Insights & Perspectives Hypotheses Are nicotinic acetylcholine 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 neurons. Based on proteomic, evolutionarily conserved class of cys- biophysical, and functional evidence, we hypothesize that binding to G loop containing receptor 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 ion channel; heteromeric or homomeric receptors [6] loop modeling; protein interaction; signal transduction (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 14À3À3, and of neurotransmitter 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 Bioessays 35: 1025–1034, ß 2013 WILEY Periodicals, Inc. www.bioessays-journal.com 1025 N. Kabbani et al. Insights & Perspectives..... 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 axons and dendrites [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 eukaryotes. 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 neurotransmission 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, 1026 Bioessays 35: 1025–1034, ß 2013 WILEY Periodicals, Inc. .....Insights & Perspectives N. Kabbani et al. 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