The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors
A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [125I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC50), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC50. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.
KeywordsAcetylcholine receptor Site-directed mutagenesis Xenopus oocyte Lipid-exposed residue Torpedo californica Muscle-type receptor
This work was supported in part by grants from the National Institutes of Health: 2RO1GM5637-10 and GM08102-27, as well as UPR Institutional Funds for Research awarded to J.A. Lasalde-Dominicci. G. Guzman was supported by NSF-AGEP grant HRD-9817642 and A. Ricardo by NIH-MARC grant 5T34GM07821.
- Cruz-Martin A., Mercado J.L., Rojas L.V., McNamee M.G., Lasalde-Dominicci J.A. 2001. Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor. J. Membr. Biol. 183:61–70PubMedCrossRefGoogle Scholar
- Guzman G.R., Santiago J., Ricardo A., Marti-Arbona R., Rojas L.V., Lasalde-Dominicci J.A. 2003. Tryptophan scanning mutagenesis in the alpha M3 transmembrane domain of the Torpedo californica acetylcholine receptor: Functional and structural implications. Biochemistry 42:12243–12250PubMedCrossRefGoogle Scholar
- Lasalde, J.A., Tamamizu, S., Butler, D.H., Vibat, C.R., Hung, B., McNamee, M.G. 1996. Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating. Biochemistry :14139–14148Google Scholar
- Otero-Cruz J.D., Báez-Pagán C.A., Caraballo-González I.M., Lasalde-Dominicci J.A. 2006. A lipid-exposed transmembrane domain of ligand-gated ion channel receptor displays a tilted spring motion during channel activation, comparing Torpedo versus muscle-type acetylcholine Receptors. A SPRING MODEL REVEALED. J. Biol. Chem. 282:9162–9171CrossRefGoogle Scholar
- Santiago J., Guzman G.R., Rojas L.V., Marti R., Asmar-Rovira G.A., Santana L.F., McNamee M., Lasalde-Dominicci J.A. 2001. Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes. J. Biol. Chem. 276:46523–46532PubMedCrossRefGoogle Scholar
- Tamamizu S., Guzman G.R., Santiago J., Rojas L.V., McNamee M.G., Lasalde-Dominicci J.A. 2000. Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 39:4666–4673PubMedCrossRefGoogle Scholar
- Wang H.L., Milone M., Ohno K., Shen X.M., Tsujino A., Batocchi A.P., Tonali P., Brengman J., Engel A.G., Sine S.M. 1999. Acetylcholine receptor M3 domain: Stereochemical and volume contributions to channel gating. Nat. Neurosci. 2:226–233. Erratum in Nat. Neurosci. 1999;2:485PubMedCrossRefGoogle Scholar
- Yu, L., Leonard, R.J., Davidson, N., Lester, H.A. 1991. Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes. Brain Res. Mol. Brain Res. :203–211Google Scholar