The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

Abstract

ResultsUsing PCR, we constructed chimeric subunits in which the α7 cytoplasmic loop immediately after TM3 and before TM4 was replaced with the homologous region of the α3 or α5 sequence (Fig. 1a). The N-terminus up to TM2 regulates intersubunit assembly into receptor complexes, and α7 subunits do not coassemble with nAChR subunits, as demonstrated for endogenous subunits in CG neurons and for chimeric α7 subunits expressed in Xenopus laevis oocytes 4,5,12,13 . In particular, we have observed that coexpression of chimeric α7 subunits containing the α3 cytoplasmic loop together with wild-type α3 and β4 subunits in Xenopus oocytes results in the formation of two distinct receptor types that have the pharmacological properties of Bgt-nAChRs and nAChRs, respectively (data not shown). Thus, a difference in the distribution of chimeric α7 as compared to wild-type α7 on the infected CG neuron surface would result from the added α3 or α5 cytoplasmic loop. It cannot be explained by assembly with an endogenous nAChR subunit that can target to the synapse. Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing α3 and α5 subunits are concentrated in the postsynaptic membrane, whereas α-bungarotoxin receptors composed of α7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of α3 targets chimeric α7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the α5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.1998 Nature America Inc.• http://neurosci.nature.com 1998 Nature America Inc.• http://neurosci.nature.com

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

“…We show that 14-3-3 directly binds, in vitro, to the α3 long cytoplasmic loop and 14-3-3 links the α3 loop to APC in heterologous cell expression studies. This α3 domain targets chimeric nAChRs to synapses in CG neurons in vivo (Williams et al, 1998). By bringing EB1, MACF, IQGAP1 and 14-3-3 together, APC organizes a multi-molecular complex that directs α3*nAChR surface delivery, stabilizes the local cytoskeleton, and links α3*nAChRs to the cytoskeleton at postsynaptic sites.…”

“…This preparation, therefore, provides a valuable opportunity to compare molecular mechanisms that orchestrate the assembly of the different receptor complexes. The α3*nAChRs are concentrated in postsynaptic membrane regions that oppose presynaptic terminal active zones (Jacob et al, 1986;Williams et al, 1998). GlyR clusters localize to separate but proximal postsynaptic membrane regions, all under one presynaptic terminal (Tsen et al, 2000).…”

“…8A). We speculated that the α3 subunit may mediate the binding to APC because our past studies showed that the α3 long cytoplasmic loop targets chimeric nAChRs to synapses in CG neurons in vivo (Williams et al, 1998;Williams et al, 1999). Because native α3*nAChRs are heteromers composed of α3, α5, and β4 subunits, we tested for APC interactions with the α3 loop by heterologous expression studies in MDCK cells.…”

Nicotinic α7 receptors: Synaptic options and downstream signaling in neurons

“…By exchanging the large intracellular loop of the ␣3 nAChR subunit for the homologous region in ␣7 subunits, Jacob and colleagues were able to show in the chick ciliary ganglion that the modified ␣7-nAChRs now become localized at PSDs on the neurons (Williams et al, 1998). Normally, the ␣7-nAChRs are excluded from PSDs, which instead contain heteromeric nAChRs with one or more ␣3 subunits.…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…On CG neurons, ␣3-nAChRs are concentrated in the postsynaptic membrane, whereas ␣7-nAChRs are localized perisynaptically and excluded from the synapse (see above). In vivo trafficking studies demonstrate that the ␣3 subunit, in particular the long intracellular loop, targets chimeric nAChR subunits to the interneuronal synapse in CG neurons (Williams et al, 1998;Temburni et al, 2000). In contrast, the intracellular loops of ␣5 and ␤4, the other subunits of native ␣3-nAChRs, do not.…”