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11- and Gi-coupled P2Y receptors
Molecular and Cellular Neuroscience 43 (2010) 363–369
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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m c n e
Two-pore potassium ion channels are inhibited by both Gq/11- and Gi-coupled P2Y receptors Sony Shakya Shrestha, Miralkumar Parmar, Charles Kennedy ⁎, Trevor J. Bushell Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 ONR, UK
a r t i c l e
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Article history: Received 4 November 2009 Revised 7 January 2010 Accepted 13 January 2010 Available online 22 January 2010 Keywords: Two-pore potassium channels K2P2.1 P2Y1 receptor P2Y12 receptor
a b s t r a c t Two-pore potassium (K2P) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Gαi-coupled hP2Y12 receptor, ADP and ATP significantly inhibited hK2P2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y12 antagonist AR-C69931MX, the hP2Y1 antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Gαq/11coupled hP2Y1 receptor, ADP and ATP also inhibited hK2P2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K2P2.1 channels, ADPinduced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Gαq/11- and Gαicoupled P2Y receptors inhibit K2P channels and the action of hP2Y12 receptors appears to involve co-activation of endogenous hP2Y1 receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity. © 2010 Elsevier Inc. All rights reserved.
Introduction Two-pore potassium (K2P) ion channels are the most recently identified family of K+ channels and the 15 members cloned in mammals (Mathie, 2007) are distributed widely throughout the body, including the CNS (Talley et al., 2001; Aller et al., 2005) and sensory nerves (Dobler et al., 2007). They play an important role in the “leak” or “background” current present in many neurones and so regulate neuronal excitability. In addition, they have been implicated in a variety of disorders, including pain (Alloui et al., 2006; Linden et al., 2006) and depression (Heurteaux et al., 2006) and are proposed as a target for neuroprotection (Lauritzen et al., 2000; Buckler and Honoré, 2005). Their activity is modulated by a range of physical and chemical factors, such as inhalation anaesthetics (Patel et al., 1999; Franks and Honoré, 2004; Gruss et al., 2004), and G protein-coupled receptors (GPCR) (Bushell et al., 2002; Mathie, 2007). In general, GPCR that couple to the Gαq/11 and Gαs G proteins mediate inhibition of K2P channel opening, whereas activation of those that couple to Gαi/o increase channel activity (Mathie, 2007). For example, we showed recently that currents carried via K2P2.1 (formerly known as TREK-1; see Kim, 2003; Honoré, 2007) were potentiated by activation of the Gαi/o-coupled mGlu4 receptor (Cain et al., 2008). P2Y receptors are a family of 8 GPCR (P2Y1, 2, 4, 6, 11, 12, 13, 14) that mediate the extracellular actions of endogenous nucleotides, such as
Abbreviations: ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; GPCR, G protein-coupled receptors; K2P, two-pore potassium ion channels; PTX, pertussis toxin. ⁎ Corresponding author. Fax: + 44 141 552 2562. E-mail address: [email protected] (C. Kennedy). 1044-7431/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2010.01.003
adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP) (Burnstock and Kennedy, 1985; Abbracchio et al., 2006). They are expressed throughout the body, including central, sensory, motor and peripheral neurones (Burnstock and Knight, 2004). When activated they interact with a variety of ion channels, including NMDA and TRPV1 receptors, voltage-gated Ca2+ channels and several types of K+ channel, leading to changes in action potential firing and neurotransmitter release (see Kennedy et al., 2003; Lechner and Boehm, 2004; Köles et al., 2008 for reviews). In addition, a K2P2.1-like current in rat cardiomyocytes was potentiated by ATP, possibly via the P2Y11 receptor (Aimond et al., 2000). The expression of P2Y receptors and K2P channels appears to overlap in many regions of the CNS and in sensory neurones and they are both novel targets for the treatment of neuronal disorders, but at present, little is known about how they interact. In preliminary experiments we found that P2Y receptor agonists modulate K2P-like currents in central neurones (Kennedy and Bushell, unpublished observations), but characterisation of this interaction was complicated by the expression of multiple P2Y receptors and K2P channels in these neurones (Watkins and Mathie, 1996; Aller et al., 2005; Mathie, 2007). Therefore, in the present study we coexpressed recombinant human K2P channels and P2Y receptors in the tsA201 cell line, as a first step in characterising the basic properties of the interaction. K2P2.1 channels were used as they are present in both the CNS (Talley et al., 2001) and sensory neurones (Alloui et al., 2006). Since K2P channels are differentially modulated by GPCR that couple to Gαq/11 and Gαi/o (Mathie, 2007), we used both P2Y1 receptors, which couple to Gαq/11, but not Gαi/o (Waldo and Harden, 2004), and P2Y12 receptors, which couple to Gαi, but not Gαq/11 (Bodor et al., 2003). In addition, unlike some P2Y
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subtypes, selective antagonists are now available for P2Y1 and P2Y12 receptors. Finally, K2P2.1 channel mutants lacking potential C terminal phosphorylation sites were used to characterise how the P2Y receptors modulated channel activity. Results Depolarisation of tsA201 cells expressing hK2P2.1 channels evoked outwardly rectifying currents of up to several nA peak amplitude (Figs. 1B, 4B, 5A). As a subpopulation of HEK293 cells, the parent cell line of tsA201 cells, express endogenous P2Y receptors (Schachter et al., 1997; Moore et al., 2001; Fischer et al., 2003), in initial experi-
Fig. 1. Modulation of hK2P2.1 currents by ADP. A. Time-course of modulation of currents evoked by a voltage ramp from −120 mV to +40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y12 receptors and hK2P2.1 channels. The current amplitude at + 20 mV for each ramp is shown. ADP (0.1 µM) was added as indicated by the horizontal bar. B. Current traces from the same cell, evoked at time-points 1 and 2 in panel A. C. The mean effects of ADP (0.1 μM) on currents in cells in which the hK2P2.1 channel was expressed alone (n = 6), with hP2Y12 receptors (n = 9) and after incubation with PTX (200 ng/ml) for at least 18 h (n = 6) are shown. Horizontal lines indicate S.E.M. **p b 0.01 for the effect of ADP in cells expressing hK2P2.1 channels and hP2Y12 receptors, compared with hK2P2.1 only. #p b 0.01 for the effect of ADP in cells expressing hK2P2.1 channels and hP2Y12 receptors with and without PTX pretreatment.
