Co-expression of the neuronal alpha7 and L247T alpha7 mutant subunits yields hybrid nicotinic receptors with properties of both wild-type alpha7 and alpha7 mutant homomeric receptors.
Injection of cDNA encoding the neuronal alpha7 subunit into Xenopus oocytes yields homomeric receptors showing responses to AcCho that have low affinity, fast desensitization, nonlinear current-voltage (I-V) relation, and sensitivity to alpha-bungarotoxin (alpha-BTX) and 5-hydroxytryptamine (5HT), both substances acting as antagonists. Mutation of the Leu-247, located in the channel domain, changes 5HT from an antagonist to an agonist, slows the rate of desensitization, renders the I-V relation linear, and increases the affinity for acetylcholine (AcCho). A study was made of receptors expressed after injecting Xenopus oocytes with mixtures of cDNAs encoding the wild-type alpha7 (WT alpha7) and the L247T alpha7 mutated nicotinic AcCho receptors (nAcChoRs). The receptors expressed were again blocked by alpha-bungarotoxin (100 nM) but exhibited both WT alpha7 and alpha7 mutant functional characteristics. Out of eight different types of hybrid receptors identified, most were inhibited by 5HT (1 mM) and showed low sensitivity to AcCho, like the WT alpha7 receptors, but exhibited a slow rate of desensitization and an I-V relation similar to those of alpha7 mutant receptors. Together, these findings indicate that the increased nAcChoR affinity and the decreased nAc-ChoR desensitization after Leu-247 mutation are uncoupled events. We propose that receptor diversity is predicted by permutations of WT alpha7 and L247T alpha7 subunits in a pentameric symmetrical model and that even partial replacement of Leu-247 with a polar residue within the leucine ring in the channel domain considerably influences the properties of neuronal alpha7 nAcChoRs.
Palma
E
E
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Sperimentale Universita di Roma La Sapienza, Rome, Italy.
Eusebi
F
F
Miledi
R
R
D000818:Animals; D002038:Bungarotoxins; D002462:Cell Membrane / Q000502:physiology*; D002645:Chickens; D004553:Electric Conductivity; D004594:Electrophysiology / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009865:Oocytes; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; D009419:Nerve Tissue Proteins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor