Reporter Mutation Studies Show That Nicotinic Acetylcholine Receptor (nAChR) α5 Subunits and/or Variants Modulate Function of α6-nAChR

• Published in: The Journal of biological chemistry 2011-11, Vol.286 (44), p.37905-37918
• Main Authors: Dash, Bhagirathi, Chang, Yongchang, Lukas, Ronald J.
• Format: Article
• Language: English
• Subjects: Animals; Cytoplasm - metabolism; DNA, Complementary - metabolism; Electrophysiology - methods; Female; Humans; Ion Channels; Ligand-binding Protein; Ligands; Mutation; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurobiology; Neurotransmitter Receptors; Nicotine - metabolism; Nicotinic Acetylcholine Receptors; Oocytes - metabolism; Point Mutation; Protein Structure, Tertiary; Receptor Structure-function; Receptors, Nicotinic - genetics; Receptors, Nicotinic - metabolism; Recombinant Fusion Proteins - chemistry; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.264044

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 α5V9′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/β3V9′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/β3V9′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/β3V9′S subunits has a gain-of-function effect more pronounced than that in the presence of α5V9′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. Background: We asked how partner subunits influence α6*-nicotinic receptor (nAChR) function. Results: We found several, novel ways to manipulate effects of α5 subunits on α6*-nAChR function. Conclusion: Extracellular domains in α6 and cytoplasmic/transmembrane domains in α6/α3/α5/β3 subunits have unexpected influences on α6*-nAChR function. Significance: We found factors that influence assembly and function of α6*-nAChRs, which play important roles in mood, reward, and nicotine dependence.