ments tsA201 cells were transfected with hK2P2.1 channels alone. The P2Y agonist ADP (1 µM and 10 μM) significantly inhibited evoked currents by 29.0 ± 5.6% (n = 4, p b 0.05) and 45.1 ± 9.8% (n = 5, p b 0.01) respectively, indicating the presence of endogenous P2Y receptors. However, 0.1 µM ADP had no significant effect (4.6 ± 4.5% inhibition, n = 6, Fig. 1C) and so ADP (0.1 µM) was used in the subsequent experiments. Modulation of hK2P2.1 currents by the hP2Y12 receptor In cells coexpressing the hP2Y12 receptor and hK2P2.1 channels, ADP (0.1 µM) significantly inhibited the evoked currents by 27.8 ± 2.9% (n = 9, p b 0.01, Figs. 1A–C). The inhibition tended to show some reversal in the continued presence of agonist and reversed slowly on agonist washout (Fig. 1A). This inhibition of hK2P currents was unexpected, as the hP2Y12 receptor couples to Gαi (Bodor et al., 2003) and activation of Gαi-coupled receptors reportedly potentiates K2P currents (Lesage et al., 2000; Cain et al., 2008). So, to confirm that the inhibitory effects of ADP were indeed mediated by Gαi, cells were exposed to the Gαi inhibitor PTX (200 ng/ml). Under these conditions ADP (0.1 µM) had no effect on hK2P2.1 current amplitude (1.3 ± 6.0% inhibition, n = 6), which was significantly different from its action in the absence of PTX (p b 0.01, Fig. 1C). Thus, tsA201 cells express a low level of endogenous, ADP-sensitive P2Y receptors, which inhibit the opening of K2P2.1 channels. Transfection with recombinant hP2Y12 receptors greatly increases the inhibition in a PTX-sensitive manner. Protein kinases play a crucial role in GPCR-induced modulation of K2P channel activity (Patel et al., 1998; Koh et al., 2001; Murbartian et al., 2005; Cain et al., 2008), therefore, we determined the effects of ADP on currents carried by hK2P2.1 channels in which the potential C terminal phosphorylation sites Ser300, Ser333 and Ser351 were mutated to alanine. ADP (0.1 µM) had no significant effect on currents carried by the S300A (2.6 ± 2.6% inhibition, n = 10, Fig. 2C), S333A (1.0 ± 1.6% inhibition, n = 6, Figs. 2A,C) and S330A/S333A (4.7 ± 2.4% potentiation, n = 8, Fig. 2C) mutants (p b 0.01 for each compared to wild-type). In contrast, the effect of ADP (0.1 µM) on the S351A mutant (19.1 ± 3.2% inhibition, n = 6, Figs. 2B,C) was not significantly different from that on the wild-type hK2P2.1 channel. Thus, intact phosphorylation sites at Ser300 and Ser333, but not Ser351, are essential for the inhibitory effect of hP2Y12 receptors on hK2P2.1 channel activity. Next, the effects of P2Y receptor antagonists on the actions of ADP on wild-type hK2P2.1 channels were investigated. In the presence of the hP2Y12 antagonist AR-C69931MX (1 µM), ADP (0.1 µM) had no significant effect (7.3 ± 4.9% inhibition, n = 12, p b 0.01 for ADP effect in the presence of antagonist compared with in its absence, Figs. 3A,C). Surprisingly, the hP2Y1 antagonist MRS2179 (10 µM) also abolished the effects of ADP (3.1 ± 2.1% inhibition, n = 10, p b 0.01 for ADP effect in the presence of MRS2179 compared with in its absence, Figs. 3B,C). ATP is an agonist at hP2Y1, but not hP2Y12 receptors (Abbracchio et al., 2006) and so its actions were determined. In cells expressing K2P2.1 channels only, ATP (0.1 µM) had no significant effect on the currents (0.2 ± 2.2% inhibition, n = 8), but when the hP2Y12 receptor was coexpressed, ATP significantly inhibited K2P2.1 currents by 23.3 ± 4.6% (n = 12, p b 0.01), with a time-course similar to that seen with ADP. In addition, UTP (10 µM), an agonist at P2Y2 and P2Y4 receptors, but not P2Y1 or P2Y12 receptors (Abbracchio et al., 2006), had no significant effect on currents in cells expressing K2P2.1 channels only (5.9 ± 1.5% inhibition, n = 5). Thus, these results suggest that the inhibitory effect of hP2Y12 receptors on K2P2.1 currents involves co-activation of endogenous hP2Y1 receptors. Modulation of hK2P2.1 currents by the hP2Y1 receptor Next, the interaction between the Gαq/11-coupled hP2Y1 receptor and hK2P2.1 channels was studied. In cells coexpressing these proteins,
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Fig. 2. Ser300 and Ser333 play a role in hP2Y12 receptor-mediated inhibition of hK2P2.1 currents. Time-course of modulation of currents evoked by a voltage ramp from −120 mV to +40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y12 receptors and hK2P2.1 channels carrying A. the S333A and B. the S351A mutations. The current amplitude at +20 mV for each ramp is shown. ADP (0.1 µM) was added as indicated by the horizontal bars. C. The mean effects of ADP (0.1 μM) on hK2P2.1 currents in cells in which the wild-type channel (n = 9), S333A (n = 6), S300A (n = 10), S300A/S333A (n = 8) or S351A (n = 6) mutants were expressed are shown. Horizontal lines indicate S.E. M. **p b 0.01 for the effect of ADP in cells expressing hK2P2.1 mutants compared with the wild-type channel.
Fig. 3. The inhibitory effects of ADP are inhibited by P2Y receptor antagonists. Timecourse of modulation of currents evoked by a voltage ramp from − 120 mV to + 40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y12 receptors and hK2P2.1 channels in the presence of ADP (0.1 µM) plus, A. AR-C69931MX (1 µM) and B. MRS2179 (10 µM). The current amplitude at + 20 mV for each ramp is shown. ADP and the antagonists were added as indicated by the horizontal bars. C. The graph shows the mean effects of ADP (0.1 µM) on hK2P currents in the absence of antagonist (n = 9) and in the presence of AR-C69931MX (1 µM) (n = 12) or MRS2179 (10 µM) (n = 10). Horizontal lines indicate S.E.M. **p b 0.01, ***p b 0.001 for the effects of ADP in the absence compared with in the presence of antagonist.
ADP (0.1 µM) significantly inhibited the evoked currents by 26.0 ± 4.2% (n = 6, p b 0.01, Fig. 4C). This action was abolished by MRS2179 (10 µM) (4.2± 3.0% inhibition, n= 4, pb 0.05 for action of ADP in the presence of MRS2179 compared with in its absence, Fig. 4C), but unaffected by AR-C69931MX (1 µM) (43.9 ± 12.6% inhibition, n = 4, Fig. 4C). In cells coexpressing the recombinant hP2Y1 receptor, ATP (0.1 µM) significantly inhibited currents carried via hK2P2.1 channels by 34.4 ± 4.2% (n = 4, p b 0.05, Figs. 4A–C). PTX (200 ng/ml) pretreatment did not suppress the inhibitory actions of ATP (0.1 µM) (47.1 ± 8.7% inhibition, n = 5, p b 0.05, Fig. 4C), consistent with the hP2Y1 receptor coupling to
Gαq/11 rather than Gαi/o (Waldo and Harden, 2004) and confirming the specificity of action of PTX. Modulation of hK2P2.1 currents by the coexpressed hP2Y1 and hP2Y12 receptors Finally, having established that inhibition of hK2P2.1 currents via recombinant hP2Y12 receptors appears to involve the co-activation of endogenous hP2Y1 receptors, we studied the effect of coexpressing recombinant hP2Y1 and hP2Y12 receptors on hK2P2.1 activity. Under these conditions, ADP (0.1 μM) significantly inhibited currents by 27.5±
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Fig. 5. Activation of coexpressed hP2Y1 and hP2Y12 receptors inhibits hK2P2.1 currents. A. Representative current traces showing the inhibitory effect of ADP application in a tsA201 cell coexpressing hK2P2.1 channels with both hP2Y1 and hP2Y12 receptors. B. The graph shows the mean effects of ADP (0.1 μM) on hK2P2.1 currents in the absence (n = 12) and presence of MRS2179 (10 μM) (n = 6) or AR-C69931MX (1 μM) (n = 6) or after incubation with PTX (200 ng/ml) for at least 18 h (n = 10). Vertical bars indicate S.E.M. **p b 0.01 for inhibition by ADP of hK2P2.1 currents in the presence compared with in the absence of antagonist.
Discussion
Fig. 4. Activation of hP2Y1 receptors inhibits hK2P2.1 currents. A. Time-course of modulation of currents evoked by a voltage ramp from − 120 mV to + 40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y1 receptors and hK2P2.1 channels. The current amplitude at + 20 mV for each ramp is shown. ATP (0.1 µM) was added as indicated by the horizontal bar. B. Current traces from the same cell, evoked at time-points 1 and 2 in panel A. C. The graph shows the mean effects of ADP (0.1 μM) on hK2P2.1 currents in the absence (n = 6) and presence of MRS2179 (10 μM) (n = 4) or AR-C69931MX (1 μM) (n = 4). Also shown are the mean effects of ATP (0.1 μM) on hK2P2.1 currents (n = 4) and after incubation of cells with PTX (200 ng/ml) for at least 18 h (n = 5). Vertical bars indicate S.E.M. **p b 0.01 for inhibition by ADP of hK2P2.1 currents in the absence compared with in the presence of MRS2179.
