Metabotropic signaling by kainate receptors

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1 Metabotropic signaling by kainate receptors Ricardo J. Rodrigues and Juan Lerma Kainate receptors (KARs) are members of the ionotropic glutamate receptor family. Despite their ubiquitous presence in the central nervous system, and in contrast to the better characterized N-methyl-D-aspartates (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), the contribution of KARs to synaptic transmission has only been demonstrated in a few central synapses. However, there is now accumulating evidence that KARs are present on both sides of the synapse, where they play distinct and diverse roles. In addition to their contribution to synaptic transmission, KARs can regulate synaptic activity and plasticity either by presynaptically modulating neurotransmitter release at GABAergic and glutamatergic synapses, or by postsynaptically regulating neuronal excitability. This prominent neuromodulatory role of KARs has been further highlighted by the finding that these glutamate-gated ion-channels can also signal through G-proteins and other second messengers. This noncanonical metabotropic signaling of KARs was firmly established by demonstrating it to be independent of ion flux. The discovery of this dual signaling capacity of KARs constituted a breakthrough in understanding how they function and since then, an increasing number of metabotropic actions of KARs have been reported. It is now clear that this dual signaling underlies the diverse functions of KARs and defining this metabotropic component of the signaling system operated by KARs will be necessary to understand the physiological contributions of glutamate receptors WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. How to cite this article: WIREs Membr Transp Signal 2012, 1: doi: /wmts.35 INTRODUCTION Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS), can signal through the activation of two families of glutamate receptors: ionotropic glutamate receptors (iglurs) that belong to the superfamily of ligandgated ion channels; and metabotropic glutamate receptors (mglurs) that belong to the superfamily of G-protein-coupled receptors (GPCRs) with the general signature of seven membrane spanning regions. Ionotropic glutamate receptors are further divided into three receptor families (although a fourth class of ionotropic glutamate receptors of unknown function also exists: delta receptors), each of which carrying the name of the agonists that Correspondence to: jlerma@umh.es Instituto de Neurociencias de Alicante, CSIC-UMH, San Juan de Alicante, Spain activate them: N-methyl-d-aspartate (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPARs), and kainate receptors (KARs). KARs are tetrameric assemblies of subunits that have a similar structure to AMPARs and NMDARs. Five KAR subunits have been cloned to date: GluK1-5 (formerly named GluR5-7, KA1, and KA2, respectively; see Lerma et al. 1 for a review). Whereas GluK1-3 can form functional homomeric or heteromeric channels, GluK4 and GluK5 only participate in heteromeric receptors and they confer higher agonist affinities to the assemblies with GluK1-3. 2,3 KARs are expressed ubiquitously in the CNS, and they are found in both presynaptic and postsynaptic elements. Postsynaptically, KARs mediate synaptic transmission and they may be involved in the regulation of neuronal excitability, whereas presynaptically they regulate neurotransmitter release in both GABAergic and glutamatergic synapses. 2 4 KARs are Volume 1, July/August WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 399

2 wires.wiley.com/mts also known to support phenomena associated with synaptic plasticity, such as short- and long-term potentiation and long-term depression, considered to be the cellular basis of learning and memory. 2 4 In addition, KARs play key roles in nociception, neuronal development, synaptogenesis, and synaptic maturation. 2,3 Indeed, as the original demonstration of the existence of KARs in neurons of the CNS 5 and as postsynaptic receptors mediating a small component of the synaptic current in mossy fibers (MF)-CA3 synapses, 6,7 it has become more evident that the role of KARs in synaptic integration and their capacity to modulate synaptic activity may be more significant than that of conveying information transfer at synapses. Therefore, the excitability of brain networks may be tightly controlled through these actions of KARs. One prominent neuromodulatory role of KARs is evident through the coupling of these receptors to heterotrimeric G-proteins and second messengers, a metabotropic non-canonical mode of signaling for these ionotropic receptors. 8 Such signaling was initially identified when the presynaptic inhibition of GABA release at inhibitory hippocampal synapses 9,10 was seen to be dependent on G-protein activation and PKC activity. 8 Subsequently, such non-canonical metabotropic signaling was more firmly established and demonstrated to be independent of ion flux. 11 However, it is still not clear how KARs, receptors that adopt the typical topology of ligand-gated ion channels, can trigger G-protein activation and second messenger synthesis. KARs do not share the seven transmembrane domains structure of GPCRs. Nevertheless, AMPAR 12 and L-type Ca 2+ -channels 13 have also seen to be capable of activating G-proteins, as well as PLC, which may trigger endoplasmic release of Ca 2+. However, the capacity of an ion channel to directly convey a signal in the absence of ion flux is still difficult to reconcile. An increasing number of metabotropic actions triggered by KAR activation have been identified in a variety of synapses and CNS regions, clear evidence of the relevance of such KAR triggered noncanonical signaling in the CNS. This dual signaling capacity also demonstrates that KARs are endowed with a variety of mechanisms to fine tune neuronal activity and network activity (Figure 1). Therefore, understanding the structure function relationships and the physiological role of this metabotropic KAR signaling is necessary to fully comprehend their role in the CNS. In this review, we will examine the evidence for the metabotropic role of KARs, paying particular attention to the most recent findings. We will also discuss structural-functional issues, as well as considering FIGURE 1 Overview of the metabotropic effects of kainate receptors (KARs). Presynaptic KARs can bidirectionally modulate neurotransmitter release, both at glutamatergic and GABAergic synapses. The inhibitory but not the facilitatory activity of KARs is most likely linked to G-protein coupled mechanisms. 8,14,19,25,34,41,45,46,61,62 The inhibition of voltage-gated Ca 2+ -channels by KARs through G i/o -protein and PKC activation, independent of ion flux, 11 may contribute to the inhibitory action of KARs on neurotransmitter release. In addition to the regulation of synaptic activity by the presynaptic modulation of neurotransmitter release, KARs can also control of network activity by regulating neuronal excitability. In addition to the ionotropic contribution to synaptic transmission, postsynaptically KARs regulate neuronal excitability by inhibiting slow afterhyperpolarization, as well as through G i/o protein and PKC dependent mechanisms. 63,65 69 which elements KARs subunits, G-proteins and second messengers are involved in such non-canonical KAR signaling. METABOTROPIC ACTION OF KARs In the absence of a suitable molecular model to account for such activity, determining whether KAR-mediated responses are due to ionotropic or metabotropic actions essentially depends on pharmacological data that defines whether or not G-proteins and protein kinases are implicated in such signaling. Using these criteria, evidence for the metabotropic action of KARs has accumulated, acting both pre- and postsynapticaly in several regions of the CNS. Presynaptic Metabotropic Actions of KARs In addition to the involvement of metabotropic KAR signaling in controlling GABA release in the hippocampus, 8 some evidence suggested that metabotropic signaling was also involved in the control of glutamate release by presynaptic KARs. 14 A purely presynaptic metabotropic role of KARs is difficult to demonstrate, sometimes due WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Volume 1, July/August 2012

3 WIREs Membrane Transport and Signaling Metabotropic signaling by kainate receptors to the technical difficulties in directly assessing presynaptic phenomena using electrophysiological recordings. Nonetheless, there is convincing evidence that KARs fulfil a presynaptic metabotropic role in regulating neurotransmitter release. For example, functional KARs have been identified in synaptosomal preparations devoid of functional somato-dendritic compartments, 15 and KAR activation controls voltage-gated Ca 2+ -channels in a G-protein- and PKC-dependent manner. 11 Therefore, we will examine all the evidences currently available that metabotropic mechanisms are involved in the regulation of both GABA and glutamate release by presynaptic KAR activation. Presynaptic Control of GABA Release Although kainate (KA) was first proposed to decrease inhibition in the hippocampus in 1981, 16 it took another 16 years to demonstrate that KARs rather than AMPARs were involved in the KA-induced inhibition of GABA release in the hippocampus. 9,10 This was finally possible due to the development of antagonists that selectively discriminate between AMPARs and KARs, 17,18 and in these studies, application of KA was seen to cause the depression of GABA-mediated postsynaptic currents in CA1 pyramidal cells induced by stimulating either the stratum oriens 9 or stratum radiatum. 