By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) ␣4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3, known to interact directly with neuronal AChRs. 14-3-3 is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3 interacted with the recombinant ␣4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3 with recombinant ␣4 subunits was abolished when serine 441 of the ␣4 subunit was mutated to alanine (␣4 S441A ). The surface levels of recombinant wild-type ␣4␤2 AChRs were ϳ2-fold higher than those of mutant ␣4 S441A ␤2 AChRs. The interaction significantly increased the steady state levels of the ␣4 subunit and ␣4␤2 AChRs but not that of the mutant ␣4 S441A subunit or mutant ␣4 S441A ␤2 AChRs. The EC 50 values for activation by acetylcholine were not significantly different for ␣4␤2 AChRs and ␣4 S441A ␤2 AChRs coexpressed with 14-3-3 in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native ␣4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of ␣4␤2 AChRs through its interaction with the ␣4 subunit. Neuronal nicotinic acetylcholine receptors (AChR)1 are a family of ligand-gated, cation-selective, homo-or heteropentameric ion channels expressed in the peripheral and central nervous system (1, 2). A multitude of neuronal AChR subtypes assembled from different combinations of ␣2-␣9 and ␤2-␤4 subunits have been identified (3,4 (7), and show attenuated self-administration of nicotine (8) suggesting that ␣4␤2 AChRs have a role in mediating addiction to nicotine. The normal and pathophysiological functions mediated by ␣4␤2 AChRs are of significant importance to human health. Some inherited forms of epilepsy, such as the autosomal dominant nocturnal frontal lobe epilepsies, are caused by ␣4␤2 AChRs harboring at least two separate mutations within their ␣4 subunit (9 -12). Most recently, ␣4␤2 AChRs, among other ␤2 subunit-containing AChRs, have been implicated in neuronal survival during aging, as surmised from the neurodegeneration observed in ␤2-subunit knock-out mice (13).The ␣4 subunit, like the other AChR subunits, consists of an extracellular N-terminal domain, followed by three transmembrane domains (M1-M3), a large cytoplasmic domain, a fourth transmembrane domain (M4), and a short extracellular C terminus. The large cytoplasmic domain is highly divergent among the various subunits, and this sequence divergence presumably provides the diversity necessary for different AChR subtypes to interact directly with cytosolic proteins of different function. To identify such proteins associated with ␣4␤2 AChRs, we used the large cytoplasmic domain of the ␣4 subunit as a bait to screen a mouse brain cDNA yeast two-hybrid library. Here we describe the isolation of a known protein termed 14-3-3. The 14-3-3 proteins family consists of sev...

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast twohybrid library using the large cytoplasmic domain of the ␣4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The ␣4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant ␣4␤2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native ␣4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant ␣4␤2 AChRs up-regulated their surface expression levels ϳ2-fold and increased their agonist sensitivity to acetylcholine ϳ3-fold. The modulation of the recombinant ␣4␤2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of ␣4␤2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.Neuronal nicotinic acetylcholine receptors (AChRs) 1 are members of a gene superfamily of ligand-gated ion channels. In vertebrates, neuronal AChRs are composed of subunits ␣2-␣10 and ␤2-␤4 (for review, see Ref. 1). AChR subunits have a large cytoplasmic domain between their third and fourth transmembrane domain whose amino acid sequence is highly divergent among the various subunits (2). The full functional unit of AChRs, like those of the N-methyl-D-aspartate receptors (3), is likely to include proteins that associate with this large cytoplasmic domain and modulate AChR functions.To identify proteins associated with ␣4 AChRs, we used bait consisting of the large cytoplasmic domain of the ␣4 subunit to screen a mouse brain cDNA yeast two-hybrid library. In this paper, we describe the isolation of VILIP-1, a member of the visinin-like protein family of calcium sensor proteins, by such a screen. VILIP-1 is a member of a superfamily of neuronal calcium sensor proteins. This superfamily has been classified into the five subfamilies termed group I-V. The recoverins belong to group I, the frequenins and neuronal calcium sensor (NCS-1) to group II, the VILIPs, hippocalcin, and neurocalcins to group III, NCS-2 to group IV, and guanylyl cyclase-activating proteins and GC-inhibiting proteins to group V (for review, see Ref. 4).The VILIP family is comprised of three members, VILIP-1, VILIP-2, and VILIP-3 (5-10). The members of this family contain 4 EF-hand motifs, of which only EF-hand 2, 3, and 4 are thought to be functional because EF-hand 1 lacks two oxygencontaining side chain residues crucial for binding calcium. A glycine residue at the second position on the polypeptide chain is myristoylated. Interestingly, within most, but not all members of this calcium sensor protein family, the myristoyl moiety is sequestered and exposed through a rapid conformational change that unmasks it in response to alterations in cellular levels of c...

The structural determinants of nicotinic acetylcholine receptor (AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum (ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the ␣4␤2 AChR subunits were required for cell surface expression of ␣4␤2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the ␣4 AChR subunit attenuates cell surface expression of mutant ␣4␤2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the ␤2 AChR subunit abolishes cell surface expression of mutant ␣4␤2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the ␤2 AChR subunit only poorly inhibits trafficking of mutant ␣4␤2 AChR to the cell surface. All mutant ␣4␤2 AChRs exhibit high-affinity binding for [ 3 H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type ␣4␤2 AChRs colocalize with the Golgi marker giantin, whereas mutant ␣4␤2 AChRs fail to do so. The striking difference between mutant ␣4 versus mutant ␤2 AChR subunits on cell surface expression of mutant ␣4␤2 AChRs points to a cooperative or regulatory role for the ␣4 AChR subunit and an obligatory role for the ␤2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of ␣4␤2 AChR ER export.

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between ␣4␤2 AChRs and the presynaptic cell adhesion molecule, neurexin-1␤. In non-neuronal tsA 201 cells, recombinant neurexin-1␤ and mature ␣4␤2 AChRs form complexes. ␣4␤2 AChRs and neurexin-1␤ also coimmunoprecipitate from rat brain lysates. When exogenous ␣4␤2 AChRs and neurexin-1␤ are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1␤. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1␤ lacking the extracellular domain. Additionally, when ␣4␤2 AChRs, ␣7 AChRs, and neurexin-1␤ are coexpressed in the same neuron, only the ␣4␤2 AChR colocalizes with neurexin-1␤ at presynaptic terminals. Collectively, these data suggest that neurexin-1␤ targets ␣4␤2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on ␣4␤2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre-and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs) 5 regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of ␣2-␣10 and ␤2-␤4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The ␣4␤2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for ␣4␤2 AChRs in nicotine addiction. ␣4␤2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7,8). Furthermore, ␤2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). ␣4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing ␣4␤2 AChRs hypersensitive to nicotine demonstrate that ␣4␤2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sen...

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