3.8% (n = 12, p b 0.01, Figs. 5A,B), which was not significantly different from its effects when hP2Y1 or hP2Y12 receptors were expressed singly. Also similar to when the hP2Y12 receptor was expressed singly, ADP (0.1 μM) had no significant effect in the presence of AR-C69931MX (1 µM) (5.8 ± 3.3% inhibition, n = 6, Fig. 5B) and MRS2179 (10 μM) (3.1 ± 1.9% inhibition, n = 6, Fig. 5B) (p b 0.01 for both for the action of ADP in the presence of antagonist compared with in its absence). In contrast, PTX (200 ng/ml) did not depress the inhibitory action of ADP (10 μM) on hK2P2.1 currents (24.0 ± 5.1% inhibition, n = 10, Fig. 5B). Thus, when hP2Y1 and hP2Y12 receptors are coexpressed, the pharmacological profile is the same as when only the hP2Y12 receptor is expressed and the coupling to G proteins is the same as when only the hP2Y1 receptor is expressed.
These results demonstrate for the first time that activation of P2Y receptors inhibits ionic currents conducted by K2P ion channels. Interestingly and unusually, the K2P currents were depressed by both Gαq/11and Gαi-coupled P2Y receptors. In tsA201 cells cotransfected with hP2Y12 receptors, ADP inhibited currents through hK2P2.1 channels and this was abolished by PTX and the P2Y12 antagonist AR-C69913MX (Ingall et al., 1999; Abbracchio et al., 2006; Ding et al., 2006). Surprisingly, ADP's effect was also abolished by the selective P2Y1 antagonist MRS2179 (Boyer et al., 1998; Abbracchio et al., 2006), which has no action at P2Y12 receptors at the concentration used here (Savi et al., 2001). Furthermore, ATP, an agonist at P2Y1, but not P2Y12 receptors (Bodor et al., 2003; Kauffenstein et al., 2004) also inhibited hK2P2.1 currents in cells expressing the hP2Y12 receptor. In cells cotransfected with hP2Y1 receptors and hK2P2.1 channels, ADP and ATP significantly inhibited the evoked currents, and this was abolished by MRS2179, but unaffected by PTX or AR-C69913MX, confirming the subtypespecificity of AR-C69913MX. The simplest explanation for these data is that the recombinant hP2Y12 receptors formed functional heterodimers with endogenous P2Y1 receptors, which are known to be expressed at a low level in the parent HEK293 cell line (Schachter et al., 1997; Moore et al., 2001; Fischer et al., 2003). Furthermore, the resulting heterodimers are sensitive to antagonists of both P2Y subtypes. Consistent with this proposal, P2Y1 receptors have previously been reported to form heterodimers with P2Y11 receptors (Ecke et al., 2008) and A1 adenosine receptors (Yoshioka et al., 2001). Formation of GPCR oligomers is now recognised as being
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common and perhaps even crucial for the trafficking, membrane expression and functional activity of GPCR and such oligomers often have novel pharmacological and signalling properties (Milligan, 2004). Indeed, when the P2Y1 agonist ADP-β-S was applied to cells coexpressing the Gαq-coupled P2Y1 receptor and the Gαi/o-coupled A1 adenosine receptor, it induced a PTX-sensitive decrease in cAMP, which was reversed by an A1 antagonist (Yoshioka et al., 2001). This appearance of a novel pharmacological and signalling profile is analogous to that reported in the present study. In an attempt to demonstrate heterodimerisation between P2Y1 and P2Y12 receptors we began co-immunoprecipitation studies, but multiple (N10) bands were seen in control Western blots with a commercially-available anti-P2Y1 antibody (Kennedy and Bushell, unpublished observations). Similar non-specificity of anti-P2Y1 antibody has been reported previously (Vial et al., 2006). In this study the Gαi/o inhibitor PTX had no effect on the inhibition of hK2P2.1 currents mediated by recombinant hP2Y1 receptors, consistent with the report that the purified hP2Y1 receptor interacts with Gαq and Gα11, but not Gαi1,2,3 (Waldo and Harden, 2004) and with numerous studies showing that Gαq/11-coupled receptors inhibit K2P channels (see Mathie, 2007). Several mechanisms have been proposed to underlie the inhibition of K2P channels by Gαq/11-coupled receptors, but as yet the exact mechanism remains conjecture (Mathie, 2007). In contrast, the inhibitory effects of P2Y12 receptor stimulation on K2P currents were abolished by PTX. Gαi may be more likely than Gαo to mediate the inhibition, as the purified, reconstituted hP2Y12 receptor interacts with all three subtypes of Gαi, but not at all with Gαo (Bodor et al., 2003), although it should be noted that in some cases other P2Y subtypes appear to show different G protein coupling when functionally expressed (Filippov et al., 2004). The inhibitory effects of P2Y12 receptor stimulation in the present study were also abolished by mutating Ser330 and Ser333 in the C terminal of the K2P2.1 channel, to alanine. In contrast, mutation of Ser351, a target for protein kinase G, had no effect. Ser330 and Ser333 have been implicated in modulation of K2P channel activity by protein kinases A and C (Patel et al., 1998; Koh et al., 2001; Murbartian et al., 2005; Cain et al., 2008). For example, we showed previously that pharmacological inhibition of protein kinases A and C potentiated currents carried by wild-type K2P2.1 channels, but was ineffective against the S300A, S333A or S330A/S333A K2P2.1 mutants and also inhibited mGluR4 potentiation of the wild-type currents (Cain et al., 2008). It is thought that Ser330 and Ser333 can be constitutively phosphorylated and that increasing the level of phosphorylation inhibits channel activity, whereas decreasing phosphorylation potentiates it (Mathie, 2007). Thus, the S300A and S333A mutants, but not the S351A mutant, displayed increased current densities compared to the wild-type channel (Cain et al., 2008), consistent with the dephosphorylated state of the channel being more active. Furthermore, the potentiation of K2P2.1 currents by mGlu4 receptor stimulation was abolished in the S300A/ S333A double mutant. The present study further indicates an essential role for S300 and S333 in the modulation of K2P channel activity by GPCR. At present it is unclear if changes in the phosphorylation state modulates directly the ability of the channel to open or if it acts indirectly by altering the binding of accessory proteins, which may in turn modulate channel activity. The negative effect of hP2Y12 receptor stimulation on K2P currents was a surprise, as the Gαi-coupled P2Y12 receptor would be predicted to depress protein kinase A activity and so potentiate K2P2.1 currents. Consistent with this prediction, activation of the Gαi/o-coupled mGlu2 and mGlu4 receptors potentiated K2P currents (Lesage et al., 2000; Cain et al., 2008). We suggest that in addition to having novel pharmacological properties, the coupling of the heterodimer to G proteins also appears to be novel, in that functional Gαi is required (inhibition is PTX-sensitive) for downstream signalling, but the downstream pathway activated is more likely to be Gαq/11-dependent (inhibition of K2P currents is seen). The role of Gαi may be to stabilise the receptor-G protein complex and this is disrupted by ADP-ribosylation of Gαi by
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PTX. However, the possibility that Gαi activates the downstream signalling per se cannot be ruled out, although this in itself would represent a novel mechanism for modulation of K2P channel activity. To further investigate this model we coexpressed P2Y1 receptors along with P2Y12 receptors and K2P2.1 channels. Under these conditions the same pharmacological profile was seen as when only the P2Y12 receptor was expressed, i.e. antagonism by both AR-C69913MX and MRS2179. However, the coupling to G proteins differed, as the inhibition was now insensitive to PTX, the same as when only the hP2Y1 receptor was expressed. Thus, the P2Y1 receptor appears to dominate the signalling pathway, though not the pharmacological profile, when it is overexpressed. Further studies are required to clarify the potential heterodimer-mediated effects seen in the present study. At present, there are no published reports on the interaction of native P2Y receptors and K2P channels in neurones, even though they appear to be coexpressed in many regions of the brain and in sensory neurones. The present data suggest that they could interact to regulate neuronal excitability. Thus, this is an intriguing area for further investigation, especially as both are potentially novel targets for the treatment of neuronal disorders. Experimental methods Culture and transfection of tsA201 cells tsA201 cells, a modified HEK293 cell line, were maintained in 5% CO2, 95% O2 in a humidified incubator at 37 °C, in growth media containing Minimum Essential Media (MEM) (with Earle's, without L-glutamine), 10% foetal calf serum, 1% non-essential amino acids, 1% penicillin (10,000 U/ml) and streptomycin (10 mg/ml). When the cells were 90% confluent they were split and plated for transfection onto 13 mm glass coverslips coated with poly-L-lysine (0.1 mg/ml). 2 h later the cells were flooded with serum-free MEM and transfected using Lipofectamine 2000 (1.5 µl/1 µg of cDNA) with cDNA (1 µg of each/ 3 coverslips) for hK2P2.1 (GenBank accession number AF171068) and the hP2Y 1 or hP2Y12 receptors (GenBank accession numbers NM_002563.2, AF313449 respectively), as appropriate, together with cDNA encoding green-fluorescent protein (0.5 µg/3 coverslips). All cDNAs were contained in the pcDNA 3.1 vector. 2 h later the transfection media was aspirated and replaced with growth media. Cells were then incubated at 34 °C, 5% CO2, 95% O2 for 48 h before being used for electrophysiological studies. To determine the role of pertussis toxin (PTX)-sensitive G proteins in the modulation of K2P currents by P2Y receptor agonists, PTX (200 ng/ml) was included in the growth media bathing transfected cells for at least 18 h prior to recordings. Electrophysiological recording A coverslip with tsA201 cells was placed in the recording chamber and the cells were superfused at room temperature with a solution composed of (mM): NaCl 140; KCl 2.5; MgCl2 2; HEPES 10; D-glucose 10; CaCl2 1, titrated to pH 7.4 with NaOH and 310 mOsm/kg osmolality with sucrose. Transmembrane ionic currents were recorded in the whole-cell, perforated-patch mode with amphotericin B (200 μg/ ml) added to a pipette solution of the following composition (mM): KCH3SO4 125; KCl 20; MgATP 4; HEPES 10; LiGTP 0.3; NaPCr 5; ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.5, titrated to pH 7.2 with KOH and 290 mOsm/kg osmolality with sucrose. Pipette resistance was 4–6 MΩ and currents were recorded using an Axopatch 1D amplifier (Axon Instruments, USA), connected to a personal computer interfaced with an ITC-18 A/D converter (Instrutech Corp; USA) and analysed using WinWCP software (Dr. J. Dempster, University of Strathclyde, UK). Transfected tsA201 cells were identified by green fluorescence and voltage-clamped at − 70 mV. K2P channel expression and modulation was investigated by stepping to −120 mV for 50 ms, followed
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by a voltage ramp from − 120 mV to +40 mV over 500 ms, then returning to − 70 mV, every 30 s, as described previously (Gruss et al., 2004; Cain et al., 2008). Modulation of the K2P currents was quantified by measuring the amplitude at + 20 mV. Once the amplitude of the evoked currents had stabilised, P2Y receptor agonists were applied in the superfusate for 5 min. Only one agonist was applied to each cell. Site-directed mutagenesis S300A, S333A, S300A/S333A and S351A mutants of the K2P2.1 channel were constructed as described previously (Cain et al., 2008) using specific primers (MWG-Biotech, Ebersberg, Germany) and pfu Ultra DNA polymerase (Stratagene, Amsterdam, The Netherlands). The products were sequenced to confirm correct insertion of each mutation (Dr. R. Tate, University of Strathclyde). Data analysis Values in the text and figures refer to mean ± S.E.M. Data were compared by paired and unpaired t-tests, or one-way analysis of variance and Tukey's comparison as appropriate. Differences were considered significant when p b 0.05. Drugs, solutions and cDNAs ATP (disodium salt), ADP (sodium salt), UTP (sodium salt) (Sigma/ RBI, UK), 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate (tetrasodium salt) (MRS2179) (Tocris, UK) and [(2R,3S,4R,5R)-3,4-dihydroxy5-[6-(2-methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl) purin-9-yl]oxolan-2-yl]methyl dihydrogen phosphate (AR-C69931MX) (a generous gift from The Medicines Company, New Jersey, USA) were dissolved in deionised water as 10 mM stock solutions and diluted in a buffer before application to the cells. PTX (Calbiochem, UK) was dissolved in deionised water (200 µg/ml). The cDNAs encoding the hP2Y1 and hP2Y12 receptors were a gift from Professors T.K. Harden and R.A. Nicholas, (University of North Carolina, Chapel Hill, NC, USA) and that for hK2P2.1 was donated by Glaxo Smith Kline, UK. References Abbracchio, M.P., Burnstock, G., Boeynaems, J.M., Barnard, E.A., Boyer, J.L., Kennedy, C., Fumagalli, M., Gachet, C., Jacobson, K.A., Weisman, G.A., International Union of Pharmacology, 2006. Update of the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev. 58, 281–341. Aimond, F., Rauzier, J.M., Bony, C., Vassort, G., 2000. Simultaneous activation of p38 MAPK and p42/44 MAPK by ATP stimulates the K+ current ITREK in cardiomyocytes. J. Biol. Chem. 275, 39110–39116. Aller, M.I., Veale, E.L., Linden, A.M., Sandu, C., Schwaninger, M., Evans, L.J., Korpi, E.R., Mathie, A., Wisden, W., Brickley, S.G., 2005. Modifying the subunit composition of TASK channels alters the modulation of a leak conductance in cerebellar granule neurons. J. Neurosci. 25, 11455–11467. Alloui, A., Zimmermann, K., Mamet, J., Duprat, F., Noel, J., Chemin, J., Guy, N., Blondeau, N., Voilley, N., Rubat-Coudert, C., Borsotto, M., Romey, G., Heurteaux, C., Reeh, P., Eschalier, A., Lazdunski, M., 2006. TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 25, 2368–2376. Bodor, E.T., Waldo, G.L., Hooks, S.B., Corbitt, J., Boyer, J.L., Harden, T.K., 2003. Purification and functional reconstitution of the human P2Y12 receptor. Mol. Pharmacol. 64, 1210–1216. Boyer, J.L., Mohanram, A., Camaioni, E., Jacobson, K.A., Harden, T.K., 1998. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′, 5′-bisphosphate. Br. J. Pharmacol. 124, 1–3. Buckler, K.J., Honoré, E., 2005. The lipid-activated two-pore domain K+ channel TREK-1 is resistant to hypoxia: implication for ischaemic neuroprotection. J. Physiol. 562, 213–222. Burnstock, G., Kennedy, C., 1985. Is there a basis for distinguishing two types of P2purinoceptor? Gen. Pharmacol. 16, 433–440. Burnstock, G., Knight, G., 2004. Cellular distribution and functions of P2 receptor subtypes in different systems. Int. Rev. Cytol. 240, 31–310. Bushell, T.J., Clarke, C., Mathie, A., Robertson, B., 2002. Pharmacological characterization of a non-inactivating outward current observed in mouse cerebellar Purkinje neurones. Br. J. Pharmacol. 135, 705–712.