10 Changes in electrophysiological properties, such as an increase in synaptic failure rate, and in the coefficient of variation and paired-pulse ratio, strongly pointed toward a presynaptic locus of action. These presynaptic KARs most probably involved receptors containing GluK1, since ATPA mimicked the effects of KA, 10 an agonist of GluK1-containing receptors. Soon afterwards, it was proposed that the mechanism underlying this inhibition was sensitive to Pertussis toxin (PTx) and inhibitors of PKC. 8 The depression of evoked inhibitory postsynaptic currents (eipsc) by KA was believed to occur through the activation of KARs at inhibitory terminals, which activate G i/o protein (PTx-sensitive) and the subsequent stimulation of a pool of PKC (via PLC). Indeed, KAR-induced depression of the evoked release from GABA hippocampal synaptosomes, a preparation of isolated nerve terminals, was later demonstrated, 15 and it was shown to be sensitive to PTx and PKC inhibitors. 19 Binding assays provided further evidence for the coupling of KARs and G i/o -proteins in hippocampal membranes. 20 Together, these observations strongly indicate that the inhibition of GABA release at CA1 interneurons is mediated by presynaptic GluK1-containing KARs, which trigger a metabotropic signaling pathway that involves G i/o proteins, PLC activation and downstream activation of PKC. Nonetheless, both the presynaptic site of action and the involvement of a direct metabotropic action of KARs have been disputed, and alternative explanations have been proposed that are mainly based on the expected ionotropic actions of KARs. Such a proposal was prompted by the observation that KARs were present in the somatodendritic compartment of CA1 interneurons. By depolarizing the somatodendritic membrane, KA induces a marked increase in the frequency of spontaneous inhibitory postsynaptic current (sipsc). 21,22 Therefore, it was argued that KA depresses eipsc by a barrage of GABA release, which in turn activates presynaptic GABA B receptors (GABA B R). 23 Although GABA B R-mediated feedback inhibition of GABA release is feasible, as proposed in dorsal horn neurons, 24 the KA-induced inhibition of GABAergic transmission at interneuron-ca1 pyramidal cell synapses mostly persists in the presence of GABA B R and other GPCR antagonists. 8,21,25 Besides, KA-induced depression of GABA release has also been observed in synaptosomes, a preparation devoid of functional somatodendritic compartments. The involvement of presynaptic metabotropic KARs at interneuron-ca1 pyramidal cell synapses was further supported by showing that the two main effects of KA a reduction of the inhibitory drive and an increase in interneuron firing rate could be dissociated, and that they are mediated by KARs with different pharmacological profile. 25,26 Indeed, while low concentrations of ATPA (0.3 μm) induce repetitive firing of interneurons, they are insufficient to depress the release of GABA. On the other hand, the endogenous agonist glutamate can depress GABA release at concentrations that are largely ineffective in inducing interneuron firing. 25 Furthermore, while the KA-induced depression of eipsc is abolished by PTx treatment, the increase in sipscs is preserved, demonstrating a spatial and functional compartmentalization of two populations of KARs in hippocampal CA1 interneurons. 25 The segregation of different KAR populations to presynaptic and somatodendritic compartments was further reinforced by studying mice lacking GluK1 or GluK2. Although KA (3 μm) induced an increase in the rate of sipsc three times higher in mice lacking GluK1 than in mice lacking GluK2, the degree of KA-evoked depression of eipsc remained similar. 27 Functional compensatory changes have been described in these null mutants but when combined with a pharmacological approach, KARs located pre- and postsynaptically in CA1 interneurons appear to have different subunit compositions, whereby GluK1-GluK2 or GluK1-GluK5 Volume 1, July/August WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 401

4 wires.wiley.com/mts are present in presynaptic terminals. 26 The physiological relevance of the spatial and functional compartmentalization of two KAR populations within the same interneurons remains unknown. Obviously, these receptors are characterized by distinct structural, pharmacological and signaling characteristics. Hence, it is likely that the properties of either receptor determine their physiological role, which may even be influenced by the source of glutamate activating each receptor (nearby synaptic terminals 28 or from nonsynaptic sources, such as astrocytes, 29 etc.). KARs could inhibit the release of GABA onto CA1 pyramidal cells when they are activated by endogenous glutamate. Indeed, stimulation of glutamatergic afferent fibers reduces GABAergic input to CA1 pyramidal cells through the activation of KARs at presynaptic GABA terminals. 28 Paired recordings of interneurons and CA1 pyramidal neurons show that endogenous activation of KARs may also facilitate GABA release. 