Structural model of a G protein-binding site within GlyR and a 7 nAChR. A: Effect of G a o and G bg on nAChR channel open probability in rat intrinsic cardiac ganglia neurons (mean Æ SEM; ÃÃ p < 0.01). (Adapted from [58, 68]). B : A structural model of a subunit for the human GlyR and a 7 nAChR. For the GlyR, NCBI: NP_001139512.1 was used as a query sequence, whereas the sequence NCBI: NP_000737.1 was used for the a 7 nAChR in addition to PDB: 2BG9 chain A as a template. C -scores of À 1.00 and À 2.20 were obtained for the best GlyR and a 7 nAChR I-TASSER models, respectively. The loop segments were generated using LoopyTM [22] and then energetically filtered to the top 10 candidates using Dfire [91]. A top conformation is presented. The nAChR and GlyR structures show proximity of ARG residues 344 ( Ã 16.1 A ) and 347 ( ÃÃ 15.8 A ), which are known to be involved in the G bg binding of the GlyR [75]. LYS residues 421 and 422 within the GlyR are also known to contribute to G bg binding. 

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... alignment of the a 7 nAChR and the GlyR subunits using TM-Align [83] confirms the structural homology between the two receptor subunits. The structural data support our hypothesis and suggest that nAChRs and GlyRs bind G bg via similar features of the M3–M4 loop. At this point, however, it is not clear whether these residues contribute to G protein binding. This question can be addressed directly in future studies using site-directed mutagenesis to alter the nAChR peptide sequence at these specific sites. If the residues contribute to G bg interaction with the nAChR, a next step would be to investigate if each receptor subunit can bind a G bg or do multiple nAChR subunits contribute to G bg association. Studies of G bg interactions with GIRK channels suggest that only one G bg binds to the tetrameric GIRK channel [84]. If this is also the case for the nAChR, it may explain the incremental potentiation of the nAChR initiated by adding G bg in patch clamp experiments [85]. While nAChRs are an important class of ion channels that modulate neuronal activity, evidence now suggests that they function by also turning on and off longer-lived cellular signaling events. This notion of metabotropic signaling through an ion channel surpasses the limited view that these receptor channels operate solely through ligand driven ion conduction. In non-neuronal cells such as immune cells, nAChRs can regulate inflammatory responses in the absence of a measured electrochemical signal [71]. Binding to the cellular signaling machinery is a fundamental new perspective on the function and regulation of nAChRs in neurons and other cell types. In a recent study, we demonstrated the existence of an a 7 nAChR/GPC comprising the scaffold protein Gprin1, G a o and growth associated protein 43 (GAP-43) in developing neural cells [60]. Using protein cross-linking, proteomic analysis, and immunoprecipitation methods, we isolated and characterized the functional dynamics of the a 7 nAChR/GPC complex. We also identified that a 7 nAChR receptor activation (by ACh as well as nicotine) is associated with receptor interaction with G a o and Gprin1 (Fig. 5). In the ligand activated state, the a 7 nAChR receptor is prefer- entially bound to G a o [GDP], whereas in the inactivate state, the receptor associates with G a o [GTP]. Experiments using the G a o activator mastaporan and the G a o inhibitor pertussis toxin confirm an effect of G a signaling on neurite growth (Fig. 5) [60]. Interestingly, binding to G proteins appears central for a 7 nAChR mediated effects on neurite growth. This signaling pathway is driven via the ability of GAP-43 to regulate G proteins and the assembly/disassembly of the axon cy- toskeleton. G a o in particular is enriched in the growth cone [86]. Thus by directly coupling to G proteins, the a 7 nAChR signals to regulate axon growth. While it is interesting to consider that G protein signaling via the a 7 nAChR can occur simultaneously with ion conduction, the kinetics of a 7 channel activation and deactivation are dramatically faster than those of the G protein signaling cycle [6]. Current data thus allows for an intracellular signaling mechanism of the nAChR independent of ion conduction, while suggesting that calcium influx through the open channel can also contribute to longer-lived G protein signaling. This is supported by the finding that the phosphorylation of GAP-43 by the calcium sensor calmodulin kinase II is at least in part driven by a 7 nAChR calcium entry into the neurite [60]. The emergence of protein-protein interaction domains in various molecules is suggested to be one way in which evolution accommodates adaptations in cellular signaling [87]. For various nAChRs interaction with G proteins appears to be a functional metabotropic component of the channel response, alongside its ionotropic function. The evidence put forth here is compelling and provides a new testable framework for exploring G protein interaction with nAChRs. Future experiments based on the construction of nAChR mutants with specific site directed mutations of the proposed G protein binding residues and their analysis in electrophysiologi- cal and biochemical assays will provide information on the role of G proteins in nAChR function. In the brain, nAChRs have been found in presynaptic terminals, postsynaptic compartments, and in various other non-synaptic regions of the cell [69, 70]. Pre-synaptic receptors regulate neurotransmitter release [71], while post-synaptic receptors contribute to plasticity and neuronal excitabil- ity [72]. While nAChR signaling capacity is influenced by subtype dependent desensitization to ACh [72], regulation by G proteins may modify receptor activity and critically amplify nAChR signaling within the cell. The computational models provided on the structure of the M3–M4 loop in the nAChR support intracellular loop localization but point to a structure capable of some degree of spatial mobility at equilibrium (Fig. 4). Because the presented models are based on the predicted conformation of an individual receptor subunit, the pentameric assembly of the nAChR may facilitate loop-loop interactions in the final tertiary protein. Interestingly, binding to G proteins, and or being in proximity of other cellular binding partners, may also influence the conformation and function of the M3–M4 loop in the cell [88]. This work was supported by a Jeffress Memorial Grant Award J-953 to Nadine Kabbani and an Australian Research Council (ARC) grant DP1093115 and ARC Australian Professorial Fellowship to David J. Adams. The authors have declared no conflict of ...