Amino Acid Residues That Confer High Selectivity of the α6 Nicotinic Acetylcholine Receptor Subunit to α-Conotoxin MII[S4A,E11A,L15A

Layla Azam,Doju Yoshikami,J Michael Mcintosh

doi:10.1074/jbc.m710288200

Abstract

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.

Highlights

␣6 can form receptors in combination with ␤2 through ␤4, leading to a large assortment of different receptor subtypes
␣6 Residues Glu-152, Asp-184, and Thr-195 Interact with Other ␣6-Selective ␣-Conotoxins—We investigated whether the identified residues were critical for the interaction with other ␣6*-selective conotoxins, ␣-CTX PIA and ␣-CTX MII analogs ␣-CTX MII[H9A,L15A] and ␣-CTX MII[E11A]
When tested on the mutant ␣3K152EE184DQ195T␤2 Nicotinic acetylcholine receptors (nAChRs), all three ␣-conotoxins were more potent in blocking the mutant than the WT ␣3␤2 nAChR, the gain in sensitivity was much less for ␣-CTX PIA than for the ␣-CTX MII analogs (Table 5 and Fig. 5)

Summary

Sequences of primers used to construct chimeras and point mutants

The numbering reflects the length of the ␣3 N-terminal fragment substituted into the corresponding region of the ␣6 subunit. To introduce ACh into the chamber, the perfusion was halted and 20 ␮l of ACh was manually applied to the chamber via the small circular hole upstream from the oocyte. Upon resumption of perfusion (which was started immediately following the introduction of ACh into the chamber), the bolus of ACh rapidly engulfed the oocyte and washed past it in a matter of seconds, as judged by the time course of ACh response. This process was repeated with different concentrations of ACh with a time interval between applications long enough to avoid desensitization. For ACh dose-response curves, the response to a given ACh concentration was normalized to the response to 100 ␮M ACh, which served as an internal control

RESULTS

Mutant nAChR

Findings

DISCUSSION