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Interactions between Ligand-Gated Ion Channels: A New Regulation Mechanism for Fast Synaptic Signaling?
Eric Boué-Grabot
To cite this version:
Eric Boué-Grabot. Interactions between Ligand-Gated Ion Channels: A New Regulation Mechanism for Fast Synaptic Signaling?. Amino Acid Receptor Reseach, 2008, Amino Acid Receptor Reseach, 978-1-60456-283-5. �hal-01146669�
Interactions between ligand-gated ion channels: a new regulation mechanism for fast synaptic signaling ?
Eric Boué-Grabot
Université Bordeaux 2, CNRS, UMR 5227, 33076 Bordeaux cedex, France
Address correspondence: Dr. Eric Boué-Grabot, e-mail: eric.boue-grabot@u-bordeaux2.fr, CNRS UMR 5227, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France, Tel.
+33 (0)5 5757-1686; Fax. +33 (0)5 5690-1421;
Acknowledgements: Our research is supported by CNRS, Université Bordeaux 2 and by the ANR grant # JC-05-44799.
ABSTRACT
Transmitter-gated ion channels are integral membrane protein complexes that modulate synaptic neurotransmission directly through the binding of a transmitter and the opening of a pore permeable to specific ions. Ligand-gated channels can be divided in three major families: the cys loop receptors including nicotinic, GABA, glycine and 5-HT3A receptors, glutamate-gated channels (AMPA, NMDA and kainate) and ATP P2X receptor-channels. In view of the clear structural differences between ligand-gated channel families, it has been assumed that each receptor type acts independently of the other. However, recent studies have challenged this principle of independence by showing that the co-activation of P2X and nicotinic receptors induces a current that is less than the sum of currents induced by applying the two transmitters separately. Activity-dependent cross-inhibition was also observed between P2X2 and 5-HT3A or between several P2X and ionotropic GABA receptors. The close proximity of P2X2 and 42 nicotinic channels, the physical association between P2X2 and 5-HT3 or GABA gated-channels as well as the involvement of the intracellular domain of both receptors strongly suggested that a molecular coupling underlies their activity-dependent cross-inhibition. In addition, the interaction between ATP and GABA-gated channels may also regulate receptor trafficking and targeting.
However, the functional interaction between distinct ligand-gated channels appears to be a complex molecular process and the identification of the precise underlying mechanisms and regulatory factors requires further studies. Asymetrical cross-inhibition has also been observed between AMPA and NMDA receptors and between GABAA and glycine receptors, although in the latter case, the interaction was dependent on intracellular phosphorylation pathways triggered by glycine receptor activation. Therefore, interactions between distinct ligand-gated channels
represent a new mechanism for receptor regulation, which may be essential for the integration of fast synaptic signaling.
Synaptic transmission between neurons is achieved through the release of one or more neurotransmitters or modulators from the same presynaptic terminal, resulting in the activation of different classes of receptors co-localized at the same post-synaptic site.
Receptors are classified by their transduction mechanisms into two main families: G protein- coupled receptors (GPCRs) and ligand-gated ion channels (LGICs). In response to the binding of the ligand, GPCRs mediate their activation through the activation of G proteins which engage second messenger pathways; whereas ligand-gated channels lead to the opening of a central pore permeable to selected ions. GPCRs are typically monomeric proteins with seven transmembrane domains, an extracellular N-terminal domain and an intracellular COOH-terminal domain.
Genome and cDNA sequencing analysis has revealed that there is three major families of ligand- gated channels, each with an unique architecture. Members of the nicotinic acetylcholine receptor family - also called “cys loop” receptors - include cationic receptor-channels for acetylcholine (Ach), serotonin (5-HT) and anionic receptor-channels for -aminobutyric acid (GABA) and glycine (Sine and Engel, 2006). These receptors are heteropentamers of subunits with four transmembrane domains (TM), a large extracellular domain, an intracellular loop between TM3 and TM4, and a short C terminal tail. Glutamate receptors are cation-selective channels divided into AMPA, NMDA and kainate receptors formed by a tetrameric association of subunits with three transmembrane domains and a reentrant loop between TM1 and TM2 (Mayer, 2005). ATP- and proton-gated-channels constitute the recently discovered receptor family. Althought these are unrelated in terms of sequences, both channel families consist of trimeric homo- or heteromeric
association of subunits with two transmembrane domains, a large extracellular domain, and both intracellular NH2 and COOH termini (Khakh et al., 2001)(Fig. 1).