30 Moreover, the depression of transient eipscs in CA1 pyramidal cells by short trains of Schaffer collateral fiber stimulation is dependent on presynaptic GluK1-containing receptors in interneurons expressing CB1Rs. 31 By contrast, neurons not expressing CB1Rs display GluK1-dependent potentiation of uipsc. The evidence provided suggests that the train-induced depression of inhibition (t-di) observed upon the release of glutamate requires GluK1-dependent presynaptic depolarization, which in turn activates CB1R signaling through the endocannabinoid 2-AG, mobilized by postsynaptic activation of mglurs. These data raise questions regarding the direct involvement of presynaptic KARs and attribute this action to CB1Rs, although experiments with several CB1R antagonists and in CB1R deficient mice demonstrate that KA-induced inhibition of GABA release persists (Paternain and Lerma, unpublished data). In accordance with our data, recordings from pairs of cholecystokinin-containing basket cells and CA1 pyramidal cells show that KAR activation with exogenous KA (1 μm) presynaptically reduces eipscs, even in the presence of CB1R antagonists. 32 Moreover, KA-induced depression of eipscs is resistant to the blockage of mglurs and can also be observed in the presence of tetrodotoxin 8 when activation of CB1R is unlikely. Indeed, it was recently shown that the inhibition of eipscs in CA1 pyramidal cells by pharmacological activation and by endogenous activation of KARs is mechanistically distinct. 33 Nevertheless, KA-induced inhibition of eipscs in CA1 pyramidal cells is reduced by the simultaneous blockade of GABA B Rs and CB1Rs, which suggests that at least part of the metabotropic effect induced by exogenous activation of KARs may be due to coincident CB1R and GABA B R signaling, while not excluding the direct metabotropic action of KARs. 33 More surprisingly was the observation that the inhibition of eipscs by KA is independent of presynaptic GluK1-containing receptors, relying rather on the activation of postsynaptic GluK2-containing receptors. 33 Thus, although a metabotropic effect of KARs seems to be consensual, there is still some controversy regarding the direct triggering of the metabotropic signaling by KARs and even, regarding the KARs involved in the KA-induced inhibition of GABA release onto CA1 pyramidal cells. Nonetheless, metabotropic signaling does appear to be involved in the inhibitory action of GABA released by endogenously activated KARs in the developing hippocampus. Indeed, tonic activation of presynaptic GluK1 KARs produces a depression of GABA release from mossy fibers in a G-protein- and PLC-dependent manner. 34 As mentioned above, activation of presynaptic KARs can also enhance GABA release. Indeed, facilitation of GABA release by exogenous application of KAR agonists has been observed at interneuronprincipal cell synapses in several regions of the CNS (neocortex, 35 hypothalamus, 36 and hippocampus 30 ), as well as between hippocampal interneurons. 27,37 Facilitation of GABA release by endogenous activation of KARs has also been reported. 30,31 While the inhibitory effects so far described involve direct metabotropic signaling 8,25,38 or an indirect metabotropic influence, 24,39 the facilitatory effects are more consistent with the ionotropic properties of KARs. A straightforward explanation is that these latter effects involves ionotropic KAR induced depolarization of terminals that lead to the opening of voltage-dependent Ca 2+ -channels or direct Ca 2+ - entry. The observation that KARs might be situated in the axons of GABA interneurons, where they could bring the axon closer to the firing threshold, 40 further favors the ionotropic mode of action. However, it is noteworthy that the facilitation by KAR activation at interneuron interneuron synapses is not sensitive to Ca 2+ -channel blockers. 37 KARs can therefore facilitate and inhibit presynaptic GABA release, even when KA is applied exogenously within the same GABAergic synapses, as evident in recordings of interneuron-pyramidal cell pairs in the CA1 hippocampal region 30 and in the basolateral amygdala. 38 What seems to be constant is that facilitation is observed at low concentrations of KA and inhibition at higher concentrations. Therefore, the effect of activating presynaptic KARs with endogenous glutamate may depend on the glutamate concentrations actually reaching these heteropresynaptic KARs. Indeed, glutamate spill over from nearby WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Volume 1, July/August 2012

5 WIREs Membrane Transport and Signaling Metabotropic signaling by kainate receptors synaptic terminals may be distinct under different conditions. A recent study showed that the increase in the ambient levels of glutamate, either that associated to a physiological reduction in astrocyte coverage or that due to the blockade of glutamate transporters, switches presynaptic facilitation into inhibition of GABAergic transmission in the hypothalamic supraoptic nucleus. 41 Interestingly, the facilitatory effect was inhibited by philanthotoxin, a blocker of Ca 2+ -permeable receptors, whereas the inhibitory effect involved a PLC-dependent metabotropic pathway. These observations reinforce the idea that bidirectional modulation of GABA release by presynaptic KARs may be achieved by their dual signaling, that is GABA release would be facilitated through canonical ionotropic KAR activity while depression would be achieved by a non-canonical metabotropic pathway. In summary, there is growing evidence that presynaptic KARs modulate GABA release in a bidirectional manner, ionotropic activity generally coinciding with stimulation of GABA release while inhibition is mediated by triggering metabotropic signaling. It is clear that the KAR signaling system is endowed with a variety of mechanisms to fine tune synaptic inhibition. However, understanding how and when this dual neuromodulatory role of presynaptic KARs comes into play under physiological conditions awaits further elucidation. Presynaptic Control of Glutamate Release Presynaptic regulation of excitatory transmission by KARs has been studied extensively and there is now compelling evidence that KARs play crucial roles in regulating glutamate release. 2 4 Pharmacological activation of KARs can either facilitate or inhibit glutamate release, depending on the synapse type and agonist concentration. However, endogenous activation by synaptically released glutamate mainly facilitates neurotransmission (as reviewed in Pinheiro and Mulle 3 ), although inhibitory actions have also been reported The mechanism underlying the facilitation of glutamate release is generally consistent with the classic ionotropic activity. At MF-CA3 pyramidal synapses, presynaptic KARs are implicated in the characteristic frequency-dependent facilitation of synaptic responses, where synaptic activation of presynaptic KARs must be quite fast (<10 ms) since KAR antagonists attenuate the potentiation of the second EPSC during high-frequency trains (e.g., 25 Hz 47 ). This fast time-course of action of presynaptic KARs is indicative of an ionotropic rather than a G-protein coupled mechanism. Indeed, there is indirect evidence indicating that KARs induce depolarization of MF terminals. 47,48 It is assumed that such depolarization would enhance action-potential driven Ca 2+ -influx 49 and that it might underlie robust short-term synaptic facilitation at these synapses. In addition, a direct contribution of presynaptic KARs to Ca 2+ -influx through the receptor itself is also likely given the marked sensitivity of synaptic facilitation to the blocker of Ca 2+ -permeable receptors, phylantotoxin. 49,50 It has also been suggested that the Ca 2+ -influx promoted by presynaptic KAR activation is further enhanced by inducing Ca 2+ mobilization from intracellular stores. All these phenomena may contribute to use-dependent facilitation at MF-CA3 pyramidal cell synapses. 49,51 Curiously enough, the enhancement of synaptic transmission at MF-CA3 synapses caused by exogenous activation of KARs with low concentrations of KA ( 50 nm) seems to involve the activation of adenylyl cyclase (AC) and PKA, in the absence of G-protein activation. 52 The activation of the AC/cAMP/PKA pathway by KARs was argued to be the consequence of the intraterminal rise in Ca 2+, activating Ca 2+ /calmodulin-sensitive AC isotypes and thus contingent to the ionotropic activitiy of KARs. 53 Nevertheless, it was also argued that presynaptic KARs are insufficient to facilitate transmitter release at MF-CA3 synapses. Indeed, it has been proposed that the facilitatory action generally attributed to presynaptic KARs may rather be due to activation of recurrent CA3 network activity. 53 On the other hand, there is growing evidence that the inhibitory effects of presynaptic KARs on glutamate release are mediated by G-protein coupling. At synapses between hippocampal Schaffer collaterals (SCh) and CA1 pyramidal cells, pharmacological activation of KARs depresses synaptic transmission with a presynaptic locus of action. 14,54 56 This inhibition is accompanied by a reduction in presynaptic Ca 2+55 and several lines of evidence suggest that there is no involvement of presynaptic depolarization. 14 Rather this process appears to involve a G-protein coupled mechanism since inhibition was abolished by the G-protein inhibitors, N-ethylmaleimide (NEM) and PTx, and indirect action of other neuromodulators has been excluded. 14 Interestingly, in these synapses a broad spectrum protein kinase inhibitor (H-7) fails to affect such inhibition, suggesting that there may be no involvement of protein kinases but rather, a membrane-delimited action most likely involving G βγ subunits which appears to directly inhibit presynaptic Ca 2+ -channels. 57 The metabotropic action of presynaptic KARs that inhibits synaptic transmission at SCh-CA1 Volume 1, July/August WILEY-VCH Verlag GmbH & Co. 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6 wires.wiley.com/mts synapses is most evident in the developing hippocampus. During early development, GluK1-containing receptors are tonically activated by endogenous glutamate, inhibiting glutamate release through the presynaptic activation of G i/o -protein and PKC. 46,58 This tonic inhibitory effect of KARs is developmentally regulated and accordingly, it is not observed in mature CA1 neurons (P14 P16). Changes in the affinity of presynaptic KARs may be responsible for this phenomenon, 46 which could play a significant role in synaptic maturation. 59 Similar tonic inhibition of glutamate release has also been observed at CA3 glutamatergic synapses in the developing hippocampus. 45 The inhibition of excitatory signals to pyramidal cells, together with the facilitation of glutamate release onto CA3 interneurons also mediated by KARs, demonstrates that KARs can control the early network activity characterized by spontaneous glutamatergic activity that is crucial for the development of synaptic circuitry. 45 Interestingly, the inhibition of glutamate release is dependent on G i/o -proteins and PKC, while facilitation onto CA3 interneurons does not involve G- protein activation. The tonic control of glutamatergic inputs to CA3 neurons by tonic activation of KARs is also developmentally regulated. Moreover, in the adult CA3 region the inhibition of synaptic transmission by the exogenous activation of KARs with high concentrations of KA 42,60 is also PTx-sensitive, albeit contingent on the inhibition of adenylate cyclase, camp synthesis and PKA activity. 61 Evidence for the involvement of metabotropic signaling in the inhibitory effect of presynaptic KARs on glutamate release is not restricted to the hippocampus. In the rat globus pallidus, the synaptic activation of KARs decreases glutamate release through a presynaptic G i/o -protein coupled and PKC-dependent mechanism. 62 In spinal cord slices, KAR activation produces the inhibition of glutamate release evoked by dorsal root stimulation onto dorsal horn neurons, which was attenuated by the G-protein inhibitor, NEM. 11 As observed in the presynaptic control of GABA release, presynaptic KARs can also facilitate or inhibit glutamate release. Moreover, the inhibitory but not the facilitatory activity of KARs is most probably linked to non-canonical KAR signaling. Postsynaptic Metabotropic Actions of KARs Postsynaptic KARs were first identified at MF-CA3 pyramidal cell synapses in the hippocampus, 6 where KAR-mediated excitatory postsynaptic currents (EPSC KA ) represent only a small part of the EPSC. Although functional postsynaptic KARs can be activated pharmacologically in several cell types of other different brain regions, EPSC KA has been observed in only a few synapses. 4 Indeed, no EPSC KA could be elicited at Schaffer collateral-ca1 pyramidal cell synapses. 6,21,22 However, a role for postsynaptic KARs was identified at these synapses in relation to the inhibition of slow afterhyperpolarization current (sahp) in CA1 pyramidal cells. 63 The sahp, activated upon short bursts of action potentials is generated by a voltage-independent, Ca 2+ -dependent K + -channel. This current has a slow decay time (I sahp ), lasting up to several seconds (5 10 seconds), it is activated in proportion to the number and frequency of action potentials, 64 and it underlies spike frequency adaptation. Nanomolar concentrations of KA cause the long-lasting inhibition of I sahp, 63 which was not due to secondary activation of other receptors or to a circuit-driven effect. Pharmacological manipulations indicate that this inhibition involves G-protein and PKC activation 63 and significantly, this effect was reproduced by synaptic glutamate released from excitatory afferents in the CA1 region. 65 More recently, the involvement of G i/o protein and MAP Kinase activation downstream of PKC has also been reported. Indeed, it has been proposed that the longterm effect of MAP Kinase dependent phosphorylation may underlie the long-lasting, near irreversible effect of KARs on the I sahp in CA1 pyramidal cells. 66 These findings clearly establish another direct metabotropic action of KARs, now at the postsynaptic level. The physiological consequence of reducing I sahp is an increase in neuronal excitability. Thus, this metabotropic action of KARs may be physiologically relevant in the temporal integration of excitatory signals and synaptic plasticity, and it may at least be partially responsible for the epileptogenic activity of KA. Postsynaptic regulation of neuronal excitability involving KAR activation through non-canonical metabotropic signaling has also been observed in CA3 pyramidal cells. 67 At these synapses, KAR activation decreases both slow (I sahp ) and medium (I mahp ) AHP currents, leading to an increase in the firing frequency of CA3 pyramidal neurons. 67 The KA-induced decrease of sahp is also G-protein and PKC dependent, and it seems to involve receptors containing GluK2 since it was preserved in GluK1 / mice but not in GluK2 / mice. 67,68 As KAR-mediated EPSCs can be recorded in MF-CA3 pyramidal cell synapses, 6,7 both modes of KAR signaling can coexist within the same synapses. 68 Both these events could be dissociated in mice lacking GluK2 or GluK5. In GluK2 / mice, both the inhibition of I sahp and KAR ionotropic synaptic transmission are lost, while in GluK5 / mice only the inhibition of I sahp is absent. 68 These results suggest that the high-affinity WILEY-VCH Verlag GmbH & Co. 