Gating of alpha3beta4 neuronal nicotinic receptor can be controlled by the loop M2-M3 of both alpha3 and beta4 subunits.
Previous studies have shown that the gating mechanism of alpha3beta4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the beta4 subunit. We have extended the study of the same location to the alpha3 subunit. Bovine alpha3beta4 receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e. leucine in alpha3 and asparagine in beta4, for an aspartate. Wild-type and mutated alpha3 and beta4 subunits were combined to form four different receptors. We have measured macroscopic currents in Xenopus oocytes elicited by nicotine, and related them to surface receptor expression measured with an epibatidine-binding essay. We also obtained single-channel recordings of the receptors to study their kinetic behaviour. The results were analysed in terms of an allosteric model with three states. We found that the effect of the mutation in the alpha3 subunit on the gating of the receptor was similar to the corresponding mutation in the beta4 subunit. The effect when both subunits were mutated was additive, suggesting that the contribution of each subunit to the gating mechanism is independent.
Rovira
J C
JC
Instituto de Neurociencias, Universidad Miguel Hernández, San Juan de Alicante, Spain.
Vicente-Agulló
F
F
Campos-Caro
A
A
Criado
M
M
Sala
F
F
Sala
S
S
Ballesta
J J
JJ
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002417:Cattle; D004558:Electric Stimulation; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D018722:Nicotinic Agonists / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017354:Point Mutation / Q000235:genetics; D011725:Pyridines / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis
D019086:Bridged Bicyclo Compounds, Heterocyclic; D018722:Nicotinic Agonists; D011725:Pyridines; D011978:Receptors, Nicotinic; C403530:nicotinic receptor alpha3beta4; C082748:epibatidine