In view of the clear structural differences between neurotransmitter-gated channels, it was assumed that each receptor type acts independently of the other. However the principle of independence has now been challenged by several studies that provide strong evidence for functional interactions between several distinct ligand-gated channels.
Nakazawa et al first observed in rat phaeochromocytoma (PC12) cells, that the combined effect of ATP and nicotine was less than the linear sum of the individual ATP and nicotinic currents and initially proposed that ATP and nicotine activated the same channels formed by an association of P2X and the nicotinic subunit around the same pore (Nakazawa et al., 1991). Cross-inhibition between P2X and the nicotinic receptor was confirmed by several groups studying sympathetic, myenteric neurons and in oocytes co-expressing P2X2 and 34 nicotinic receptors (Barajas- Lopez et al., 1998; Khakh et al., 2000; Searl et al., 1998; Zhou and Galligan, 1998). Together these studies clearly demonstrated that P2X2 and nicotinic receptors form separate channels and that the co-activation of both receptors results in non-additive responses owing to an inhibition of both channel types. The authors also found that the current inhibition was bidirectional and did not involve ligand binding sites, a calcium-dependent mechanism, a change in the driving force, or a cytoplasmic signaling mechanism. In addition, decreasing the level of expression of P2X2 and 44 nicotinic receptors resulted in additive responses suggesting that a close proximity of the receptors is required for the inhibition (Khakh et al., 2000). Similar inhibitory cross-talk was also demonstrated between P2X2 and 5-HT3, and between P2X2 and GABAA/C receptor-channels through co-expression studies and in native myenteric neurons (Fig. 2) (Boue-Grabot et al., 2003;
Boue-Grabot et al., 2004a; Boue-Grabot et al., 2004b). The investigation of P2X2 and GABAA receptor co-activation revealed that direction of the inhibition was dependent on a specific GABAA subunit composition (Boue-Grabot et al., 2004b). In cells expressing GABA receptors containing and subunits, the channels showed reciprocal inhibition, i.e. non-additive responses were recorded when ATP and GABA were co-applied, when GABA was administered during exposure to ATP, or when ATP was administered during exposure to GABA. In oocytes expressing , and GABAA receptor subunits, GABA inhibited the response to ATP whereas ATP did not inhibit the response to GABA. Regardless of the GABA receptor composition, current occlusion between P2X2 and GABAA/C receptors was independent of the ion flow direction, calcium or voltage, thereby suggesting that a molecular process was involved.
Co-purification experiments indicated that P2X2 interacts physically with 5-HT3 and 1 GABAc receptor channels (Boue-Grabot et al., 2003; Boue-Grabot et al., 2004a). In addition, the co- transfection of 1 and P2X2 receptors revealed a co-clustering of these receptors in transfected hippocampal neurons and that P2X2 receptors modified the addressing of GABAC receptors in a dominant way by inducing their translocation from an internal vesicular compartment to surface clusters common to both receptors. The close spatial arrangement between P2X2 and 42 nicotinic receptors in transfected hippocampal neurons was also demonstrated by fluorescence resonance energy transfer analysis using fluorescent-tagged subunits (Khakh et al., 2005), suggesting that activity-dependent cross-inhibition between P2X2 and “cys loop” receptors including nicotinic, 5-HT3 or GABA receptors may arise from molecular interactions.
Indeed, it was recently discovered that G-protein coupled receptors can interact directly with ligand-gated channels leading to a functional and reciprocal modulation of each receptor type.
Such a direct interaction was first demonstrated between dopamine D5 and GABAA receptors.