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Koh, S.D., Monaghan, K., Sergeant, G.P., Ro, S., Walker, R.L., Sanders, K.M., Horowitz, B., 2001. TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. J. Biol. Chem. 276, 44338–44346. Köles, L., Gerevich, Z., Oliveira, J.P., Zadori, Z.S., Wirkner, K., Illes, P., 2008. Interaction of P2 purinergic receptors with cellular macromolecules. Naunyn Schmied Arch. Pharmacol. 377, 1–33. Lauritzen, I., Blondeau, N., Heurteaux, C., Widmann, C., Romey, G., Lazdunski, M., 2000. Polyunsaturated fatty acids are potent neuroprotectors. EMBO J. 19, 1784–1793. Lechner, S.G., Boehm, S., 2004. Regulation of neuronal ion channels via P2Y receptors. PUSI 1, 31–41. Lesage, F., Terrenoire, C., Romey, G., Lazdunski, M., 2000. Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors. J. Biol. Chem. 275, 28398–28405. 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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m c n e
Two-pore potassium ion channels are inhibited by both Gq/11- and Gi-coupled P2Y receptors Sony Shakya Shrestha, Miralkumar Parmar, Charles Kennedy ⁎, Trevor J. Bushell Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 ONR, UK
a r t i c l e
i n f o
Article history: Received 4 November 2009 Revised 7 January 2010 Accepted 13 January 2010 Available online 22 January 2010 Keywords: Two-pore potassium channels K2P2.1 P2Y1 receptor P2Y12 receptor
a b s t r a c t Two-pore potassium (K2P) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Gαi-coupled hP2Y12 receptor, ADP and ATP significantly inhibited hK2P2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y12 antagonist AR-C69931MX, the hP2Y1 antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Gαq/11coupled hP2Y1 receptor, ADP and ATP also inhibited hK2P2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K2P2.1 channels, ADPinduced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Gαq/11- and Gαicoupled P2Y receptors inhibit K2P channels and the action of hP2Y12 receptors appears to involve co-activation of endogenous hP2Y1 receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity. © 2010 Elsevier Inc. All rights reserved.
Introduction Two-pore potassium (K2P) ion channels are the most recently identified family of K+ channels and the 15 members cloned in mammals (Mathie, 2007) are distributed widely throughout the body, including the CNS (Talley et al., 2001; Aller et al., 2005) and sensory nerves (Dobler et al., 2007). They play an important role in the “leak” or “background” current present in many neurones and so regulate neuronal excitability. In addition, they have been implicated in a variety of disorders, including pain (Alloui et al., 2006; Linden et al., 2006) and depression (Heurteaux et al., 2006) and are proposed as a target for neuroprotection (Lauritzen et al., 2000; Buckler and Honoré, 2005). Their activity is modulated by a range of physical and chemical factors, such as inhalation anaesthetics (Patel et al., 1999; Franks and Honoré, 2004; Gruss et al., 2004), and G protein-coupled receptors (GPCR) (Bushell et al., 2002; Mathie, 2007). In general, GPCR that couple to the Gαq/11 and Gαs G proteins mediate inhibition of K2P channel opening, whereas activation of those that couple to Gαi/o increase channel activity (Mathie, 2007). For example, we showed recently that currents carried via K2P2.1 (formerly known as TREK-1; see Kim, 2003; Honoré, 2007) were potentiated by activation of the Gαi/o-coupled mGlu4 receptor (Cain et al., 2008). P2Y receptors are a family of 8 GPCR (P2Y1, 2, 4, 6, 11, 12, 13, 14) that mediate the extracellular actions of endogenous nucleotides, such as
Abbreviations: ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; GPCR, G protein-coupled receptors; K2P, two-pore potassium ion channels; PTX, pertussis toxin. ⁎ Corresponding author. Fax: + 44 141 552 2562. E-mail address: [email protected] (C. Kennedy). 1044-7431/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2010.01.003
adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP) (Burnstock and Kennedy, 1985; Abbracchio et al., 2006). They are expressed throughout the body, including central, sensory, motor and peripheral neurones (Burnstock and Knight, 2004). When activated they interact with a variety of ion channels, including NMDA and TRPV1 receptors, voltage-gated Ca2+ channels and several types of K+ channel, leading to changes in action potential firing and neurotransmitter release (see Kennedy et al., 2003; Lechner and Boehm, 2004; Köles et al., 2008 for reviews). In addition, a K2P2.1-like current in rat cardiomyocytes was potentiated by ATP, possibly via the P2Y11 receptor (Aimond et al., 2000). The expression of P2Y receptors and K2P channels appears to overlap in many regions of the CNS and in sensory neurones and they are both novel targets for the treatment of neuronal disorders, but at present, little is known about how they interact. In preliminary experiments we found that P2Y receptor agonists modulate K2P-like currents in central neurones (Kennedy and Bushell, unpublished observations), but characterisation of this interaction was complicated by the expression of multiple P2Y receptors and K2P channels in these neurones (Watkins and Mathie, 1996; Aller et al., 2005; Mathie, 2007). Therefore, in the present study we coexpressed recombinant human K2P channels and P2Y receptors in the tsA201 cell line, as a first step in characterising the basic properties of the interaction. K2P2.1 channels were used as they are present in both the CNS (Talley et al., 2001) and sensory neurones (Alloui et al., 2006). Since K2P channels are differentially modulated by GPCR that couple to Gαq/11 and Gαi/o (Mathie, 2007), we used both P2Y1 receptors, which couple to Gαq/11, but not Gαi/o (Waldo and Harden, 2004), and P2Y12 receptors, which couple to Gαi, but not Gαq/11 (Bodor et al., 2003). In addition, unlike some P2Y
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subtypes, selective antagonists are now available for P2Y1 and P2Y12 receptors. Finally, K2P2.1 channel mutants lacking potential C terminal phosphorylation sites were used to characterise how the P2Y receptors modulated channel activity. Results Depolarisation of tsA201 cells expressing hK2P2.1 channels evoked outwardly rectifying currents of up to several nA peak amplitude (Figs. 1B, 4B, 5A). As a subpopulation of HEK293 cells, the parent cell line of tsA201 cells, express endogenous P2Y receptors (Schachter et al., 1997; Moore et al., 2001; Fischer et al., 2003), in initial experi-
Fig. 1. Modulation of hK2P2.1 currents by ADP. A. Time-course of modulation of currents evoked by a voltage ramp from −120 mV to +40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y12 receptors and hK2P2.1 channels. The current amplitude at + 20 mV for each ramp is shown. ADP (0.1 µM) was added as indicated by the horizontal bar. B. Current traces from the same cell, evoked at time-points 1 and 2 in panel A. C. The mean effects of ADP (0.1 μM) on currents in cells in which the hK2P2.1 channel was expressed alone (n = 6), with hP2Y12 receptors (n = 9) and after incubation with PTX (200 ng/ml) for at least 18 h (n = 6) are shown. Horizontal lines indicate S.E.M. **p b 0.01 for the effect of ADP in cells expressing hK2P2.1 channels and hP2Y12 receptors, compared with hK2P2.1 only. #p b 0.01 for the effect of ADP in cells expressing hK2P2.1 channels and hP2Y12 receptors with and without PTX pretreatment.
