Neuronal two-pore-domain potassium channels and their regulation by G protein-coupled receptors

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1 J Physiol (2007) pp Topical Review Neuronal two-pore-domain potassium channels and their regulation by G protein-coupled receptors Alistair Mathie Biophysics Section, Blackett Laboratory, Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, UK Leak potassium currents in the nervous system are often carried through two-pore-domain potassium (K2P) channels. These channels are regulated by a number of different Gprotein-coupled receptor (GPCR) pathways. The TASK subfamily of K2P channels are inhibited following activation of the G protein Gα q. The mechanism(s) that transduce this inhibition have yet to be established but there is evidence to support a role of phosphatidylinositol 4,5-bisphosphate (PIP 2 )hydrolysis products, depletion of PIP 2 itself from the membrane, or a direct action of activated Gα q on TASK channels. It seems possible that more than one pathway may act in parallel to transduce inhibition. By contrast, TRESK channels are stimulated following activation of Gα q. This is due to stimulation of the protein phosphatase, calcineurin, which dephosphorylates TRESK channels and enhances their activity. TREK channels are the most widely regulated of the K2P channel subfamilies being inhibited following activation of Gα q and Gα s but enhanced following activation of Gα i. The multiple pathways activated and the apparent promiscuous coupling of at least some K2P channel types to different G protein regulatory pathways suggests that the excitability of neurons that express K2P channels will be profoundly sensitive to variations in GPCR activity. (Received 21 September 2006; accepted after revision 26 October 2006; first published online 26 October 2006) Corresponding author A. Mathie: Biophysics Section, Blackett Laboratory, Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK. a.mathie@imperial.ac.uk Two-pore-domain potassium channels (K2P) underlie leak K + currents and are expressed throughout the central nervous system (Talley et al. 2001; Aller et al. 2005). Currents through these channels contribute to the resting membrane potential of neurons and regulate their excitability. There are 15 members of the K2P channel family, in mammals, which can be divided into six subfamilies based on their structural and functional properties, the TWIK (TWIK1, TWIK2, KCNK7), TASK (TASK1, TASK3, TASK5), TREK (TREK1, TREK2, TRAAK), TALK (TALK1, TALK2, TASK2), THIK (THIK1, THIK2) and TRESK subfamilies (Goldstein et al. 2001; O Connell et al. 2002; Lesage, 2003). K2P channels are highly regulated by pharmacological agents and physiological mediators and by a number of G protein-coupled receptor (GPCR)-activated pathways and protein kinases (Goldstein et al. 2001). To date, only three of these six subfamilies have been shown to be regulated by GPCR pathways, the TASK subfamily, the TREK subfamily and TRESK, although there is evidence of GPCR-mediated modulation of neuronal leak potassium conductances which are not easily assigned to any of these three subfamilies (e.g. Bushell et al. 2002). By far the best studied GPCR pathway is the (differential) regulation of all three of these subfamilies following activation of the G protein Gα q.inthis short review, I will consider the regulation of these channels by GPCRs, detailing the multiple regulatory pathways for K2P channels that have been described thus far. Regulation of the TASK subfamily of K2P channels following activation of Gα q The TASK subfamily of K2P channels (TASK1, TASK3 and the non-functional TASK5) underlie leak currents in avariety of cell types (Buckler et al. 2000; Czirjak et al. 2000; Millar et al. 2000; Talley et al. 2000; Brickley et al. 2001; Han et al. 2002; Clarke et al. 2004; Kang et al. 2004a; Alleret al. 2005). TASK1 and TASK3 channels have been shown to contribute to background currents in many neuronal populations throughout the CNS, including thalamocortical neurons, cerebellar granule neurons (CGNs), dorsal vagal neurons, spinal cord DOI: /jphysiol

2 378 A. Mathie J Physiol neurons, hippocampal neurons and a number of different motoneurons. These conductances have been shown to be inhibited by a wide variety of Gα q -coupled receptors, including those for thyrotropin-releasing hormone, serotonin (5-HT), glutamate and acetylcholine (e.g. Millar et al. 2000; Talley et al. 2000; Bayliss et al. 2001; Sirois et al. 2002; Chemin et al. 2003; Kettunen et al. 2003; Meuth et al. 2003; Perrier et al. 2003; Hopwood & Trapp, 2005; Larkman & Perkins, 2005). In some instances, these conductances may be carried predominantly by one TASK channel. For example, in rat dorsal vagal neurons the leak K + current may be best attributed to TASK1 homodimers (Hopwood & Trapp, 2005). In other neurons, both TASK1 and TASK3 may function together. Indeed there is evidence for TASK heterodimeric channels in a number of neurons such as somatic motoneurons (Berg et al. 2004) and it is well established that heterologously expressed TASK1 and TASK3 subunits can form heterodimeric channels (Czirjak &Enyedi, 2002; Berg et al. 2004; Clarke et al. 2004). Perhaps the most extensively studied leak K + conductance, thought to be carried predominantly by TASK K2P channels, is the leak current seen in CGNs termed I K(SO) (for standing outward K + current) (Watkins &Mathie, 1996). I K(SO) is known to develop postnatally (Watkins & Mathie, 1996; Brickley et al. 2001; Han et al. 2002; Lauritzen et al. 2003; Aller et al. 2005) and its appearance correlates well with the development of ahyperpolarized resting membrane potential in CGNs (Watkins & Mathie, 1996). Application of muscarine or acetylcholine inhibits I K(SO) (Fig. 1A) (Watkins & Mathie, 1996; Boyd et al. 2000; Millar et al. 2000; Han et al. 2002; Lauritzen et al. 2003; Takayasu et al. 2003). This results in a depolarization of the membrane and an increased probability of action potential firing (Fig. 1B)(Watkins & Mathie, 1996; Takayasu et al. 2003). Thus this inhibition of I K(SO) by muscarine is an important mechanism regulating the excitability of CGNs, and in turn, the firing properties of Purkinje neurons (Takayasu et al. 2003). Although, initially, TASK1 channels were suggested to underlie I K(SO) in CGNs (Millar et al. 2000), subsequently, several other K2P channels have been identified in these neurons. It is now known that CGNs express high levels of TWIK1, TASK1, TASK3, TREK2 and THIK2 channel subunits, and lower levels of TREK1, TRAAK and TWIK2 (see, for example, Talley et al. 2001; Mathie et al. 2003; Aller et al. 2005). Of those channels that are highly expressed, three of them, TASK1, TASK3 and TREK2 have been shown, at both the whole-cell and single-channel level, to contribute to I K(SO) in CGNs (Han et al. 2002; Lauritzen et al. 2003; Kang et al. 2004a). As stated above for motoneurons, there is, however, evidence that TASK1 and TASK3 can form heterodimers and that these heterodimers also contribute to I K(SO) (Kang et al. 2004a; Alleret al. 2005). Indeed a recent study with TASK1 knockout mice suggests that much of the ph-sensitive component of I K(SO) is likely to be mediated by TASK1/TASK3 heterodimers (Aller et al. 2005). Figure 1. Activation of muscarinic receptors inhibits the native K + leak current, I K(SO),incerebellar granule neurons A, inhibition of I K(SO) in rat cerebellar granule neurons following activation of muscarinic receptors (from Millar et al. 2000). B, muscarinic inhibition of I K(SO) enhances the excitability of cerebellar granule neurons. In current clamp recordings the depolarizing current injections give a larger depolarization in the presence of muscarine and initiate action potential firing (from Watkins & Mathie, 1996).

3 J Physiol G protein regulation of K2P channels 379 The inhibition of I K(SO) by muscarine was shown to be through activation of M 3 receptors which couple, primarily, to the G protein Gα q.inaddition to regulation of leak K + currents in cerebellar granule cells and elsewhere in the CNS, regulation by Gα q -coupled receptors has also been demonstrated in cells outside the CNS and in several expression systems for TASK1, TASK3 and TASK1/TASK3 heterodimers (Czirjak et al. 2000, 2001; Czirjak & Enyedi, 2002; Talley & Bayliss, 2002; Chemin et al. 2003; Besana et al. 2004; Lopes et al. 2005). Following activation of the GPCR, Gα q stimulates the enzyme phospholipase C (PLC), which hydrolyses the membrane phospholipid PIP 2 into the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP 3 ). IP 3 is known to stimulate the release of calcium from intracellular stores, whilst DAG activates protein kinase C (PKC). The G protein dependence of receptor-mediated inhibition of TASK channels was confirmed by the intracellular application of GTPγ S(anon-hydrolysable form of GTP) which both inhibited channel current and prevented the recovery from receptor-mediated inhibition of TASK1 and TASK3 (Fink et al. 1996; Kim et al. 2000; Czirjak et al. 2001; Chemin et al. 2003). The sequence of events that occur subsequent to activation of Gα q that leads to TASK3 channel inhibition is not well established. There are three primary hypotheses as to how channel inhibition may occur (Fig. 2). The involvement of PLC in the regulatory pathway has been suggested by several studies (Czirjak et al. 2001; Chemin et al. 2003; Kettunen et al. 2003) and thus the first hypothesis concerns the role of one or more of the hydrolysis products of PIP 2 generated following activation of PLC. There is little evidence to suggest a role for IP 3 and calcium mobilization. Application of IP 3 does not inhibit TASK channels (Duprat et al. 1997; Czirjak et al. 2001; Chemin et al. 2003) in excised patches, and whilst there is some evidence that suggests a role of intracellular calcium (Kettunen et al. 2003) most studies argue against such a role, at least as the primary transducer of inhibition following activation of PLC (Duprat et al. 1997; Talley et al. 2000; Czirjak et al. 2001; Chemin et al. 2003; Lopes et al. 2005). Evidence for the involvement of the DAG/PKC pathway is more equivocal. Whilst DAG itself does not inhibit TASK channels directly (Chemin et al. 2003; Lopes et al. 2005), the role of PKC is much less clear. TASK channels contain consensus sites for PKC-mediated phosphorylation (Kim et al. 2000; Rajan et al. 2000); however, it is not clear whether phosphorylation of these sites leads to functional changes in TASK channel currents. A number of studies have reported that activators of PKC, such as phorbol-12-myristate-13-acetate (PMA), failed to affect TASK1 (Duprat et al. 1997; Leonoudakis et al. 1998; Czirjak Figure 2. Three hypotheses as to how activation of Gα q leads to inhibition of TASK channels (see text for details) A, inhibition of TASK channels is mediated through one or more of the products of PIP 2 hydrolysis. B, inhibition of TASK channels is mediated through depletion of PIP 2 which normally maintains TASK channel activity. C, inhibition of TASK channels occurs through a direct action of activated Gα q on the channel.

4 380 A. Mathie J Physiol et al. 2001), or TASK3 (Kim et al. 2000; Meadows & Randall, 2001; Chemin et al. 2003). Furthermore PKC inhibitors such as staurosporine or bisindolyl maleimide were often found not to prevent receptor-mediated inhibition (Czirjak et al. 2001; Chemin et al. 2003; Lopes et al. 2005). On the other hand, other studies have been able to show a PKC-mediated inhibition of TASK1 (Lopes et al. 2000; Besana et al. 2004) and TASK3 (Vega-Saenz de Miera et al. 2001; Veale et al. 2006). For example, TASK1 seems to be modestly inhibited following activation of PKCε and this underlies the platelet activity factor (PAF) receptor-mediated inhibition of TASK1 current in cardiac cells (e.g. Barbuti et al. 2002; Besana et al. 2004). It is possible that at least some of these differences may be attributed to differences between cell types and channel and regulatory protein expression levels, nevertheless the regulation of TASK channels by PKC, in particular, needs further investigation. Takentogether, it is apparent that data in support of an involvement of any of the classical second messengers in the Gα q -mediated regulation of TASK channels are contradictory and inconclusive. This lack of definitive evidence for a role of one or more of the hydrolysis products of PIP 2 in TASK channel regulation has led to a broadening of the search for other possible mechanisms. In addition to generating PIP 2 hydrolysis products, activation of PLC actively depletes cell membranes of PIP 2 itself. Although traditionally regarded as simply a substrate for PLC, it has recently become apparent that PIP 2 can act as a signalling molecule in its own right. PIP 2 is known to regulate the function of a wide variety of ion channels, including several different families of K + channels (e.g. Hilgemann et al. 2001; Suh & Hille, 2005). PIP 2 is thought to interact directly with channels to modulate gating and has been shown to stabilize the open confirmation of K IR channels (Huang et al. 1998; Xiao et al. 2003; Enkvetchakul et al. 2005). Additionally, PIP 2 depletion is currently the most favoured theory to explain Gα q receptor-coupled inhibition of M current carried through KCNQ channels (Delmas & Brown, 2005). Thus the second hypothesis is that depletion of PIP 2 levels in the membrane, rather than the generation of downstream effectors, is responsible for the Gα q -mediated inhibition of TASK channels. It has been shown that PIP 2 is required for normal TASK channel activity and that TASK channels and other K2P channels are further activatedbypip 2 and inhibited by PIP 2 scavengers such as polylysine or antibodies (Chemin et al. 2003; Lopes et al. 2005). Furthermore, recovery of K2P channel currents following receptor-induced inhibition is slowed or prevented in the presence of wortmannin (Czirjak et al. 2001; Lopes et al. 2005), which at relatively high concentrations inhibits phosphatidylinositol 4-kinase and therefore the re-synthesis of PIP 2 following hydrolysis. On the other hand, some studies have shown that PLC blockers such as U have failed to completely prevent inhibition of TASK channels (Boyd et al. 2000; Talley & Bayliss, 2002; Chemin et al. 2003). An excellent recent study has provided convincing evidence which seems to demonstrate that receptor-mediated TASK channel inhibition can proceed independently of either PLC activity or PIP 2 depletion (Chen et al. 2006). In this study Chen et al. showed that Gα q -mediated regulation of TASK3 and TASK1 channels persisted when these channels were expressed in cell lines with very low levels of PIP 2. Moreover,amutatedversionofGα q,deficient at activating PLC, was still able to transduce inhibition of TASK3 channels. These data led Chen et al. to propose a third hypothesis whereby activation of PLC is not required for TASK3 channel inhibition and, instead, activated Gα q acts directly (or via an alternative, as yet unknown, second messenger pathway) to inhibit TASK3 channels (Chen et al. 2006). Whilst this is an attractive hypothesis, since activated Gα q can associate with TASK3 channels in immune complexes and inhibit those channels in excised patches (Chen et al. 2006), it remains to be demonstrated that Gα q (or an intermediary second messenger) does indeed bind directly to TASK3 channels to alter their activity and, if so, where exactly on the channel Gα q (or the intermediary molecule) binds. In fact, there is no reason, a priori, why several components of the signalling cascade may not act together during Gα q -mediated inhibition of TASK3 current, as would appear to be the case for inhibition of TREK1 (see section on TREK channel modulation below; Murbartian et al. 2005; Chemin et al. 2005; see also Beech et al. 1991; Hille, 1992). Regulation of TRESK K2P channels following activation of Gα q TRESK K2P channels were initially detected only in spinal cord (Sano et al. 2003) but subsequent studies across different species have established that they are, in fact, expressed in a number of neuronal populations in different regions of CNS and peripheral nervous system including the cerebellum, dorsal root ganglia and cerebral cortex (Czirjak et al. 2004; Kang et al. 2004b;Kang & Kim, 2006). TRESK channels show a large calcium-dependent increase in current following Gα q receptor activation (Czirjak et al. 2004). This enhancement of current following Gα q activation is unique to TRESK channels compared to other K2P channels studied to date, and is in stark contrast to the inhibition seen of TASK K2P channels (above) and TREK K2P channels (below) following activation of Gα q. The enhancement of TRESK current involves activation of calcineurin (calcium calmodulin-dependent

5 J Physiol G protein regulation of K2P channels 381 phosphatase 2B) following the rise in intracellular calcium that occurs subsequent to Gα q activation (Fig. 3). Calcineurin has been shown to bind directly to a nuclear factor of activated T cells (NFAT)-like docking site on TRESK channels (Czirjak & Enyedi, 2006) and acts to alter the phosphorylation state of the channel through dephosphorylation. Dephosphorylation of TRESK channels by calcineurin at one or more of three adjacent serine residues in the C terminus (S274, S276, S279) gives the observed increase in TRESK current. Thus, by implication, steady-state, basal TRESK channel activity is down-regulated by channel phosphorylation, although it is unknown, at present, which pathways act on TRESK channels to phosphorylate them at rest and keep their basal activity low. Multiple G protein-mediated regulation of the TREK subfamily of K2P channels Similar to Gα q -coupled receptor-mediated inhibition of TASK channels (above) regulation by Gα q -coupled receptors has also been demonstrated for TREK1 channels (Czirjak & Enyedi, 2002; Chemin et al. 2003; Enyeart et al. 2005; Lopes et al. 2005; Murbartian et al. 2005) and TREK2 channels (Lesage et al. 2000; Chemin et al. 2003). By contrast comparatively little inhibition of TRAAK channels by this same pathway is observed (Chemin et al. 2003; Lopes et al. 2005). As for Gα q -mediated inhibition of TASK channels (above) there is conflicting evidence regarding the pathway linking G protein activation and channel inhibition. For TREK1 and TREK2 channels whilst neither IP 3 nor manipulations in intracellular calcium directly affect current (Fink et al. 1996; Lesage et al. 2000; Chemin et al. 2003) it has been suggested that DAG may cause a direct inhibition of TREK channels independently of PKC (Chemin et al. 2003, 2005). However, other experimental observations suggest that DAG produces little inhibition of TREK1 channels when applied directly (Lopes et al. 2005). Again there are conflicting views as to the relative importance of PKC activation and PIP 2 depletion in Gα q -mediated inhibition of TREK channels (Fig. 4A). Whilst some studies provide good evidence in favour of the PIP 2 depletion hypothesis (Chemin et al. 2005; Lopes et al. 2005), there is strong evidence to suggest that at least part of the inhibition of TREK1 and TREK2 following activation of Gα q depends on PKC-mediated phosphorylation of the channel (see below and Murbartian et al. 2005; Kang et al. 2006). In addition to inhibition through activation of Gα q, TREK1 and TREK2 channels can also be inhibited by activation of the G protein Gα s which leads to elevations of camp (Fig. 4B) (Patelet al. 1998; Bang et al. 2000; Lesage et al. 2000). A single site on the C terminus of TREK1 (S333) has been identified as necessary for Gα s and camp-mediated inhibition of the channel (Patel et al. 1998; Bockenhauer et al. 2001). Phosphorylation of TREK1 channels by camp also leads to a profound change in the kinetic properties of TREK1 channels, altering channel behaviour from that of a voltage-independent Figure 3. Activation of Gα q leads to an enhancement of TRESK channel currents A, activation of Gα q stimulates the activity of calcineurin which dephosphorylates TRESK channels and enhances their activity (see text for details). B, TRESK channel currents are enhanced by stimulation of muscarinic receptors. TRESK inward current amplitude (in 80 mm K + )inxenopus oocytes is enhanced by carbachol (based on part of a figure in Czirjak et al. 2004, with permission).

