screened by patch clamping. The mutants (Kir3.1F137S and Kir3.4S143T) that generate currents as homomeric channels (43) were constructed with a QuickChange kit (Stratagene). Mutations were confirmed by automated sequencing.

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 1.5–2.5 M{Omega} 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{Omega}) 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 agonist—we termed this "desensitization." After removal of agonist, currents returned to baseline—we 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 {tau}) (3).



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Fig. 1. Fast desensitization occurs through all studied G protein-coupled receptors (GPCRs). A: example of a quinpirole-activated current in the KIR3.1/3.2/D2 cell line recorded at –60 mV in the whole cell patch-clamp configuration. The 20-s application of a maximal dose of agonist (10 mM) is indicated by the horizontal bar (in this and all subsequent figures). Current desensitization (as indicated) was measured as shown by measuring current amplitudes at time points 1, 5, 10, and 20 s from the peak. Data are expressed as %desensitization from peak. B: summary of the current desensitization observed over 20 s in all receptor + channel cell lines studied. Data are from the following numbers of cells: HKIR3.1/3.2/A1, n = 32; HKIR3.1/3.2/{alpha}2A, n = 18; HKIR3.1/3.2/D2, n = 24; HKIR3.1/3.2/GGB, n = 26; HKIR3.1/3.2/M4, n = 9. HKIR3.1/3.2/A1, HEK-293 stable cell line expressing Kir3.1+3.2A channel complex and adenosine A1; NECA, 5'-(N-ethylcarboxamido)adenosine; HKIR3.1/3.2/D2, HEK-293 stable cell line expressing Kir3.1+3.2A channel complex and dopamine D2S; HKIR3.1/3.2/{alpha}2, HEK-293 stable cell line expressing Kir3.1+3.2A channel complex and adrenergic {alpha}2A.

 
Calcium imaging. HEK-293 cells were cultured on poly-L-lysine-coated coverslips and were loaded with 5 µM fura 2-AM for 30 min at 37°C. Cells were perfused with an extracellular buffer containing (in mM) 140 NaCl, 2.5 KCl, 0.5 MgCl2, 1.2 CaCl2, 10 HEPES, and 5 glucose. The solution was buffered at pH 7.4 with NaOH. Fluorescence microscopy was performed with a polychrome II monochromator as an epifluorescent light source (T.I.L.L. Photonics) connected to a Zeiss microscope. Images were collected and analyzed with a digital microscopy system (Openlab; Improvision, Coventry, UK). Cells were excited alternately at 340 and 380 nm, and intracellular calcium was assessed from the ratio of the respective emitted intensity of fluorescent signals at 470–550 nM (the dichroic was 400DCLP and the emission filter 510/80; Chroma Technology).

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-{beta}-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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kir3.1+3.2A desensitization after stimulation of a diverse array of Gi/o-coupled receptors. We used a HEK-293 stable cell line that expresses the Kir3.1+3.2A channel complex (HKIR3.1/3.2) and dual Gi/o-coupled receptor plus channel stable lines (adenosine A1: HKIR3.1/3.2/A1, adrenergic {alpha}2A: HKIR3.1/3.2/{alpha}2, dopamine D2S: HKIR3.1/3.2/D2, muscarinic M4: HKIR3.1/3.2/M4, and the heterodimeric GABAB1b/2: HKIR3.1/3.2/GGB) (27, 28). Cells were studied with the whole cell configuration of the patch-clamp technique, and drugs were applied with a rapid agonist application system (see MATERIALS AND METHODS). Figure 1A shows how current desensitization was measured during agonist application. We next investigated whether currents, which were activated via a diverse array of receptors with a saturating concentration of agonist (A1: 1 µM NECA, {alpha}2A: 3 µM norepinephrine, D2S: 10 µM quinpirole, M4: 10 µM carbachol, GABAB1b/2: 100 µM baclofen), exhibited desensitization in a qualitatively and quantitatively similar fashion (representative recordings from the different channel + receptor cell lines are shown in Figs. 2 and 5). The current desensitization was quantified at the time points indicated, and the mean data are summarized in Fig. 1B and in Table 1. We previously (2) used radioligand binding to measure levels of receptor expression in the HKIR3.1/3.2/A1, HKIR3.1/3.2/{alpha}2, and HKIR3.1/3.2/D2 cell lines and found that receptors were expressed to similar levels. It is clear that the rapid desensitization of currents was quantitatively similar for the {alpha}2A and D2S receptors, whereas the A1 receptor exhibited more profound desensitization that was seemingly accounted for by an initial rapid phase not present with the other receptors. In the KIR3.1/3.2/A1 line we also saw a transient reactivation in current on removal of agonist. These unique kinetic effects were observed consistently in two different clonal isolates of A1 and Kir3.1/3.2 channel-expressing cell lines and in recordings from HKIR3.1/3.2-expressing cells transiently transfected with the adenosine A1 receptor (data not shown). A potential explanation for this behavior is given below.