ments tsA201 cells were transfected with hK2P2.1 channels alone. The P2Y agonist ADP (1 µM and 10 μM) significantly inhibited evoked currents by 29.0 ± 5.6% (n = 4, p b 0.05) and 45.1 ± 9.8% (n = 5, p b 0.01) respectively, indicating the presence of endogenous P2Y receptors. However, 0.1 µM ADP had no significant effect (4.6 ± 4.5% inhibition, n = 6, Fig. 1C) and so ADP (0.1 µM) was used in the subsequent experiments. Modulation of hK2P2.1 currents by the hP2Y12 receptor In cells coexpressing the hP2Y12 receptor and hK2P2.1 channels, ADP (0.1 µM) significantly inhibited the evoked currents by 27.8 ± 2.9% (n = 9, p b 0.01, Figs. 1A–C). The inhibition tended to show some reversal in the continued presence of agonist and reversed slowly on agonist washout (Fig. 1A). This inhibition of hK2P currents was unexpected, as the hP2Y12 receptor couples to Gαi (Bodor et al., 2003) and activation of Gαi-coupled receptors reportedly potentiates K2P currents (Lesage et al., 2000; Cain et al., 2008). So, to confirm that the inhibitory effects of ADP were indeed mediated by Gαi, cells were exposed to the Gαi inhibitor PTX (200 ng/ml). Under these conditions ADP (0.1 µM) had no effect on hK2P2.1 current amplitude (1.3 ± 6.0% inhibition, n = 6), which was significantly different from its action in the absence of PTX (p b 0.01, Fig. 1C). Thus, tsA201 cells express a low level of endogenous, ADP-sensitive P2Y receptors, which inhibit the opening of K2P2.1 channels. Transfection with recombinant hP2Y12 receptors greatly increases the inhibition in a PTX-sensitive manner. Protein kinases play a crucial role in GPCR-induced modulation of K2P channel activity (Patel et al., 1998; Koh et al., 2001; Murbartian et al., 2005; Cain et al., 2008), therefore, we determined the effects of ADP on currents carried by hK2P2.1 channels in which the potential C terminal phosphorylation sites Ser300, Ser333 and Ser351 were mutated to alanine. ADP (0.1 µM) had no significant effect on currents carried by the S300A (2.6 ± 2.6% inhibition, n = 10, Fig. 2C), S333A (1.0 ± 1.6% inhibition, n = 6, Figs. 2A,C) and S330A/S333A (4.7 ± 2.4% potentiation, n = 8, Fig. 2C) mutants (p b 0.01 for each compared to wild-type). In contrast, the effect of ADP (0.1 µM) on the S351A mutant (19.1 ± 3.2% inhibition, n = 6, Figs. 2B,C) was not significantly different from that on the wild-type hK2P2.1 channel. Thus, intact phosphorylation sites at Ser300 and Ser333, but not Ser351, are essential for the inhibitory effect of hP2Y12 receptors on hK2P2.1 channel activity. Next, the effects of P2Y receptor antagonists on the actions of ADP on wild-type hK2P2.1 channels were investigated. In the presence of the hP2Y12 antagonist AR-C69931MX (1 µM), ADP (0.1 µM) had no significant effect (7.3 ± 4.9% inhibition, n = 12, p b 0.01 for ADP effect in the presence of antagonist compared with in its absence, Figs. 3A,C). Surprisingly, the hP2Y1 antagonist MRS2179 (10 µM) also abolished the effects of ADP (3.1 ± 2.1% inhibition, n = 10, p b 0.01 for ADP effect in the presence of MRS2179 compared with in its absence, Figs. 3B,C). ATP is an agonist at hP2Y1, but not hP2Y12 receptors (Abbracchio et al., 2006) and so its actions were determined. In cells expressing K2P2.1 channels only, ATP (0.1 µM) had no significant effect on the currents (0.2 ± 2.2% inhibition, n = 8), but when the hP2Y12 receptor was coexpressed, ATP significantly inhibited K2P2.1 currents by 23.3 ± 4.6% (n = 12, p b 0.01), with a time-course similar to that seen with ADP. In addition, UTP (10 µM), an agonist at P2Y2 and P2Y4 receptors, but not P2Y1 or P2Y12 receptors (Abbracchio et al., 2006), had no significant effect on currents in cells expressing K2P2.1 channels only (5.9 ± 1.5% inhibition, n = 5). Thus, these results suggest that the inhibitory effect of hP2Y12 receptors on K2P2.1 currents involves co-activation of endogenous hP2Y1 receptors. Modulation of hK2P2.1 currents by the hP2Y1 receptor Next, the interaction between the Gαq/11-coupled hP2Y1 receptor and hK2P2.1 channels was studied. In cells coexpressing these proteins,
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Fig. 2. Ser300 and Ser333 play a role in hP2Y12 receptor-mediated inhibition of hK2P2.1 currents. Time-course of modulation of currents evoked by a voltage ramp from −120 mV to +40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y12 receptors and hK2P2.1 channels carrying A. the S333A and B. the S351A mutations. The current amplitude at +20 mV for each ramp is shown. ADP (0.1 µM) was added as indicated by the horizontal bars. C. The mean effects of ADP (0.1 μM) on hK2P2.1 currents in cells in which the wild-type channel (n = 9), S333A (n = 6), S300A (n = 10), S300A/S333A (n = 8) or S351A (n = 6) mutants were expressed are shown. Horizontal lines indicate S.E. M. **p b 0.01 for the effect of ADP in cells expressing hK2P2.1 mutants compared with the wild-type channel.
Fig. 3. The inhibitory effects of ADP are inhibited by P2Y receptor antagonists. Timecourse of modulation of currents evoked by a voltage ramp from − 120 mV to + 40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y12 receptors and hK2P2.1 channels in the presence of ADP (0.1 µM) plus, A. AR-C69931MX (1 µM) and B. MRS2179 (10 µM). The current amplitude at + 20 mV for each ramp is shown. ADP and the antagonists were added as indicated by the horizontal bars. C. The graph shows the mean effects of ADP (0.1 µM) on hK2P currents in the absence of antagonist (n = 9) and in the presence of AR-C69931MX (1 µM) (n = 12) or MRS2179 (10 µM) (n = 10). Horizontal lines indicate S.E.M. **p b 0.01, ***p b 0.001 for the effects of ADP in the absence compared with in the presence of antagonist.
ADP (0.1 µM) significantly inhibited the evoked currents by 26.0 ± 4.2% (n = 6, p b 0.01, Fig. 4C). This action was abolished by MRS2179 (10 µM) (4.2± 3.0% inhibition, n= 4, pb 0.05 for action of ADP in the presence of MRS2179 compared with in its absence, Fig. 4C), but unaffected by AR-C69931MX (1 µM) (43.9 ± 12.6% inhibition, n = 4, Fig. 4C). In cells coexpressing the recombinant hP2Y1 receptor, ATP (0.1 µM) significantly inhibited currents carried via hK2P2.1 channels by 34.4 ± 4.2% (n = 4, p b 0.05, Figs. 4A–C). PTX (200 ng/ml) pretreatment did not suppress the inhibitory actions of ATP (0.1 µM) (47.1 ± 8.7% inhibition, n = 5, p b 0.05, Fig. 4C), consistent with the hP2Y1 receptor coupling to
Gαq/11 rather than Gαi/o (Waldo and Harden, 2004) and confirming the specificity of action of PTX. Modulation of hK2P2.1 currents by the coexpressed hP2Y1 and hP2Y12 receptors Finally, having established that inhibition of hK2P2.1 currents via recombinant hP2Y12 receptors appears to involve the co-activation of endogenous hP2Y1 receptors, we studied the effect of coexpressing recombinant hP2Y1 and hP2Y12 receptors on hK2P2.1 activity. Under these conditions, ADP (0.1 μM) significantly inhibited currents by 27.5±
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Fig. 5. Activation of coexpressed hP2Y1 and hP2Y12 receptors inhibits hK2P2.1 currents. A. Representative current traces showing the inhibitory effect of ADP application in a tsA201 cell coexpressing hK2P2.1 channels with both hP2Y1 and hP2Y12 receptors. B. The graph shows the mean effects of ADP (0.1 μM) on hK2P2.1 currents in the absence (n = 12) and presence of MRS2179 (10 μM) (n = 6) or AR-C69931MX (1 μM) (n = 6) or after incubation with PTX (200 ng/ml) for at least 18 h (n = 10). Vertical bars indicate S.E.M. **p b 0.01 for inhibition by ADP of hK2P2.1 currents in the presence compared with in the absence of antagonist.