6 382 A. Mathie J Physiol leak conductance to a voltage-dependent current with much lower over-all open probability (Bockenhauer et al but see Maingret et al. 2002). A second camp-dependent phosphorylation site in the C terminus of TREK1 channels (S300) has recently been identified by Murbartian et al. (2005). Mutation of this amino acid, rather surprisingly, attenuated both camp- and PKC-dependent inhibition of TREK1 currents, suggesting possible interactions between the two regulatory pathways. Furthermore, S333 phosphorylation was shown to be a prerequisite for S300 phosphorylation to occur in a regulatory cascade (Murbartian et al. 2005). Thus there is an apparent convergence on serine residues in the C terminus of TREK channels of both Gα q - and Gα s -mediated inhibitory pathways. Recently, enhanced 5-HT receptor-mediated inhibition of TREK1 channels through the Gα s pathway has been suggested to contribute to the action of antidepressant agents such as fluoxetine when used in the treatment of depression (Heurteaux et al. 2006). Since stimulation of Gα s and subsequent increase in camp leads to inhibition of TREK channels, it might be anticipated that stimulation of Gα i (and subsequent decreases in camp levels) will, at least in some circumstances, lead to an increase in TREK current (Fig. 4B). For both TREK2 and TREK1 channels, such an increase has indeed been observed following activation of Gα i by mglur 2 and mglur 4 receptor stimulation, respectively (Lesage et al. 2000; Cain & Bushell, 2006). There is other evidence for regulatory pathways acting to enhance current through TREK channels. Both ATP-mediated stimulation of the p38 MAPK and P42/44 MAPK pathways (Aimond et al. 2000) and phosphorylation of TREK1 channels by protein kinase Gfollowing activation by cgmp or sodium nitroprusside (Koh et al. 2001) have been shown to enhance TREK channel activity in both expression systems and native cells. Conclusions A number of G protein-coupled pathways have been shown to regulate the activity of K2P channels. The most commonly occurring regulation seems to be Gα q -mediated inhibition which is seen for both TASK and TREK channels. There does not, however, appear to be agreement as to how this regulation is transduced and as more data emerges, it seems increasingly likely that a number of different potential regulatory pathways are stimulated following Gα q activation. It is possible that these may act in parallel to ensure robust channel regulation. Alternatively, the dominant mechanism may depend on the particular receptor stimulated, the relative expression levels of the different proteins involved, or the cell type studied. It is of interest that regulation of M current by muscarinic receptors or bradykinin receptors (both of which activate Gα q ) is transduced through different pathways, even in the same neuron type (Cruzblanca et al. 1998). Additional complexity is revealed by the apparent promiscuous coupling of particular K2P channel types to different G protein regulatory pathways. This is perhaps best illustrated for TREK2 channel currents which are inhibited following activation of the Gα s -coupled 5HT 4 receptor or the Gα q -coupled mglur 1 receptor but enhanced following activation of the Gα i -coupled mglur 2 receptor (Lesage et al. 2000). The excitability of neurons that express TREK2 channels will therefore be profoundly sensitive to variations in GPCR activity. Figure 4. Multiple GPCR pathways act on TREK channels to regulate their activity A, inhibition of TREK channels following activation of Gα q is thought to be mediated either through PKC or through depletion of PIP 2 (see text for details). B, TREK channel activity can be up- or down-regulated by Gα i and Gα s,respectively, by altering the levels of intracellular camp.

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