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Table 1. Degree of desensitization present at point of drug removal

 
Desensitization in our system did not seem to be accounted for by K+ accumulation in the cell and reduction of driving force. We measured the reversal potential (Erev) before and after agonist application. There was only a small change: in the HKIR3.1/3.2/D2 line (10 µM quinpirole), {Delta}Erev = –2.18 ± 6.6 mV (n = 5). We examined desensitization at another holding potential (–100 mV) and observed no statistical difference in the magnitude of the effect. In the HKir3.1/3.2A/D2 line, using 10 µM quinpirole and measuring desensitization 10 s into the response, we found that at –60 mV there was 16.7 ± 1.9% desensitization (n = 19) compared with 12.8 ± 1.4% desensitization at –100 mV (n = 6; not significant).

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.



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Fig. 2. Desensitization occurs with other Kir3.0 channel isoforms. A: representative example traces of carbachol-activated currents (10 µM) in the HKIR3.1/3.2/M4 cell line, the HKIR3.1/3.4 cell line transiently expressing the muscarinic M4 receptor, and Kir3.1F137S+M4 and Kir3.4S143T+M4 transiently expressed in HEK293 cells. During the 30-s application of carbachol, each channel type clearly undergoes some degree of desensitization. B: summary of desensitization data for the channels described in A activated after muscarinic M4 receptor stimulation. Data are from the following numbers of cells: HKIR3.1/3.2/M4, n = 9; HKIR3.1/3.4+M4, n = 12; Kir3.1F137S+M4, n = 15; Kir3.4S143T+M4, n = 4. *P ≤ 0.05.

 
Desensitization does not solely result from activation of a Gq/11-coupled pathway. We investigated the possibility that the observed current desensitization is due solely to a concomitant activation of an inhibitory receptor-mediated Gq/11 pathway (20) by using fura 2 ratiometric imaging to examine changes in intracellular Ca2+, which may result from phospholipase C{beta} activation and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ mobilization. We investigated whether a change in intracellular Ca2+ could be detected when a maximal concentration of the appropriate agonist was applied to each of our HEK-293 cell lines as described above. In two of these cell lines (HKIR3.1/3.2/M4 and HKIR3.1/3.2/{alpha}2A) we found a significant increase in Ca2+ on agonist application (Fig. 3A, top), suggesting that HEK-293 cells are likely to contain endogenous muscarinic (e.g., M1 and M3) and adrenergic receptors (e.g., {alpha}1) receptors coupled to Gq/11. Another possibility is activation of phospholipase C by release of G{beta}{gamma} (38). We further studied the carbachol-mediated Ca2+ rise and found that it occurred in all clonal HEK-293 cell isolates studied, and thus we used it as a positive control. In contrast, no Ca2+ response was detected in the A1 or GABAB cell lines after the application of NECA and baclofen, respectively (Fig. 3A, bottom). Figure 3B shows mean 340 nm-to-380 nm ratios from a number of cells in each of the cell lines. It is clear that cell lines that display desensitization of Kir3.1/3.2A currents need not necessarily coactivate phospholipase C{beta} signaling pathways, as detected by intracellular Ca2+ mobilization.



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Fig. 3. Ca2+ responses in HEK-293 cell stable lines. A: representative recordings of the time course of the changes in the 340 nm-to-380 nm ratio in a series of Kir3.1+3.2A+receptor-expressing stable cell lines. Agonist concentrations: norepinephrine 3 µM, carbachol 10 µM, baclofen 100 µM, and 5'-(N-ethylcarboxamido)adenosine (NECA) 1 µM. In those cell lines with no response to the appropriate agonist (HKIR3.1/3.2/A1 and HKIR3.1/3.2/GGB), carbachol, presumably acting through an endogenous Gq/11-coupled muscarinic receptor, was used a positive control. B: pooled data. ***P ≤ 0.001. NS, not significant.