Discussion
Fig. 4. Activation of hP2Y1 receptors inhibits hK2P2.1 currents. A. Time-course of modulation of currents evoked by a voltage ramp from − 120 mV to + 40 mV over 500 ms, every 30 s, in a tsA201 cell coexpressing recombinant hP2Y1 receptors and hK2P2.1 channels. The current amplitude at + 20 mV for each ramp is shown. ATP (0.1 µM) was added as indicated by the horizontal bar. B. Current traces from the same cell, evoked at time-points 1 and 2 in panel A. C. The graph shows the mean effects of ADP (0.1 μM) on hK2P2.1 currents in the absence (n = 6) and presence of MRS2179 (10 μM) (n = 4) or AR-C69931MX (1 μM) (n = 4). Also shown are the mean effects of ATP (0.1 μM) on hK2P2.1 currents (n = 4) and after incubation of cells with PTX (200 ng/ml) for at least 18 h (n = 5). Vertical bars indicate S.E.M. **p b 0.01 for inhibition by ADP of hK2P2.1 currents in the absence compared with in the presence of MRS2179.
3.8% (n = 12, p b 0.01, Figs. 5A,B), which was not significantly different from its effects when hP2Y1 or hP2Y12 receptors were expressed singly. Also similar to when the hP2Y12 receptor was expressed singly, ADP (0.1 μM) had no significant effect in the presence of AR-C69931MX (1 µM) (5.8 ± 3.3% inhibition, n = 6, Fig. 5B) and MRS2179 (10 μM) (3.1 ± 1.9% inhibition, n = 6, Fig. 5B) (p b 0.01 for both for the action of ADP in the presence of antagonist compared with in its absence). In contrast, PTX (200 ng/ml) did not depress the inhibitory action of ADP (10 μM) on hK2P2.1 currents (24.0 ± 5.1% inhibition, n = 10, Fig. 5B). Thus, when hP2Y1 and hP2Y12 receptors are coexpressed, the pharmacological profile is the same as when only the hP2Y12 receptor is expressed and the coupling to G proteins is the same as when only the hP2Y1 receptor is expressed.
These results demonstrate for the first time that activation of P2Y receptors inhibits ionic currents conducted by K2P ion channels. Interestingly and unusually, the K2P currents were depressed by both Gαq/11and Gαi-coupled P2Y receptors. In tsA201 cells cotransfected with hP2Y12 receptors, ADP inhibited currents through hK2P2.1 channels and this was abolished by PTX and the P2Y12 antagonist AR-C69913MX (Ingall et al., 1999; Abbracchio et al., 2006; Ding et al., 2006). Surprisingly, ADP's effect was also abolished by the selective P2Y1 antagonist MRS2179 (Boyer et al., 1998; Abbracchio et al., 2006), which has no action at P2Y12 receptors at the concentration used here (Savi et al., 2001). Furthermore, ATP, an agonist at P2Y1, but not P2Y12 receptors (Bodor et al., 2003; Kauffenstein et al., 2004) also inhibited hK2P2.1 currents in cells expressing the hP2Y12 receptor. In cells cotransfected with hP2Y1 receptors and hK2P2.1 channels, ADP and ATP significantly inhibited the evoked currents, and this was abolished by MRS2179, but unaffected by PTX or AR-C69913MX, confirming the subtypespecificity of AR-C69913MX. The simplest explanation for these data is that the recombinant hP2Y12 receptors formed functional heterodimers with endogenous P2Y1 receptors, which are known to be expressed at a low level in the parent HEK293 cell line (Schachter et al., 1997; Moore et al., 2001; Fischer et al., 2003). Furthermore, the resulting heterodimers are sensitive to antagonists of both P2Y subtypes. Consistent with this proposal, P2Y1 receptors have previously been reported to form heterodimers with P2Y11 receptors (Ecke et al., 2008) and A1 adenosine receptors (Yoshioka et al., 2001). Formation of GPCR oligomers is now recognised as being
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common and perhaps even crucial for the trafficking, membrane expression and functional activity of GPCR and such oligomers often have novel pharmacological and signalling properties (Milligan, 2004). Indeed, when the P2Y1 agonist ADP-β-S was applied to cells coexpressing the Gαq-coupled P2Y1 receptor and the Gαi/o-coupled A1 adenosine receptor, it induced a PTX-sensitive decrease in cAMP, which was reversed by an A1 antagonist (Yoshioka et al., 2001). This appearance of a novel pharmacological and signalling profile is analogous to that reported in the present study. In an attempt to demonstrate heterodimerisation between P2Y1 and P2Y12 receptors we began co-immunoprecipitation studies, but multiple (N10) bands were seen in control Western blots with a commercially-available anti-P2Y1 antibody (Kennedy and Bushell, unpublished observations). Similar non-specificity of anti-P2Y1 antibody has been reported previously (Vial et al., 2006). In this study the Gαi/o inhibitor PTX had no effect on the inhibition of hK2P2.1 currents mediated by recombinant hP2Y1 receptors, consistent with the report that the purified hP2Y1 receptor interacts with Gαq and Gα11, but not Gαi1,2,3 (Waldo and Harden, 2004) and with numerous studies showing that Gαq/11-coupled receptors inhibit K2P channels (see Mathie, 2007). Several mechanisms have been proposed to underlie the inhibition of K2P channels by Gαq/11-coupled receptors, but as yet the exact mechanism remains conjecture (Mathie, 2007). In contrast, the inhibitory effects of P2Y12 receptor stimulation on K2P currents were abolished by PTX. Gαi may be more likely than Gαo to mediate the inhibition, as the purified, reconstituted hP2Y12 receptor interacts with all three subtypes of Gαi, but not at all with Gαo (Bodor et al., 2003), although it should be noted that in some cases other P2Y subtypes appear to show different G protein coupling when functionally expressed (Filippov et al., 2004). The inhibitory effects of P2Y12 receptor stimulation in the present study were also abolished by mutating Ser330 and Ser333 in the C terminal of the K2P2.1 channel, to alanine. In contrast, mutation of Ser351, a target for protein kinase G, had no effect. Ser330 and Ser333 have been implicated in modulation of K2P channel activity by protein kinases A and C (Patel et al., 1998; Koh et al., 2001; Murbartian et al., 2005; Cain et al., 2008). For example, we showed previously that pharmacological inhibition of protein kinases A and C potentiated currents carried by wild-type K2P2.1 channels, but was ineffective against the S300A, S333A or S330A/S333A K2P2.1 mutants and also inhibited mGluR4 potentiation of the wild-type currents (Cain et al., 2008). It is thought that Ser330 and Ser333 can be constitutively phosphorylated and that increasing the level of phosphorylation inhibits channel activity, whereas decreasing phosphorylation potentiates it (Mathie, 2007). Thus, the S300A and S333A mutants, but not the S351A mutant, displayed increased current densities compared to the wild-type channel (Cain et al., 2008), consistent with the dephosphorylated state of the channel being more active. Furthermore, the potentiation of K2P2.1 currents by mGlu4 receptor stimulation was abolished in the S300A/ S333A double mutant. The present study further indicates an essential role for S300 and S333 in the modulation of K2P channel activity by GPCR. At present it is unclear if changes in the phosphorylation state modulates directly the ability of the channel to open or if it acts indirectly by altering the binding of accessory proteins, which may in turn modulate channel activity. The negative effect of hP2Y12 receptor stimulation on K2P currents was a surprise, as the Gαi-coupled P2Y12 receptor would be predicted to depress protein kinase A activity and so potentiate K2P2.1 currents. Consistent with this prediction, activation of the Gαi/o-coupled mGlu2 and mGlu4 receptors potentiated K2P currents (Lesage et al., 2000; Cain et al., 2008). We suggest that in addition to having novel pharmacological properties, the coupling of the heterodimer to G proteins also appears to be novel, in that functional Gαi is required (inhibition is PTX-sensitive) for downstream signalling, but the downstream pathway activated is more likely to be Gαq/11-dependent (inhibition of K2P currents is seen). The role of Gαi may be to stabilise the receptor-G protein complex and this is disrupted by ADP-ribosylation of Gαi by
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PTX. However, the possibility that Gαi activates the downstream signalling per se cannot be ruled out, although this in itself would represent a novel mechanism for modulation of K2P channel activity. To further investigate this model we coexpressed P2Y1 receptors along with P2Y12 receptors and K2P2.1 channels. Under these conditions the same pharmacological profile was seen as when only the P2Y12 receptor was expressed, i.e. antagonism by both AR-C69913MX and MRS2179. However, the coupling to G proteins differed, as the inhibition was now insensitive to PTX, the same as when only the hP2Y1 receptor was expressed. Thus, the P2Y1 receptor appears to dominate the signalling pathway, though not the pharmacological profile, when it is overexpressed. Further studies are required to clarify the potential heterodimer-mediated effects seen in the present study. At present, there are no published reports on the interaction of native P2Y receptors and K2P channels in neurones, even though they appear to be coexpressed in many regions of the brain and in sensory neurones. The present data suggest that they could interact to regulate neuronal excitability. Thus, this is an intriguing area for further investigation, especially as both are potentially novel targets for the treatment of neuronal disorders. Experimental methods Culture and transfection of tsA201 cells tsA201 cells, a modified HEK293 cell line, were maintained in 5% CO2, 95% O2 in a humidified incubator at 37 °C, in growth media containing Minimum Essential Media (MEM) (with Earle's, without L-glutamine), 10% foetal calf serum, 1% non-essential amino acids, 1% penicillin (10,000 U/ml) and streptomycin (10 mg/ml). When the cells were 90% confluent they were split and plated for transfection onto 13 mm glass coverslips coated with poly-L-lysine (0.1 mg/ml). 2 h later the cells were flooded with serum-free MEM and transfected using Lipofectamine 2000 (1.5 µl/1 µg of cDNA) with cDNA (1 µg of each/ 3 coverslips) for hK2P2.1 (GenBank accession number AF171068) and the hP2Y 1 or hP2Y12 receptors (GenBank accession numbers NM_002563.2, AF313449 respectively), as appropriate, together with cDNA encoding green-fluorescent protein (0.5 µg/3 coverslips). All cDNAs were contained in the pcDNA 3.1 vector. 2 h later the transfection media was aspirated and replaced with growth media. Cells were then incubated at 34 °C, 5% CO2, 95% O2 for 48 h before being used for electrophysiological studies. To determine the role of pertussis toxin (PTX)-sensitive G proteins in the modulation of K2P currents by P2Y receptor agonists, PTX (200 ng/ml) was included in the growth media bathing transfected cells for at least 18 h prior to recordings. Electrophysiological recording A coverslip with tsA201 cells was placed in the recording chamber and the cells were superfused at room temperature with a solution composed of (mM): NaCl 140; KCl 2.5; MgCl2 2; HEPES 10; D-glucose 10; CaCl2 1, titrated to pH 7.4 with NaOH and 310 mOsm/kg osmolality with sucrose. Transmembrane ionic currents were recorded in the whole-cell, perforated-patch mode with amphotericin B (200 μg/ ml) added to a pipette solution of the following composition (mM): KCH3SO4 125; KCl 20; MgATP 4; HEPES 10; LiGTP 0.3; NaPCr 5; ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.5, titrated to pH 7.2 with KOH and 290 mOsm/kg osmolality with sucrose. Pipette resistance was 4–6 MΩ and currents were recorded using an Axopatch 1D amplifier (Axon Instruments, USA), connected to a personal computer interfaced with an ITC-18 A/D converter (Instrutech Corp; USA) and analysed using WinWCP software (Dr. J. Dempster, University of Strathclyde, UK). Transfected tsA201 cells were identified by green fluorescence and voltage-clamped at − 70 mV. K2P channel expression and modulation was investigated by stepping to −120 mV for 50 ms, followed
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by a voltage ramp from − 120 mV to +40 mV over 500 ms, then returning to − 70 mV, every 30 s, as described previously (Gruss et al., 2004; Cain et al., 2008). Modulation of the K2P currents was quantified by measuring the amplitude at + 20 mV. Once the amplitude of the evoked currents had stabilised, P2Y receptor agonists were applied in the superfusate for 5 min. Only one agonist was applied to each cell. Site-directed mutagenesis S300A, S333A, S300A/S333A and S351A mutants of the K2P2.1 channel were constructed as described previously (Cain et al., 2008) using specific primers (MWG-Biotech, Ebersberg, Germany) and pfu Ultra DNA polymerase (Stratagene, Amsterdam, The Netherlands). The products were sequenced to confirm correct insertion of each mutation (Dr. R. Tate, University of Strathclyde). Data analysis Values in the text and figures refer to mean ± S.E.M. Data were compared by paired and unpaired t-tests, or one-way analysis of variance and Tukey's comparison as appropriate. Differences were considered significant when p b 0.05. Drugs, solutions and cDNAs ATP (disodium salt), ADP (sodium salt), UTP (sodium salt) (Sigma/ RBI, UK), 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate (tetrasodium salt) (MRS2179) (Tocris, UK) and [(2R,3S,4R,5R)-3,4-dihydroxy5-[6-(2-methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl) purin-9-yl]oxolan-2-yl]methyl dihydrogen phosphate (AR-C69931MX) (a generous gift from The Medicines Company, New Jersey, USA) were dissolved in deionised water as 10 mM stock solutions and diluted in a buffer before application to the cells. PTX (Calbiochem, UK) was dissolved in deionised water (200 µg/ml). The cDNAs encoding the hP2Y1 and hP2Y12 receptors were a gift from Professors T.K. Harden and R.A. Nicholas, (University of North Carolina, Chapel Hill, NC, USA) and that for hK2P2.1 was donated by Glaxo Smith Kline, UK. References Abbracchio, M.P., Burnstock, G., Boeynaems, J.M., Barnard, E.A., Boyer, J.L., Kennedy, C., Fumagalli, M., Gachet, C., Jacobson, K.A., Weisman, G.A., International Union of Pharmacology, 2006. Update of the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev. 58, 281–341. Aimond, F., Rauzier, J.M., Bony, C., Vassort, G., 2000. Simultaneous activation of p38 MAPK and p42/44 MAPK by ATP stimulates the K+ current ITREK in cardiomyocytes. J. Biol. Chem. 275, 39110–39116. Aller, M.I., Veale, E.L., Linden, A.M., Sandu, C., Schwaninger, M., Evans, L.J., Korpi, E.R., Mathie, A., Wisden, W., Brickley, S.G., 2005. Modifying the subunit composition of TASK channels alters the modulation of a leak conductance in cerebellar granule neurons. J. Neurosci. 25, 11455–11467. Alloui, A., Zimmermann, K., Mamet, J., Duprat, F., Noel, J., Chemin, J., Guy, N., Blondeau, N., Voilley, N., Rubat-Coudert, C., Borsotto, M., Romey, G., Heurteaux, C., Reeh, P., Eschalier, A., Lazdunski, M., 2006. TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 25, 2368–2376. Bodor, E.T., Waldo, G.L., Hooks, S.B., Corbitt, J., Boyer, J.L., Harden, T.K., 2003. Purification and functional reconstitution of the human P2Y12 receptor. Mol. Pharmacol. 64, 1210–1216. Boyer, J.L., Mohanram, A., Camaioni, E., Jacobson, K.A., Harden, T.K., 1998. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′, 5′-bisphosphate. Br. J. Pharmacol. 124, 1–3. Buckler, K.J., Honoré, E., 2005. The lipid-activated two-pore domain K+ channel TREK-1 is resistant to hypoxia: implication for ischaemic neuroprotection. J. Physiol. 562, 213–222. Burnstock, G., Kennedy, C., 1985. Is there a basis for distinguishing two types of P2purinoceptor? Gen. Pharmacol. 16, 433–440. Burnstock, G., Knight, G., 2004. Cellular distribution and functions of P2 receptor subtypes in different systems. Int. Rev. Cytol. 240, 31–310. Bushell, T.J., Clarke, C., Mathie, A., Robertson, B., 2002. Pharmacological characterization of a non-inactivating outward current observed in mouse cerebellar Purkinje neurones. Br. J. Pharmacol. 135, 705–712.
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