Through a series of biochemical approaches, Liu et al. convincingly demonstrated that the intracellular COOH-terminal tail of the D5 receptor specifically binds directly to the main intracellular loop of the GABAA 2 subunit (Liu et al., 2000). D5 receptor activity was downregulated upon GABA receptor activation and the reciprocal activation of D5 receptors reduced the amplitude of GABA-induced currents. The reduction was abolished with the intracellular administration of peptides corresponding either to the D5 receptor or 2 interacting domains showing that their physical coupling underlied the functional cross-modulation. A direct interaction was also demonstrated between D1 and NMDA receptors (Lee et al., 2002; Pei et al., 2004). In this case, the authors identified not one but two distinct sites of interaction: one between the C-terminal domain of the D1 and NR1 subunits and a second between the C-terminal domain of D1 and NR2A with different consequences for NMDA receptor function. By similar approaches, theses authors have also shown that the interaction between D1 and NR2A leads to an inhibition of the NMDA-induced current by D1 stimulation, whereas the interaction between D1 and NR1 seems to suppress NMDA receptor-mediated cell death upon D1 activation.
Reciprocally, NMDA receptor activation increased the number of D1 receptors on the plasma membrane surface, and consequently enhanced the response to dopamine.
The first evidence that molecular interactions lead to the functional cross-talk between ligand- gated channels came from experiments using a C-terminal truncated form of P2X2 subunits.
Truncation of this domain abolishes the functional cross-talk between P2X2 and 5-HT3, nicotinic or GABA receptor-channels (Boue-Grabot et al., 2003; Boue-Grabot et al., 2004a). Likewise, expression of a minigene encoding either the distal C-terminal portion of the P2X2 receptor or the main intracellular loop located between the third and fourth transmembrane domains of 5-HT3 or
1 subunits reduced the current inhibition between P2X2 and 5-HT3 or 1 GABA receptor- channels, respectively. These results indicated that an interaction between the intracellular domains of each receptor is required for the functional cross-talk. For GABAA receptors which comprise heteromeric associations of , or , and subunits, it has been problematic to identify the actual subunits involved in the molecular coupling. This problem has been overcome by using chimeric 1-GABAA receptors with 2, 3 or 2 sequences downstream from the third
transmembrane domain (Boue-Grabot et al., 2004b). Each 1-GABAA receptor forms a functional homomeric receptor similar to the homomeric 1 wild-type. Non-additive responses and co-clustering were observed between P2X2 and 1-3 chimeras, but not with 1-2 or 1-
2, indicating that P2X2 interacts mainly with subunits of GABAA receptors. This is consistent
with FRET experiments between P2X2 and 42 nicotinic receptors which showed a higher fluorescence transfer efficiency between P2X2 and 2 subunits, rather than 4 subunits (Khakh et al., 2005).
Although, most research has focused on the interaction of native and recombinant P2X2 subtypes, several P2X channels have the ability to interact functionally with distinct ion channels. A cross- inhibition between P2X3 or P2X2/3 and GABAA was described in dorsal root ganglia neurons but, in constrast to other studies, the authors suggested an ion-dependent mechanism (Sokolova et al., 2001). It was proposed that chloride efflux through GABA-gated channels inhibits P2X channels and conversely, calcium influx though P2X inhibits GABAA receptors. This was in contrast to studies involving P2X2 and “cys loop” receptors in particular with experiments showing that cation-selective GABAA receptor-channels, generated by a mutation of 3 subunits, did not prevent functional cross-inhibition with P2X2 (Boue-Grabot et al., 2004b). These findings clearly demonstrated that chloride is not involved in this coupling, although it could be argued that P2X2
or P2X3 interact with other channels by distinct mechanisms. Recently Toulmé et al. (2007) provide convincing evidence for a molecular interaction between P2X3 and GABAA receptors that was also responsible for the ion-independent cross-inhibition in both dorsal root ganglia neurons and a recombinant expression system (Toulme et al., 2007). The authors identified a specific intracellular motif of three consecutive amino-acids QST at positions 386-388 in the C- terminal tail of P2X3 subunits required for the functional coupling with GABAA receptors. The current occlusion between native P2X3 and GABAA receptors in DRG neurons was abolished either by infusion of a peptide containing the QST motif or a viral infection of the main intracellular loop of GABAA 3 subunits.