 
G protein-independent inhibition of Kir3.1+3.2A currents in HKIR3.1/3.2/A1 cell line. Recently there was a report describing channel inhibition by some receptors in a G protein-independent fashion (5). We investigated this finding further, and our data are shown in Fig. 4. With a whole cell patch-clamp configuration in which guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) replaced GTP in the pipette solution, inwardly rectifying currents steadily increased until a stable baseline was reached. In Fig. 4A we show that GTP{gamma}S-loaded cells of the HKIR3.1/3.2/A1 cell line exhibit a direct and reversible inhibition of current in response to NECA. As the concentration of NECA was increased the rate of inhibition increased, whereas it did not affect the relief of inhibition, suggesting a bimolecular reaction. Figure 4B shows that NECA applied to the KIR3.1/3.2 cell line does not cause any inhibition in the absence of the A1 receptor and that NECA applied to the HKIR3.1/3.2/D2 cell line during desensitization after quinpirole application does not lead to any additional desensitization. In a similar experiment using the HKIR3.1/3.2/GGB cell line, we showed that baclofen (100 mM) applied to GTP{gamma}S-loaded cells does not induce current inhibition (Fig. 4C). Similarly, current mediated by GTP{gamma}S-loaded cells of the HKIR3.1/3.2/D2 and HKIR3.1/3.2/M4 cell lines did not display inhibition in response to agonists, either (data not shown). Finally, we showed that another agonist (adenosine) at the A1 receptor can cause this phenomenon (Fig. 4D; n = 7 cells). This process can be blocked by the A1 receptor antagonist DPCPX (n = 2; not shown). Thus our data are consistent with a recent report (5) but show that this phenomenon is confined to the A1 receptor.



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Fig. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S)-loaded cells reveal an inhibitory effect of A1 adenosine agonists. A: HKIR3.1/3.2/A1 cells were dialyzed for ~10 min with pipette solution containing 0.9 mM GTP{gamma}S until currents had reached steady state. Currents were recorded at a holding potential of –60 mV, and NECA (first application 1 µM and second application 10 µM) was applied as indicated. Ba2+ (1 mM) was applied at the end of the experiment to confirm the presence of a K+ current. The kinetics of the process are shown on the right. {tau}on, activation time constant; {tau}off, deactivation time constant. ***P ≤ 0.001. B: i: current trace recorded from a GTP{gamma}S-loaded HKIR3.1/3.2 cell showing that NECA-induced current inhibition was present only in the A1 receptor-expressing HKIR3.1/3.2/A1 cell line. Similar results were obtained in 2 other cells. ii: Application of NECA during quinpirole (Quin)-induced desensitization does not further increase desensitization in the KIR3.1/3.2/D2 cell line. C: traces were recorded from HKIR3.1/3.2/GGB cells loaded with GTP{gamma}S as described in A. Application of baclofen (100 µM) caused no current inhibition. Similar results were obtained in 2 other cells. D: HKIR3.1/3.2/A1 cells were dialyzed for ~10 min with pipette solution containing 0.9 mM GTP{gamma}S until currents had reached steady state. In the cell shown, G{beta}1 and G{gamma}1 were also cotransfected to further increase currents. Adenosine (Ado; 1 µM) led to an inhibition comparable to that of NECA (10 µM). This occurred in 6 other cells.

 
Desensitization is dependent on degree of channel activation. We next examined how receptor occupancy might affect the observed desensitization by varying the agonist concentration. We used the HKIR3.1/3.2/GGB and HKIR3.1/3.2/A1 cell lines and applied the relevant agonists at a low concentration (approximately EC50) and at a high, saturating concentration. Example traces for stimulation of HKIR3.1/3.2/GGB cells with 1 and 100 µM are shown in Fig. 5A. It is clear that at the lower concentration the channel took longer to reach peak activation, and the extent of channel desensitization was also significantly reduced. The data are summarized in Fig. 5B. In the HKIR3.1/3.2/A1 line, the initial rapid phase of the fast desensitization appeared to be absent at a 30 nM NECA concentration, and the accompanying current reactivation effect—usually observed on removal of 1 µM NECA—was reduced. We also examined the relationship between the magnitude of GABAB receptor stimulation (measured as peak current density, Imax) and the extent of current desensitization after 20-s exposure to varying concentrations of baclofen. It is clear that the intensity of receptor stimulation was well correlated with percent current desensitization (Fig. 5C, left). We then looked for a relationship between the time taken for current to reach peak amplitude and the extent of desensitization at different concentrations of baclofen (Fig. 5C, right). Interestingly, we found a good degree of inverse correlation between these two parameters (although linear regression was not performed because of the saturating nature of this effect at 10 and 100 µM baclofen). Clearly, the slower the current activates the less it desensitizes, and the stronger the intensity of receptor stimulation the greater the extent of current desensitization. This is consistent with previous studies in native cardiac myocytes (24).



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Fig. 5. Desensitization depends on agonist concentration. A: superimposed whole cell current traces recorded at –60 mV from the HKIR3.1/3.2/GGB cell line in standard solutions showing a response to a 20-s application of 1 and 100 µM baclofen. B. summary of data for GABAB (left) and A1 (right) receptor (30 nM and 1 µM NECA) cell lines. The 1 µM baclofen responses were significantly slower to activate, so desensitization was measured only on removal of agonist. C: graph on left shows a direct correlation between GABAB receptor stimulation [peak current densities (Imax) measured at –60 mV] and % desensitization at the end of a 20-s application of baclofen. Linear regression was performed with MicroCal Origin software (r2 = 0.97). Graph on right shows an inverse correlation between time taken for current to fully activate [lag + time to peak (TTP); described in MATERIALS AND METHODS] and the degree of desensitization observed (% desensitization).

