Dataset Information

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].

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.

SUBMITTER: Azam L

PROVIDER: S-EPMC2431060 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].

Azam Layla L Yoshikami Doju D McIntosh J Michael JM

The Journal of biological chemistry 20080225 17

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 Xeno ...[more]

PMID: 18299323

Similar Datasets

Project description:Functional heterologous expression of naturally-expressed and apparently functional mammalian ?6*-nicotinic acetylcholine receptors (nAChRs; where '*' indicates presence of additional subunits) has been difficult. Here we wanted to investigate the role of N-terminal domain (NTD) residues of human (h) nAChR ?6 subunit in the functional expression of h?6*-nAChRs. To this end, instead of adopting random mutagenesis as a tool, we used 15 NTD rare variations (i.e., Ser43Pro, Asn46Lys, Asp57Asn, Arg87Cys, Asp92Glu, Arg96His, Glu101Lys, Ala112Val, Ser156Arg, Asn171Lys, Ala184Asp, Asp199Tyr, Asn203Thr, Ile226Thr and Ser233Cys) in nAChR h?6 subunit to probe for their effect on the functional expression of h?6*-nAChRs.N-terminal ?-helix (Asp57); complementary face/inner ?-fold (Arg87 or Asp92) and principal face/outer ?-fold (Ser156 or Asn171) residues in the h?6 subunit are crucial for functional expression of the h?6*-nAChRs as variations in these residues reduce or abrogate the function of h?6h?2*-, h?6h?4- and h?6h?4h?3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal ?-helix) in h?6 subunit reduce h?6h?2*-nAChRs function those at residues Arg96 (?2-?3 loop), Asp199 (loop F) or Ser233 (?10-strand) increase h?6h?2*-nAChR function. Similarly substitution of NTD ?-helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in h?6 subunit increase the function of h?6h?4-nAChRs. All other variations in h?6 subunit do not affect the function of h?6h?2*- and h?6h?4*-nAChRs. Incorporation of nAChR h?3 subunits always increase the function of wild-type or variant h?6h?4-nAChRs except for those of h?6(D57N, S156R, R87C or N171K)h?4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in h?6 subunit drive the h?6h?4h?3-nAChRs into a nonfunctional state as at spontaneously open h?6(D57N, S156R or N171K)h?4h?3V9'S-nAChRs (V9'S; transmembrane II 9' valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of h?6h?4- and/or h?6h?4h?3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in h?6 subunit.Hence investigating functional consequences of natural variations in nAChR h?6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of h?6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in h?6 subunit that compromise h?6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.

Project description:α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

Dataset Information

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].

Publications

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets