Insights & Perspectives Hypotheses

Are nicotinic receptors coupled to G proteins?

Nadine Kabbani1)*, Jacob C. Nordman1), Brian A. Corgiat1), Daniel P. Veltri2), Amarda Shehu2), Victoria A. Seymour3) and David J. Adams3)

It was, until recently, accepted that the two classes of acetylcholine (ACh) synaptic plasticity [2, 3]. Mutations receptors are distinct in an important sense: muscarinic ACh receptors within nAChR genes are implicated signal via heterotrimeric GTP binding proteins (G proteins), whereas in a number of human disorders þ 2þ þ including drug addiction and schizo- nicotinic ACh receptors (nAChRs) open to allow flux of Na ,Ca ,andK phrenia [4]. ions into the cell after activation. Here we present evidence of direct coupling Nicotinic receptors belong to an between G proteins and nAChRs in . Based on proteomic, evolutionarily conserved class of cys- biophysical, and functional evidence, we hypothesize that binding to G loop containing channels that proteins modulates the activity and signaling of nAChRs in cells. It is includes GABAA,glycine,and5HT3 important to note that while this hypothesis is new for the nAChR, it is receptorsaswellastwonewlydiscovered channels: a zinc-activated channel and consistent with known interactions between G proteins and structurally an invertebrate GABA-gated cation chan- related ligand-gated ion channels. Therefore, it underscores an evolution- nel [5]. In mammals, genes encoding arily conserved metabotropic mechanism of G protein signaling via nAChR neuronal nAChR subunits have been channels. identified and labeled a (a1–a10) and b (b1–b4). Functional nAChRs are derived Keywords: from an arrangement of five subunits into .acetylcholine; G protein coupling; intracellular loop; ligand-gated ; heteromeric or homomeric receptors [6] loop modeling; protein interaction; (Fig. 1A). The activity of nAChRs also appears driven by direct protein-protein associations with molecules such as receptor kinases, scaffolds, and signaling Introduction motion a slower chemical signaling effectors [7]. A growing list of proteins has cascade via the binding and activation emerged as components of the nAChR of heterotrimeric GTP binding proteins signaling network. This list includes It is often said that two main types (G proteins) following ligand activa- scaffold proteins such as 1433, and of receptors exist – tion [1]. Neuronal nicotinic acetylcholine the calcium sensor visinin like protein- ionotropic ligand-gated ion channels receptors (nAChRs) are a subdivision of 1 [8, 9]. In this article, we discuss findings (LGICs), which permit rapid ion flow LGICs widely distributed in nervous on associations between nAChRs and directly across the cell membrane, and tissue and contribute to processes G proteins. These findings support the metabotropic receptors, which set in such as neurotransmitter release and hypothesis that nAChRs couple to G proteins at the plasma membrane.

DOI 10.1002/bies.201300082 Evolutionary emergence

1) Department of Molecular Neuroscience, *Corresponding author: Nadine Kabbani of an intracellular protein- Krasnow Institute for Advanced Study, Fairfax, E-mail: [email protected] protein interaction domain VA, USA 2) Department of Computer Science, George Abbreviations: in nicotinic receptors Mason University, Fairfax, VA, USA ACh, acetylcholine; GPCR, G protein coupled 3) Health Innovations Research Institute, RMIT receptor; LGIC, ligand-gated ion channel; University, Melbourne, VIC, Australia nAChR, nicotinic acetylcholine receptor; TM, Nicotinic receptor subunits share a transmembrane. topology that consists of a large

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sequence analysis reveals a key difference between prokaryotic and eukaryotic channel composition at the intracellular domain. Notably, the marked absence of the M3–M4 loop in GLIC suggests a difference in the cellular activity between GLIC and mammalian nAChRs (Fig. 2A) [13]. Without an M3–M4 loop the GLIC channel is likely unable to participate in the myriad of intracellular interac- Hypotheses tions characteristic of eukaryotic nAChRs. Protein interactions and post- translational modifications of the M3–M4 loop are now established functional features of nAChR function in cells. For example, specific residues in the M3–M4 loop of the a4b2 nAChR play a vital role in trafficking the receptor to the cell surface (Fig. 2B) [16]. Other residues in the M3–M4 loop target the nAChR to functional domains such as and [17, 18]. Lastly, a number of serine/threonine and tyrosine residues throughout the M3–M4 loop contribute to receptor Figure 1. nAChR Structure and function. A: The nAChR as viewed from above shows five kinetics and gating when phosphorylated subunits arranged around a central cation-conducting pore. A ligand-binding site is formed [19]. It seems likely that most, if not at the interface of two subunits. B: An illustration of a single nAChR subunit embedded in all, of the intracellular protein binding the membrane. C: The protein structure of the pentameric nAChR obtained from T. of the nAChR evolved through the marmorata (PDB 2BG9) in the plasma membrane. Shown are the location and function of the major receptor domains [13]. A single subunit is highlighted in purple using visual emergence of an M3–M4 loop in molecular dynamics (VMD) [89]. Mutations in the membrane and intracellular regions of the . human nAChR are shown [90]. The absence of an M3–M4 loop in the recently discovered GLIC protein crystal structure [13] leaves a knowledge gap in our structural understanding of extracellular N-terminal domain, four Fast, ionotropic nAChR intracellular protein interac- transmembrane (TM) domains, and a mediatedbyLGICsisessentialfor tions. In Fig. 1C, we present a structural single large intracellular loop located survival responses in multicellular model of the nAChR based on the between TM domains 3 and 4 (M3–M4; organisms [12]. Among the penatmeric available crystal structure of the muscle Fig. 1B). In most nAChRs, the M3–M4 LGICs, cys-loop receptors make up a nAChR from Torpedo marmorata [20]. loop contains 100 amino acid residues subfamily previously thought only to This model is obtained through the I- and shares low sequence homology existineukaryotes,butwhichwas TASSER structure prediction server [21] with other nAChRs [6]. Based on site recently found in prokaryotes [13]. and allows us to computationally esti- directed mutagenesis studies, the M3– Studies on the prokaryotic origin of mate a conformation for the M3–M4 M4 loop is found to mediate important cys-loop receptor channels reveal loop that was absent in the reported receptor properties such as export from that a functional, cation-conducting crystal structure [20]. In Fig. 2A, we use the endoplasmic reticulum (ER) and nAChR homolog exists in several LoopyTM [22] and JACKAL software [23] trafficking to the plasma membrane bacterial species and an archaea to propose a more probable structure for (Fig. 1C) [10]. In contrast, the acetylcho- genus [14]. The prokaryotic homolog, the human a7 nAChR. This type of line (ACh) binding site is highly con- Gloeobacter violaceus LGIC (GLIC), analysis generated a series of energeti- served and is formed extracellularly binds extracellular protons instead of cally favorable M3–M4 loop structures at the interface of two a subunits or ACh, but maintains most of the mem- for the human nAChR. One such one a and one b subunit (Fig. 1A). brane sensitive structural and bio- structure is presented in Fig. 2A. It is Extracellular binding of ligands, ago- physical properties of the eukaryotic not unlikely that this M3–M4 loop exists nists or antagonists, and allosteric nAChR [14, 15]. Electron microscopy in several structural conformations, modulators determines the conforma- and protein cross-linking experiments all of which were found to extend tion of the nAChR: basal, active, or confirm a homopentameric organiza- into the cytoplasm of the cell. Based desensitized [11]. tion of the GLIC protein. However, on this structural modeling evidence,

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a,5b, and 12 g subunit G proteins. The structurally diverse a subunits are grouped into functional families on the Hypotheses basis of how they signal: Gas increases cAMP synthesis, Gai inhibits it, Gaq and Ga11/12 couple to phospholipase C to release IP3 and diacylglycerol, and Ga12/13 signals via Rho kinases. Diversity within bg subunits appears to have arisen later in evolution [25]. Based on early studies using non- hydrolyzable GTP analogs, cholera toxin and a mutants, it was originally thought that heterotrimeric G protein activity required a conformational change that led to a physical dissocia- tion of the a subunit from the bg subunits. However recent work using FRET imaging and chemical crosslink- ing shows that dissociation of the subunits may not be needed for signaling [26]. The bg complex may remain associated with the a subunit and still allow for G protein signaling in the cell [27]. Indeed, a trimeric G protein appears able to bind and modulate effector targets just as well [28]. How G proteins recognize their cellular partners is not well understood. Compartmentalization of G proteins within membrane regions such as lipid rafts and focal adhesions plays an important role in localizing the G protein in the vicinity of its targets [29]. Mutagenesis studies also show that amino acids in a protein give informa- Figure 2. Evolution and structure of the nAChR. A: From left to right: First image, the tion for the binding of specific G structure of the crystallized prokaryotic GLIC channel protein [13] within the membrane. proteins [30]. An important body of Second image, a single subunit within the GLIC channel protein is shown in purple. Third image, homology modeling using I-TASSER was used to confirm the structure of a GLIC literature exists on the regulation of G subunit. Fourth image, homology modeling using I-TASSER was used to predict the protein interaction with GPCRs. The structure of the human a7 nAChR (NCBI: NP_000737.1 was used as sequence template) [21, evidence indicates that specific residues 89]. This computational model suggests an intracellular M3–M4 loop structure for the human in the GPCR intracellular loops are a7 nAChR (dotted box). The loop conformation was specifically modeled de novo using the critical for G protein binding and LoopyTM loop software [22]. Of the 100 possible conformations, the top 15 candidate loop recognition [31]. In the example of D2 structures were identified using Dfire followed by JACKAL softwares [23, 91, 92]. A receptors, the Ga activation representative M3–M4 loop conformation in the human a7 nAChR is presented. B:A i multiple sequence alignment of the M3–M4 loop for various nAChRs as well as the human site is found near the plasma membrane GlyR. Amino acids that contribute to Gbg binding to the GlyR are shown [75]. Conserved and can also bind calmodulin, suggest- and similar residues are indicated. ing that G protein-receptor interactions are influenced by association with other we predict an evolutionary emergence G proteins regulate the proteins [32]. of an intracellular protein-binding in- signaling of various G protein binding has also been terface at the M3–M4 loop of the nAChR. studied in a number of ion channels. In The M3–M4 loop region of eukaryotic receptors at the plasma voltage-gated calcium channels such as nAChRs may also contain secondary membrane Cav2 channels, up to two distinct structures formed by associations of the interaction sites for Gaq and Gai/o and individual M3–M4 loops in the channel Heterotrimeric G proteins (G proteins) several binding sites for Gbg have been pentamer (Fig. 1C). This protein-binding serve as molecular switches for various discovered in a single channel [33]. interface is a focal point for interaction GPCRs and a growing list of ion channels A consensus QXXER and a G protein between nAChRs and the signaling at the plasma membrane [24]. The interaction domain (GID) sequence elements of the cell. genome of Homo sapiens contains 23 appears to dictate G protein interaction

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Figure 3. G protein pathways regulate phosphorylation or dephosphorylation proteins in Src-family kinase mediated nAChR function. A summary of the major affect its location in the cell, ability to regulation of nAChRs (Fig. 3). interactions between nAChRs and G protein bind proteins, and degradation [34]. signaling pathways in neural cells. In addi- Most nAChRs contain at least one tion to binding G proteins, nAChRs can associate with GPCRs and G protein-gated phosphorylation site within the M3– Nicotinic receptors interact ion channels such as GIRK1 at the plasma M4 loop [19]. A cohort of kinases with GPCRs membrane. A series of residues within the linked to G protein signaling phosphor- M3–M4 loop of the nAChR confer channel ylates and directly regulates nAChRs. G protein coupled receptors (GPCRs) regulation by various G protein kinases To date, this includes Abelson family are an important gene superfamily (dotted lines). kinases (AFK), cAMP activated protein making up >4% of the human kinase A (PKA), protein kinase C genome [48]. This family of receptors with the calcium channel [33]. An (PKC) [35], and the Src family kinase can transmit information from numer- integrated view of G protein-target (Src) [36–39]. ous extracellular ligands into the cell interactions based on studies of ion Gas and Gai proteins are major via a G protein chemical signaling channels and GPCRs suggests that G activators of signaling pathways for cascade [49, 50]. A newer notion protein interaction is influenced by the non-receptor tyrosine kinase Src in in receptor biology is that GPCRs several factors: (1) structural preserva- the cell. In addition to directly binding assemble into higher order receptor tion and molecular access to a G G proteins, this class of enzymes is networks (multimers) consisting of protein-binding pocket or domain; increasingly recognized for its ability to GPCRs and their functional part- (2) a conducive proximity between modulate the function of LGICs such as ners [51, 52]. According to this theory, the G protein and the target receptor; nAChRs. Studies show that Src-family GPCRs not only bind to receptors, (3) simultaneous association with a kinases bind and phosphorylate tyro- but can also associate with structurally mutual scaffold or adaptor that facili- sine residues in the M3–M4 loop region diverse ion channels including LGICs tates the formation of the G protein of several nAChRs (Fig. 3) [40–42]. In [53, 54]. In this regard, coupling to complex (GPC) in the cell. bovine adrenal chromaffin cells, Src has GPCRs may facilitate interactions be- been found to form large multimeric tween ion channels and G proteins in complexes with nAChRs [43] and regu- the cell. Hypothesis: Nicotinic late ACh- and nicotine-induced cate- Functional and biophysical associ- receptors couple to cholamine secretion [44, 45]. Wang ations between GPCRs and LGICs in G proteins et al. [39] demonstrate that a3b4a5 neurons are well documented [55]. nAChRs in chromaffin cells and a3b4a5 In hippocampal neurons, a GABAA/D5 nAChRs in HEK cells can be phosphory- dimer is formed Nicotinic receptors are lated via c-Src. Several Src-family kin- through the binding of the second regulated by G protein ases including c-Src, Fyn, and Lyn intracellular loop of the GABAA channel activated kinases positively regulate a3b4 nAChRs [46] with the carboxy terminal tail of the and negatively regulate a7 nAChRs (see D5 dopamine receptor. This GABAA/D5 Conformational changes in the struc- Box 1) [36, 47]. In light of these findings, dimer is found to direct dopaminergic ture of the receptor as a result of it is interesting to consider the role of G modulation of inhibitory transmission

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This b2 nAChR/D2 dopamine receptor Box 1 dimer regulates dopamine release in the striatum and plays a role in nicotine Hypotheses Hippocampal neurons mediated reward behavior in rodents [57]. Co-immunoprecipitation experi- The a7 nAChR is phosphorylatd by c-Src at residues Y386 and Y442 within the ments confirmed the interaction be- M3–M4 loop. This mechanism alters the trafficking and the expression of the tween b2 nAChRs and D2 dopamine nAChR at the cell surface [47]. receptors in cells and brain tissue. Complexes of the b2 nAChR and the Parasympathetic ganglion neurons D2 dopamine receptor were also found to contain Ga proteins [58]. However, it VIP potentiation of the a7 nAChR is directly blocked by the application is not clear whether these G proteins of the Ga blocker pertussis toxin as well as the inactive GDP substrate i/o associate with the nAChR or with the GDP-b-S [67]. dopamine receptor or possibly both. Antibodies selective for Ga ,Ga , and Gbg indicate that blocking the activity o i Recent work by our laboratory and of Ga and Gbg is sufficient to entirely abolish the VIP and PACAP induced o others support the possibility of inter- potentiation of the nAChR response [68] action between G proteins and nAChRs Application of GTP-g-S was found to directly increase whole cell current independent of GPCRs [7, 59, 60]. amplitudes of the nAChR in the presence of ACh or nicotine. In inside- out patch recordings, the same application of GTP-g-S resulted in reversible fourfold increase in the open probability of the nAChR channel [58]. Functional interactions between The addition of Gbg resulted in a fivefold increase in the open probability of nicotinic receptors and G the nAChR channel, whereas the addition of Gao alone had little to no effect proteins on channel kinetics [58]. Functional interactions between nAChRs, GPCRs, and G proteins occur in a number of systems. Experiments con- ducted in mammalian parasympathet- in the hippocampus [53]. A similar complex is critical for glutamatergic ic neurons demonstrate a functional functional dimer between the D1 transmission underlying synaptic plas- role for nAChR interactions with dopamine receptor and the NR1 sub- ticity and learning [56]. pituitary adenylate cyclase-activating unit of the NMDA Coupling between D2 dopamine (PAC) and vasoactive intestinal pep- channel has been identified [55]. In receptors and nAChRs has been ob- tide (VIP) (VPAC) receptors and their hippocampal neurons, formation of served at presynaptic terminals of the associated G protein pathways. PAC1 an NMDA/D1 dopamine receptor ventral striatum (Fig. 3 and Table 1). receptors bind pituitary adenylate

Table 1. G protein interactions of the nAChR.

Nicotinic G protein Protein interaction receptor interaction Cell and tissue source detection method Reference a a7Gai/o Whole brain tissue of C57BL6 mice a-Bgtx pulldown, Western blot, [59] LC-ESI MS Cortical neurons and PC12 cells from rat IP, Western blot, LC-ESI MS [60] Gaq/12 Whole brain tissue of C57BL6 mice a-Bgtx pulldown, Western blot, [59] LC-ESI MS Gbg Cortical neurons and PC12 cells from rat IP, Western blot, LC-ESI MS [60] Gprin1 Cortical neurons and PC12 cells from rat IP, Western blot, LC-ESI MS [60] a4b2Gao Intrinsic cardiac ganglia from rat GST–M3–M4 loop pulldown, IP, [58] Western blot Gbg Intrinsic cardiac ganglia from rat GST–M3–M4 loop pulldown, IP, [58] Western blot Gai/o Whole brain tissue of C57BL6 mice IP, MALDI-TOF MS [7] Gprin1 Whole brain tissue of C57BL6 mice IP, MALDI-TOF MS [7] GIRK1 Whole brain tissue of C57BL6 mice IP, MALDI-TOF MS [7] D2R Striatal neurons from rat and HEK cells Transfection, IP, Western blot [57] a3b4a5Gao Intrinsic cardiac ganglia from rat GST–M3–M4 loop pulldown, IP, [58] Western blot Gbg GST–M3–M4 loop pulldown, IP, [58] Western blot

a Interaction between a7 nAChRs and Gai/o were not detected in Fischer et al. [58].

