Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state.
The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two alpha subunits are rotated relative to its non-alpha subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the alpha subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the alphaG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the alpha subunits.
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building 16, Room 573B, Cambridge, Massachusetts 02139, USA.
Mathew
M
Tantama
Stuart
S
Licht
D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016013:Likelihood Functions; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D018722:Nicotinic Agonists / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D018722:Nicotinic Agonists; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D002794:Choline