Tryptophan scanning mutagenesis of the gammaM4 transmembrane domain of the acetylcholine receptor from Torpedo californica

The Journal of Biological Chemistry
Alejandro Ortiz-AcevedoJ A Lasalde-Dominicci

Abstract

The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the gammaM4 transmembrane domain (gammaTM4) by introducing tryptophan, at residues 451-462, along the gammaTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [(125)I]alpha-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC(50), and Hill coefficient values for WT are 1.8 +/- 0.4 fmol, 30.3 +/- 1.1 microM, and 1.8 +/- 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 +/- 0.5, 3.6 +/- 0.6, and 3.0 +/- 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC(50) (19.5 +/- 1.7, 11.4 +/- 0.7, 16.4 +/- 3.8, and 19.1 +/- 2.6 microM, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an incr...Continue Reading

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Citations

Oct 18, 2006·Journal of Neuroscience Research·Madeline Nieves-CintrónJosé A Lasalde-Dominicci
Jul 11, 2008·Journal of Neuroscience Research·Madeline Nieves-CintrónJosé A Lasalde-Dominicci
Jan 24, 2007·The Journal of Biological Chemistry·José David Otero-CruzJosé Antonio Lasalde-Dominicci
Apr 17, 2016·Progress in Lipid Research·Francisco J Barrantes, Jacques Fantini

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