Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach.
1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, alpha5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3beta4alpha5 receptors expressed in oocytes. 2. As previously reported, the main indication of the presence of alpha5 in alpha3beta4alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3beta4 receptors). If the alpha3beta4alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, alpha5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4alpha5 vs. alpha3(LT)beta4 and by 40 fold for alpha3beta4(LT)alpha5 vs. alpha3beta4(LT)). The much greater effect observed in alpha3beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3beta4alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4. Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC(50) for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3beta4alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.
Groot-Kormelink
P J
PJ
Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AX.
Boorman
J P
JP
Sivilotti
L G
LG
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression; D005838:Genotype; D005914:Globins / Q000235:genetics; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D014982:Xenopus laevis
D018076:DNA, Complementary; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D005914:Globins; D000109:Acetylcholine