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7 WIREs Membrane Transport and Signaling Metabotropic signaling by kainate receptors kainate binding GluK5 subunit is mandatory for KAR activation to trigger metabotropic signaling. However, more recently the opposite was observed in mice lacking one or both high-affinity GluK4 and GluK5 subunits. 69 While high-affinity KARs subunits are critical for ionotropic KAR activity, the ability of KARs to trigger metabotropic signaling is retained in double GluK4-GluK5 KO. Thus, whether specific subunits are responsible for the ionotropic and metabotropic activity of KARs awaits further clarification. In neonatal hippocampal CA3 interneurons, tonic inhibition of I mahp seems to be provided by GluK1-containing KARs through a G-protein coupled mechanism. 70 This phenomenon is responsible for the high interneuronal firing rate that is so important to establish neuronal connectivity. 70 This tonic inhibitory action on the mahp is lost once development is completed. Thus, KARs also control AHP of interneurons through a metabotropic action during early development. Irrespective of the subunits involved, the findings accumulated to date clearly demonstrate that as well as carrying a part of the synaptic current, postsynaptic KARs also regulate neuronal excitability by inhibiting AHP through a metabotropic action. Furthermore, it has been confirmed that both ionotropic and metabotropic signaling of KARs can coexist and that they are processes that can be separated. Hence, it is clear that the metabotropic action of KARs is independent of their ionotropic properties, as indicated previously. 11 STRUCTURE FUNCTION OF THE METABOTROPIC ACTIONS OF KARs As indicated above, the ability of KARs to transduce a signal through non-canonical metabotropic signaling has received strong experimental support over recent years. The metabotropic actions of KARs have been described in many cell types from different regions of the CNS, and in association with the presynaptic control of neurotransmitter release or the postsynaptic regulation of neuronal excitability. However, the mechanisms that drive this non-conventional metabotropic signaling remain elusive. More precisely, how KARs activate G-proteins to trigger such metabotropic effects, or what determines the mode of action of KARs are fundamental questions that remain unanswered. The growing amount of pharmacological evidence for the G-proteins-mediated effects of KARs has not been accompanied by advances in our understanding of how KARs activate these G proteins. Indeed, evidence for an interaction between KARs and G-proteins is scarce. Prior to the description of the metabotropic actions of KARs, PTx-sensitive agonist binding to goldfish KARs was reported, indicating the existence of a functional interaction between KARs and PTx-sensitive G-proteins. 71 Similar PTx-sensitive KAR agonist-binding was also observed in hippocampal membranes 20 and there is evidence of a physical interaction between KARs and a G αq protein, 68 suggesting that these proteins may interact either directly or indirectly. The failure to co-immunoprecipitate GluK2 and G αq in GluK5 / mice indicates that this interaction might be mediated by GluK5 subunits. 68 The involvement of G αq in the modulation by KARs of I sahp in CA3 pyramidal cells is congruent with similar G αq -coupled modulation of the I sahp triggered by GPCRs. 72 However, this is at odds with the sensitivity to NEM of KA-induced I sahp modulation in both CA3 and CA1 pyramidal cells, and with the PTx-sensitivity of the metabotropic actions of KARs, which suggests the involvement of the G i or G o protein. Thus, it remains to be determined if the pharmacological evidence for the functional coupling of KARs and G i/o -proteins is supported by a physical interaction between them. Regarding the second messengers involved in the metabotropic phenomenon described so far, both G i and G o proteins could participate in these events. The sensitivity to PTx, and the involvement of PLC and PKC, is in agreement with the activation of the G o protein, whereas the effects contingent to the inhibition of adenylate cyclase and the ensuing reduction of camp would involve G i protein activation. Therefore, it is still necessary to identify the G-proteins involved in the metabotropic actions of KARs, which constitutes the first important step toward characterizing the mechanism underlying this non-canonical KAR signaling. How KARs physically interact with G-proteins remains unclear as they do not contain the conventional motifs in their C-terminal domains that would support a direct interaction with G-proteins similar to those of the classical GPCRs. This may indicate that ancillary proteins are required to form a KAR/G-protein signaling complex, a model supported by the increasing number of proteins thought to interact with KARs. However, the proteins so far identified are mainly involved in KAR trafficking and subcellular localization (reviewed in Contractor et al. 4 ), although more recently binding to proteins such as KRIP6 73 and to NETO1 and 2 74 was identified, opening new perspectives. These KAR interacting proteins directly modulate receptor channel gating. Thus, the evidence that interacting proteins modulate KAR function, in this case in the ionotropic context, make the Volume 1, July/August WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 405