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cyclase-activating polypeptide (PACAP) components of an immunoprecipitated specificity and binding affinity between with high affinity. VPAC1/2, on the other nAChR complex from the adult mouse G proteins and the M3–M4 loop of hand, are activated by the neuropep- brain. A single interaction network of the nAChR. Second, it is tempting to tides VIP and PACAP [61, 62]. In chick the b2 subunit was found to contain speculate that the minimal structure for ciliary ganglion cells that express over 20 new receptor interacting G protein binding exists not within one a7- and a3-containing nAChRs, PACAP proteins [7]. Evident among the inter- but multiple M3–M4 loop segments, only inhibits a7 containing nAChRs [63]. actions was a cohort of G protein which are rendered accessible only in VIP, on the other hand, can direct pathway molecules including Gai/o the fully formed pentameric receptor. the recruitment of both nAChRs to the and the Ga interacting protein G This notion is supported by our obser- cell surface in a G protein dependent protein regulated inducer of neurite vation of the ability to detect Gai/o manner [63–65]. Similar work by outgrowth 1 (Gprin1). In addition, b2 interaction in an immunoprecipitation Hypotheses No¨renberg et al. [66] demonstrated nAChRs associated with the G protein assay of the endogenous b2 nAChR, but that neuropeptide Y inhibits the release coupled, inwardly rectifying, Kþ chan- not in a pull-down experiment of the of catecholamines via G protein activa- nel GIRK1 [7]. M3–M4 loop of the b2 subunit [7]. tion of PKA and phosphorylation of A similar proteomic screen of the nAChRs. a7 subunit uncovered more than 50 An understanding of the mecha- new proteins that bound a7nAChRsin Identification of a G protein- nisms of nAChR regulation by G pro- thecortexofmice[59].Thisscreen binding site within the nicotinic teins in rat parasympathetic neurons revealed a number of shared a7 receptor came from several studies using and b2 nAChR interactions and whole cell patch clamp recordings. highlighted a difference in the interac- Cys-loop LGIC proteins are differentially The findings of these experiments indi- tion network between the two sub- regulated by G proteins [74]. Of note is cate that VIP and PACAP potentiate units. Common to both a7andb2 the (GlyR) because G nAChR mediated currents in a G protein subunits was the ability to physically proteins directly modulate them. An dependent manner (Box 1). Experiments associate with several G proteins important study by Yevenes et al. [75] using the broad G protein agonist GDP- including Ga12,Gai,andGao subunits shows a role for Gbg in regulating the g-S confirm the role of G proteins in the as well as Gaq and Gbg in the brain amplitude of the GlyR in cells. The effect potentiation of ACh-evoked currents in [59]. These findings strongly support of Gbg on the GlyR was phosphoryla- neurons (Box 1) [68]. Taken together, our hypothesis that nAChRs interact tion-independent but highly sensitive to these studies underscore an important with G proteins in neural cells. pertussis toxin. The same study reveals mechanism of G protein interaction and Evidence on direct coupling be- that Gbg enhances GlyR function by regulation of nAChRs in neurons. The tween G proteins and nAChRs also increasing the apparent affinity of the evidence also suggests that nAChR comes from protein pull-down experi- receptor for glycine, as measured by interaction with G proteins is direct ments using a glutathione S-transferase increased channel open probability and selective and is consistent with G (GST) fusion protein of the M3–M4 loop (Fig. 4A) [75]. Exposure to GTP-g-S or protein binding to other LGICs. Because of several nAChRs. These pull-down overexpression of Gbg in the cell can each pentameric nAChR has five poten- studies clearly show that several Ga as strongly potentiate the receptor, which tial G protein (Gbg) binding sites, well as Gbg subunits can bind the M3– is consistent with the effect of Gbg cooperative binding could be responsi- M4 loop segment [58]. Interactions on other ion channels at the plasma ble for the fivefold increase in open between nAChRs and G proteins are membrane [75]. channel probability observed in re- summarized in Table 1. As shown in Basic amino acids such as arginine sponse to Gbg application [58]. In future Table 1, the findings are not entirely (R), histidine (H), and lysine (K) studies it will be important to determine conclusive and based on a limited mediate G protein binding to targets the stoichiometry of Gbg binding to number of published observations. For such as b adrenergic kinases, GIRK 2þ the nAChR. example, Gao appears to interact with channels, Ca channels, and phos- a3, a4, a5, a7, b2, and b3 subunits [58– pholipase C [76–80]. Based on struc- 60], but Gbg can only bind a3, a4, a5, tural mutagenesis, a series of basic Proteomic discovery of a and b2, and not b3, b4, or a7 residues in the M3–M4 loop of the GlyR nicotinic receptor/G subunits [58]. Binding inconsistencies have been found to accommodate Gbg protein-signaling complex between nAChRs and G proteins may interaction [81]. As shown in Fig. 2B, stem from important experimental var- two separate amino acid sequences Isolation and characterization of protein iables within the M3–M4 pulldown (RFRRK and KK) in the M3–M4 loop of complexes from cultured cell or native assay. First, a number of studies are the a1 subunit of the GlyR appear tissue has become common using based on recombinant expression of the necessary for Gbg binding [81]. Muta- methods of immunoaffinity-, ligand-, GST M3–M4 loop fusion protein within tions at these residues not only inhibit and tag-based chromatography fol- E. coli, in which post-translational Gbg interaction with the GlyR, but lowed by mass spectrometry (MS) for modification and protein editing will also change receptor potentiation by proteomic analysis [73]. Recently, matrix not be similar to that in mammalian ethanol [82]. assisted laser desorption/ionization cells. These changes may explain a An alignment of residues in the time of flight (MALDI-TOF) MS identified significant amount of variability in M3–M4 loop of nAChRs reveals similar

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next step would be to investigate if each receptor subunit can bind a Gbg or do multiple nAChR subunits contribute to Hypotheses Gbg association. Studies of Gbg inter- actions with GIRK channels suggest that only one Gbg 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 Gbg in patch clamp experiments [85].

Nicotinic receptor association with G proteins regulates neurite growth 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 chan- nels operate solely through ligand driven ion conduction. In non-neuronal cells such as immune cells, nAChRs can Figure 4. Structural model of a G protein-binding site within GlyR and a7 nAChR. A: Effect regulate inflammatory responses in the of Gao and Gbg on nAChR channel open probability in rat intrinsic cardiac ganglia neurons absence of a measured electrochemical (mean SEM; p < 0.01). (Adapted from [58, 68]). B: A structural model of a subunit for the signal [71]. Binding to the cellular human GlyR and a7 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 a7 nAChR in signaling machinery is a fundamental addition to PDB: 2BG9 chain A as a template. C-scores of 1.00 and 2.20 were obtained new perspective on the function and for the best GlyR and a7 nAChR I-TASSER models, respectively. The loop segments were regulation of nAChRs in neurons and generated using LoopyTM [22] and then energetically filtered to the top 10 candidates using other cell types. Dfire [91]. A top conformation is presented. The nAChR and GlyR structures show proximity In a recent study, we demonstrated of ARG residues 344 ( 16.1 A) and 347 ( 15.8 A), which are known to be involved in the the existence of an a7 nAChR/GPC Gbg binding of the GlyR [75]. LYS residues 421 and 422 within the GlyR are also known to comprising the scaffold protein Gprin1, contribute to Gbg binding. Gao and growth associated protein 43 (GAP-43) in developing neural cells [60]. Using protein cross-linking, proteomic basic amino acids in nAChRs (Fig. 2B). near the plasma membrane and in the analysis, and immunoprecipitation In particular two positively charged vicinity of R344 and R347 in both the methods, we isolated and characterized amino acids (K and R) in the M3–M4 a7 nAChR and the GlyR. Structural the functional dynamics of the a7 loop of the nAChR and GlyR indicate alignment of the a7 nAChR and the nAChR/GPC complex. We also identified sequence conservation at the G protein- GlyR subunits using TM-Align [83] con- that a7 nAChR receptor activation (by binding site. Structural modeling of firms the structural homology between ACh as well as nicotine) is associated the a7 nAChR and the GlyR through the two receptor subunits. The structur- with receptor interaction with Gao and LoopyTM [22] and JACKAL software [23] al data support our hypothesis and Gprin1 (Fig. 5). In the ligand activated suggests that these putative G protein suggest that nAChRs and GlyRs bind state, the a7 nAChR receptor is prefer- binding residues within the M3–M4 Gbg via similar features of the M3–M4 entially bound to Gao[GDP], whereas loop are near the plasma membrane loop. in the inactivate state, the receptor (Fig. 4B) [21, 83]. In particular, amino At this point, however, it is not clear associates with Gao[GTP]. Experiments acids R344 and R347, which mediate whether these residues contribute to G using the Gao activator mastaporan and GlyR interaction with Gbg [75], appear protein binding. This question can be the Gao inhibitor pertussis toxin con- conserved and similarly oriented to addressed directly in future studies firm an effect of Ga signaling on neurite the plasma membrane in the a7 nAChR using site-directed mutagenesis to alter growth (Fig. 5) [60]. and GlyR structures. Residues K421 the nAChR peptide sequence at these Interestingly, binding to G proteins and K422 which also contribute to specific sites. If the residues contribute appears central for a7 nAChR mediated GlyR association with Gbg are also to Gbg interaction with the nAChR, a effects on neurite growth. This signaling