Together, these results strongly suggest that a molecular interaction between P2X and members of “cys loop” receptors leads to an activity-dependent cross-inhibition by a conformational spread mechanism in which the motion triggered by the gating of one channel type is communicated to the other channels and induces its closure (Bray and Duke, 2004).
However, presently, there is no clear evidence of a direct interaction between the intracellular domains of individual channels, and several findings also argue in favor of the involvement of protein complexes to promote an association between distinct ligand-gated channels (Fig. 2).
The fact that ATP and ACh were shown to operate through independent receptors with an additive response in visceral ganglia neurons (Reyes et al., 2006) or that nicotinic and P2X2 receptors were closely localized in hippocampal neurons but not in ventral midbrain neurons (Khakh et al., 2005), indicates that interactions between distinct ligand-gated channels may involve regulory factors.
It is also unlikely that such an interaction is direct, due to the fact that the interacting region of
“cys loop” receptors (nicotinic, or 1 GABA and 5-HT3 subunits) shows no primary sequence homology. Similarly, the COOH-terminal sequence of P2X subunits are of varying length and sequence. Indeed the QST interacting motif within the tail of P2X3 subunits is absent from other P2X subtypes including receptor-interacting P2X2.
It is now clear that neurotransmitter receptors are involved in a complex interplay with numerous submembranal protein networks that regulate their function or trafficking (Collins and Grant, 2007), this therefore raises the possibility that interactions between receptor-associated proteins promotes the physical and functional coupling between distinct ligand-gated channels.
Recent studies have also provide evidence for activity-dependent cross-talk between two different members of the “cys loop” receptor family -GABAA and glycine receptors- in spinal cord neurons or GABAA and 5-HT3 receptors in myenteric neurons, and between two different ionotropic glutamate receptors -AMPA and NMDA receptors- in hippocampal neurons (Bai et al., 2002; Li et al., 2003; Miranda-Morales et al., 2007). Inhibition of GABA-evoked currents by glycine receptor activation as well as inhibition of NMDA receptors by activation of AMPA receptors are both mediated by non-ionotropic mechanisms. The mechanism of the interaction between GABA and glycine is however different and involves the state of phosphorylation of the GABAA receptor and/or mediator proteins. Cross-talk between distinct glutamate receptors has been found G-protein or protein kinase independent, and so it would be interesting to determine if direct or indirect interactions occur between these channels.
Taken together, these data demonstrate that the concept of functional independence might not be true for many receptors and that there is a complex interplay between fast neurotransmitter receptors in the nervous system. The physiological relevance of this new level of regulation at the synaptic level remains to be established. However, because fast neurotransmitters such as GABA/glycine (Jonas et al., 1998), ATP/acetylcholine (Galligan and Bertrand, 1994; Redman and Silinsky, 1994) or ATP/GABA are co-released in the nervous system (Jo and Role, 2002; Jo and Schlichter, 1999), interactions between their respective receptor-channels may exert powerful and rapid receptor modulation which may be essential for the integration of synaptic signals.
FIGURE LEGENDS
Figure 1 : Topology of the different ionotropic receptor subunits.
Figure 2 : Functional and molecular interactions between P2X2 and GABAA-gated channels.
A. Whole-cell current responses recorded in oocytes co-expressing 23 GABAA and P2X2 receptors. Co-application of ATP + GABA (100 M each) induced currents (Actual) significantly smaller than the sum (Predicted) of the individual ATP and GABA responses (from Boue-Grabot et al., 2004). B. Direct or indirect interactions between the C-terminal tail of P2X subunits and the main intracellular loop of cys loop receptor subunit underly the current inhibition.
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