Abstract
To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/β3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human β3 subunit) almost completely abolishes the very small amount of function seen for α6β4*-nAChR and does not induce function of α6β2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6β4*-nAChR or induce function of α6β2*-nAChR. However, coexpression with mutant chimeric α5/β3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6β4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/β3(V9)'(S) subunits to have a gain-of-function effect on α6β2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either β2 or β4 subunits have some function that is modulated in the presence of α5 or α5/β3 subunits. Coexpression with α5/β3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.
Highlights
We asked how partner subunits influence ␣6*-nicotinic receptor function
Coexpression with chimeric mutant h␣5/h3V9ЈS subunits as accessory partners significantly increased nicotinic responses to 10 M nicotine (225 Ϯ 31 nA), and nearly every oocyte injected with nicotinic acetylcholine receptors (nAChR) h␣6, h4, and mutant h␣5/ h3V9ЈS subunits expressed functional nAChR (Fig. 2, Table 2)
To understand how ␣5 subunits might incorporate into ␣6*-nAChR, we exploited the reporter or gain-of-function mutant strategy [20] to reveal whether ␣5 subunits or their variants integrate into ␣6*-nAChR complexes that are on the cell surface and functional
Summary
We asked how partner subunits influence ␣6*-nicotinic receptor (nAChR) function. Results: We found several, novel ways to manipulate effects of ␣5 subunits on ␣6*-nAChR function. Aware that coexpression with mutated h␣5 subunits (M2 second transmembrane domain 9Ј position, h␣5V290S ϭ h␣5V9ЈS) do not induce spontaneous opening or increased agonist sensitivity of the nAChR subtypes tested [19] we further hypothesized that recombinant, chimeric h␣5/h3V273S subunits (in which the N-terminal, large extracellular domain of the h␣5 subunit is linked to the remaining domains of the gain-of-function h3V9ЈS subunit) would serve as a reporter mutation and, upon integration into ␣6*-nAChR, would increase their agonist sensitivity. These findings provide insight into the assembly, structure, and function of functional ␣6␣5*-nAChR, which could be exploited as models for development of new ligands to affect mood and drug dependence
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