Structure-Function Analysis of the β Subunit of Neuronal Nicotinic Acetylcholine Receptors

Citation

Figl, Antonio (1996) Structure-Function Analysis of the β Subunit of Neuronal Nicotinic Acetylcholine Receptors. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/e390-k051. https://resolver.caltech.edu/CaltechTHESIS:11072019-170728389

Abstract

Nicotinic receptors belong to the superfamily of ligand-gated ion channels. Since evidence was rapidly accumulating implicating the non-α subunits in ligand-binding events, we decided to investigate eventual contributions of the neuronal β subunit to these events by performing a series of increasingly detailed experiments on a series of chimeric β subunits. In the first set of experiments, we constructed a variety of chimeric β subunits consisting of NH₂-terminal neuronal β4 sequences and COOH-terminal β2 sequences and expressed them with the α3 subunit in Xenopus oocytes. The results showed that (a) two residues in the extracellular domain of chimeric β4•β2 subunits (108β2Phe↔β4Val, 110β2Ser↔β4Thr) account for much of the relative cytisine sensitivity; and (b) four extracellular residues of chimeric β4•β2 subunits (112β2Ala↔β4Val, 113β2Val↔β4Ile and 115β2Ser↔β4Arg, 116β2Tyr↔β4Ser) account for most of the relative tetramethylammonium sensitivity.

Encouraged by the above results, we continued our experiments with additional chimeras of the β2 and β4 neuronal nicotinic subunits to locate regions that contribute to differences between the acetylcholine dose-response relationships of α3β2 and α3β4 receptors. Substitutions within the first 120 residues convert the EC₅₀ for ACh from one wild-type value to the other, suggesting that amino acids within the first 120 residues of β2 and the corresponding region of β4 contribute to an agonist binding site that bridges the α and β subunits in neuronal nicotinic receptors.

Since the EC₅₀ phenotypes caused by the β2 and β4 subunits could be due to a difference in gating or binding properties, we attempted to unravel this question by performing voltage-jump relaxations for the series of neuronal nicotinic acetylcholine receptors we constructed previously. The chimeric β4/β2 subunits showed a transition in the concentration dependence of the relaxation rate constants in the region between residues 94 and 109, analogous to our previous observation with steady-state dose-response relationships. The data reinforce previous conclusions that the region between residues 94 and 109 on the β subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.

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