Acetylcholine receptors directed mutagenesis

The basic reactions of thiolsulfonates have been known for sometime (Field et al., 1961, 1964), but more recently, they have been applied to the study of protein interactions by site-directed modification of native cysteines or through modification of cysteines introduced at particular points in proteins by mutagenesis. Such studies have yielded insights into the structure and binding site characteristics of proteins (Kirley, 1989). Pascual et al. (1998) used AEAETS to probe the acetylcholine receptor from the extracellular side of the membrane in order to investigate the molecular accessibility and electrostatic potential within the open and closed channel. 

Mishina M, Tobimatsu T, Imoto K, Tanaka KI, Fujita Y, et al. 1985. Location of functional regions of acetylcholine receptor a-subunit by site-directed mutagenesis. Nature 313 364. 

Savarese, T. M., Wang, C. D., and Fraser, C. M. (1992) Site-directed mutagenesis of the rat ml muscarinic acetylcholine receptor. Role of conserved cysteines in receptor function. J. Biol. Chem. Thl, 11439-11448. 

Fraser CM. Wang CD, Robinson DA, Gocayne JD, Venter JC (1989) Site-directed mutagenesis of ml muscarinic acetylcholine receptors conserved aspartic adds play important roles in receptor function. Mol Pharmacol 36 840-847 Gellman RL, Kallianos JA, McNamara JO (1987) receptors mediate endogenous noradrenergic suppression of kindling development. J Pharmacol Exp Ther 241 891-898... 

Changeux JP, Galzi JL, Devilhers-Thiery A, et al The functional architecture of the acetylcholine nicotinic receptor explored by affinity labeling and site-directed mutagenesis. Q Rev Biophys 25 395-432, 1992... 

The involvement of specific residues in the determination of conductance properties of the protein is readily addressed by use of the synthetic proteins. Accordingly, a tethered tetramer of M2 8 segments in which serine-8 is replaced with alanine was synthesized. This analog, T4M28(S - A), also forms channels in PC membranes with a single-channel conductance in symmetric 0.5-M KCl of 20 pS (49), lower than the 24 pS observed with T4M28. These results are in accord with observations obtained with site-directed mutagenesis of rodent acetylcholine receptors (72). 

---