Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes.
Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.
Exley
Richard
R
School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK.
Moroni
Mirko
M
Sasdelli
Federica
F
Houlihan
Lee M
LM
Lukas
Ronald J
RJ
Sher
Emanuele
E
Zwart
Ruud
R
Bermudez
Isabel
I
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018722:Nicotinic Agonists / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014982:Xenopus laevis
D048948:14-3-3 Proteins; D018832:Molecular Chaperones; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D017868:Cyclic AMP-Dependent Protein Kinases