Abstract
Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central and peripheral nervous system and are localized at synaptic and extrasynaptic sites of the cell membrane. However, the mechanisms regulating the localization of nicotinic receptors in distinct domains of the cell membrane are not well understood. N-cadherin is a cell adhesion molecule that mediates homotypic binding between apposed cell membranes and regulates the actin cytoskeleton through protein interactions with the cytoplasmic domain. At synaptic contacts, N-cadherin is commonly localized adjacent to the active zone and the postsynaptic density, suggesting that N-cadherin contributes to the assembly of the synaptic complex. To examine whether N-cadherin homotypic binding regulates the cell surface localization of nicotinic receptors, this study used heterologous expression of N-cadherin and α3β4 nAChR subunits C-terminally fused to a myc-tag epitope in Chinese hamster ovary cells. Expression levels of α3β4 nAChRs at cell-cell contacts and at contact-free cell membrane were analyzed by confocal microscopy. α3β4 nAChRs were found distributed over the entire surface of contacting cells lacking N-cadherin. In contrast, N-cadherin-mediated cell-cell contacts were devoid of α3β4 nAChRs. Cell-cell contacts mediated by N-cadherin-deleted proteins lacking the β-catenin binding region or the entire cytoplasmic domain showed control levels of α3β4 nAChRs expression. Inhibition of actin polymerization with latrunculin A and cytochalasin D did not affect α3β4 nAChRs localization within N-cadherin-mediated cell-cell contacts. However, treatment with the Rho associated kinase inhibitor Y27632 resulted in a significant increase in α3β4 nAChR levels within N-cadherin-mediated cell-cell contacts. Analysis of α3β4 nAChRs localization in polarized Caco-2 cells showed specific expression on the apical cell membrane and colocalization with apical F-actin and the actin nucleator Arp3. These results indicate that actomyosin contractility downstream of N-cadherin homotypic binding regulates the cell surface localization of α3β4 nAChRs presumably through interactions with a particular pool of F-actin.
Highlights
Neuronal nicotinic acetylcholine receptors are ligandgated ion channels comprised of five homologous subunits arranged around a central pore
To determine whether the a3b4-myc nicotinic acetylcholine receptors (nAChRs) colocalized with microtubules, Caco-2 cells were colabeled with anti-acetylated atubulin and with anti-b-tubulin antibodies; none of these two microtubule markers appeared associated with a3b4-myc nAChRs (Figure 7). These results indicate that a3b4-myc nAChRs are expressed only on the apical surface of polarized epithelial cells and are associated with areas of the cell membrane enriched in a particular pool of F-actin, which is probably polymerized by the Arp2/3 actin nucleator complex
Homo and heteromeric neuronal nAChRs expressed throughout the nervous system are localized to synaptic and extra-synaptic sites of the cell membrane [4,5]
Summary
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels comprised of five homologous subunits arranged around a central pore. They belong to the superfamily of cys-loop receptors due to the presence of an extracellular pair of disulphide-bonded cysteines separated by a stretch of 13 amino acids [1]. Neuronal nAChRs with different subunit compositions localize to distinct domains of the cell surface including pre and postsynaptic membranes, and extrasynaptic sites [5]. Neuronal nAChRs containing a3 subunits are localized at the postsynaptic density apposed to presynaptic active zones to glutamate receptors in excitatory synapses of the central nervous system [12]. The mechanisms regulating the localization of a3b4 nAChRs on the cell membrane and their assembly into receptor clusters are not entirely understood
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.