 
We investigated the effect of competitive inhibition of GDP-GTP exchange by including guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S) in the pipette solution (in addition to the normal amount of GTP). This phosphorylation-resistant analog of GDP has been shown to attenuate many G protein-mediated signaling processes (23), probably by reversibly occupying the guanine nucleotide binding pocket on the G{alpha} subunit, thereby preventing binding of GTP and formation of active G{alpha}-GTP and G{beta}{gamma} subunits. We used the HKIR3.1/3.2/GGB cell line to examine the effect of GDP{beta}S on signaling to the channel, and representative current traces with different amounts of GDP{beta}S in the pipette solution are shown in Fig. 6A. We found that desensitization was dramatically attenuated with 3 mM GDP{beta}S present in the pipette solution (Fig. 6B) and that the rate of activation of the current was significantly slowed (Fig. 6C). Unexpectedly, the rate of deactivation of currents on removal of agonist was also significantly enhanced in the presence of GDP{beta}S (Fig. 6C).



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Fig. 6. Activation and desensitization of Kir3.1/3.2 currents are inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S). A: representative examples of baclofen-activated currents (100 µM) recorded at –60 mV from HKIR3.1/3.2/GGB cells that had been dialyzed for at least 7 min with pipette solution (P/S) containing 0.3 mM GTP and 2 mM ATP (control) plus either 1 or 3 mM GDP{beta}S as indicated. B: summary of desensitization data obtained from a number of experiments as described in A. C: activation (lag + TTP) and deactivation (deactivation {tau}) kinetics were also quantified as described in MATERIALS AND METHODS. These bar graphs summarize data obtained from experiments described in A. GDP{beta}S acted to both decelerate channel activation and accelerate deactivation rates. *P ≤ 0.05, ***P ≤ 0.001.

 
Thus, from the data shown in Figs. 5 and 6, it is apparent that the degree of desensitization correlates with the rate of activation. Our previous studies (2) showed that activation kinetics can also be affected by components of the whole ternary complex (i.e., the unique combination of agonist, receptor, and G protein isoform). To investigate the role of the specific G protein {alpha}-subunit we used two G{alpha} subunits, Gi{alpha}2 and Go{alpha}A, which had been made resistant to the actions of PTX by the mutation of the carboxy terminus –4 cysteine residue to glycine. Gi{alpha}2C352G or Go{alpha}AC351G were transiently transfected into the HKIR3.1/3.2/GGB cell line, and the cells were subsequently treated with PTX to eliminate coupling to endogenous G proteins. We previously established (2) that activation with 100 µM baclofen application was significantly slowed via Gi{alpha}2C352G compared with Go{alpha}AC351G. We found that baclofen-activated currents also exhibited significantly greater desensitization when mediated through Go{alpha}A compared with Gi{alpha}2 (Fig. 7).



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Fig. 7. The G{alpha} subunit determines the extent of GABAB-mediated desensitization. Summary of desensitization data measured from baclofen-induced currents recorded in HKIR3.1/3.2/GGB cells coexpressing either Gi{alpha}2C352G or Go{alpha}AC351G and treated with pertussis toxin (100 ng/ml for 16 h) is shown. Currents were measured at –60 mV in response to application of 100 µM baclofen. *P ≤ 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our data suggest that Kir3.0 channel desensitization is a general property of all receptors and channel isoforms. It depends on the intensity of the signal to the G protein and reflects the dynamics of the G protein cycle. Other factors may act to modulate this underlying response but are not central as a causative mechanism.

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 receptors—desensitization was less profound at lower agonist concentrations—and 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 GDP{beta}S, 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{beta}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{alpha} 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 {alpha}-subunits are simultaneously activated to the equilibrium condition in which they are actively cycling (i.e., binding and releasing G{beta}{gamma}). In the latter state, some G{beta}{gamma} 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{alpha} 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{beta}{gamma} 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 GTP{gamma}S. This was a finding unique to the A1 receptor—we 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{beta}{gamma} 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{beta}{gamma} 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
 
This work was supported by the Wellcome Trust, the Royal Society, and the British Heart Foundation. J. L. Leaney is a Royal Society Dorothy Hodgkin Fellow.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Tinker, Rm. 420, 4th Floor, BHF Laboratories and Dept. of Medicine, University College London, 5 University St., London WC1E 6JJ, UK (E-mail: a.tinker{at}ucl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* J. L. Leaney and A. Benians contributed equally to this work. Back


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