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Figure 5. Interaction with G proteins mediates a7nAChRsignalingduringaxongrowth. the construction of nAChR mutants with GAP-43 is a chief mediator of Gao and cytoskeletal proteins actin and tubulin [60]. A: In specific site directed mutations of the the inactive state, the a7 nAChR associates with Gao (GTP bound), which is associated proposed G protein binding residues with GAP-43 phosphorylation in the growth cone. This process drives cytoskeletal and their analysis in electrophysiologi- assembly and growth. B: Activation of the a7 nAChR, on the other hand, promotes cal and biochemical assays will provide the dephosphorylation of GAP-43 (by the calcium sensor PP2B) and an inhibition of Gao (GDP bound). This leads to cytoskeletal disassembly and collapse of the growth information on the role of G proteins in cone [60]. nAChR function. In the brain, nAChRs have been found in presynaptic terminals, post- synaptic compartments, and in various pathway is driven via the ability of GAP-43 by the calcium sensor calmodu- other non-synaptic regions of the GAP-43 to regulate G proteins and the lin kinase II is at least in part driven by cell [69, 70]. Pre-synaptic receptors assembly/disassembly of the axon cy- a7 nAChR calcium entry into the regulate neurotransmitter release [71], toskeleton. Gao in particular is enriched neurite [60]. while post-synaptic receptors contrib- in the growth cone [86]. Thus by directly ute to plasticity and neuronal excitabil- coupling to G proteins, the a7 nAChR ity [72]. While nAChR signaling capacity signals to regulate axon growth. While it Conclusion is influenced by subtype dependent is interesting to consider that G protein desensitization to ACh [72], regulation signaling via the a7 nAChR can occur The emergence of protein-protein inter- by G proteins may modify receptor simultaneously with ion conduction, action domains in various molecules activity and critically amplify nAChR the kinetics of a7 channel activation is suggested to be one way in which signaling within the cell. The computa- and deactivation are dramatically faster evolution accommodates adaptations in tional models provided on the structure than those of the G protein signaling cellular signaling [87]. For various of the M3–M4 loop in the nAChR cycle [6]. Current data thus allows for an nAChRs interaction with G proteins support intracellular loop localization intracellular signaling mechanism of appears to be a functional metabotropic but point to a structure capable of some the nAChR independent of ion conduc- component of the channel response, degree of spatial mobility at equilibrium tion, while suggesting that calcium alongside its ionotropic function. The (Fig. 4). Because the presented models influx through the open channel can evidence put forth here is compelling are based on the predicted conforma- also contribute to longer-lived G protein and provides a new testable framework tion of an individual receptor subunit, signaling. This is supported by the for exploring G protein interaction with the pentameric assembly of the nAChR finding that the phosphorylation of nAChRs. Future experiments based on may facilitate loop-loop interactions in

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