8 wires.wiley.com/mts FIGURE 2 Reconstitution of dual signaling by recombinant GluK1 2b. In neuroblastoma cells, the transfection of GluK1 2b alone reproduces both modes of signaling. Rapid application of kainate induces a typical ionotropic response, while activation of these receptors induces their internalization by activating non-canonical signaling. This auto-internalization is blocked by PTx and it could be induced through the direct activation of PKC by PdBu, indicating the involvement of KAR induced activation of G i/o protein and subsequently, of the downstream PKC. existence of a macromolecular complex governing KAR triggered G-protein activation more feasible. The existence of a protein complex that transduces the consequences of KAR activation to G- proteins would represent a determinant of KAR activity. All the known homo- or heteromeric subunit compositions of KARs can support ionotropic activity, and GluK1 subunits are sufficient to produce both ionotropic and metabotropic signaling in heterologous expression systems. 75 This is indicated by the repetitive activation of homomeric GluK1 receptors, which induces their internalization through the activation of PTx sensitive G-proteins and PKC. It is particularly relevant that metabotropic activities can be reproduced in neuroblastoma cells transfected with GluK1 subunits alone (Figure 2), indicating that GluK1 is sufficient to reproduce both types of receptor signaling. Indeed, KA induces an inward current in these cells, indicating that both ionotropic and metabotropic responses are induced by the same type of receptor complex. Thus, a factor other than subunit composition discriminates the mode of action of KARs, pointing to interacting proteins as the candidates to influence such effects. CONCLUSION Having developed selective pharmacological tools to discriminate AMPARs and KARs, and with the aid of mice deficient for KAR subunits, the role of KARs is now being elucidated. Accordingly, there is growing evidence that like the closely related AMPARs, KARs are designed to be key modulators of synaptic function, thereby regulating network activity. It has also emerged that KARs can signal through a nonconventional G-protein coupled pathway that triggers second messenger cascades, as well as engaging in conventional ionotropic signaling. The increasing activities thought to be mediated by KARs through G protein activation have not only firmly established the metabotropic mode of action of KARs but also, they indicate the functional relevance of such noncanonical signaling in the role played by KARs in the physiology of the nervous system. It is now clear that this non-canonical signaling is elicited directly by KARs and that it is independent of the ionotropic activity, constituting a breakthrough in our understanding of KAR function. This dual signaling capacity of KARs further emphasizes their prominent role in modulating network activity, highlighting the distinct and diverse mechanisms that KARs use to modulate synaptic function. Postsynaptically, in addition to mediating part of the synaptic current at certain central synapses, KARs regulate neuronal excitability through the modulation of after-hyperpolarizing currents by G-proteins activation. Presynaptically, KARs can bi-directionally modulate the release of GABA and glutamate. This bimodal activity is probably supported by the dual signaling ability of KARs, and it is appears that while facilitation is due to ionotropic activity, the inhibitory actions of KARs are linked to the non-canonical signaling. Together, these actions indicate that KARs participate in various mechanisms to control the excitability of neuronal networks (Figure 1). How all these actions are coordinated under conditions of physiological receptor activation, as well as the behavioral consequences of these effects, remain a major barrier to fully comprehending the role of KARs in the CNS. What is now clear is that KARs can no longer simply be considered as ligand-gated ion channels and hence, defining the metabotropic component of this KAR-operated signaling system is inherent to understanding the physiological contributions of glutamate receptors. In this regard, we anxiously await information as to how KARs activate G-proteins in the absence of ionotropic activity. Indeed, evidence for a physical interaction between KARs and G-proteins is still intriguingly scarce and perhaps, at present it is more important to determine if the activation of G-proteins by KARs involves a direct interaction or if intermediates are involved. The recent identification WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Volume 1, July/August 2012

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