ALPHA-HELICAL DISTORTING SUBSTITUTIONS DISRUPT COUPLING BETWEEN M3-MUSCARINIC-RECEPTOR AND G-PROTEINS

Acetylcholine stimulation of the m3 or m2 muscarinic receptor expresse d in Xenopus laevis oocytes induces either a fast transient or slowly oscillating calcium-sensitive chloride current. The speed of these cur rents reflects the efficiency of receptor coupling to guanine nucleoti de-binding proteins and phosphatidylinositol (PI) turnover. Point muta tions of the m3 receptor were made in a region of the third cytoplasmi c loop to test whether receptor function relied on an alpha-helical st ructure of the G protein-coupling domain. Proline substitution for glu tamate at position 257 disrupted the m3 response. Also, single alanine insertions between residues 259 and 260 disrupted the m3 receptor-sti mulated response while double alanine insertions at this site had no e ffect. Based on these results, we suggest that a region of the third c ytoplasmic loop of the m3 receptor possesses an amphipathic alpha-heli cal conformation.