Alpha-helical distorting substitution disrupt coupling between m3 muscarinic receptor and G proteins.
Acetylcholine stimulation of the m3 or m2 muscarinic receptor expressed in Xenopus laevis oocytes induces either a fast transient or slowly oscillating calcium-sensitive chloride current. The speed of these currents reflects the efficiency of receptor coupling to guanine nucleotide-binding proteins and phosphatidylinositol (PI) turnover. Point mutations of the m3 receptor were made in a region of the third cytoplasmic loop to test whether receptor function relied on an alpha-helical structure of the G protein-coupling domain. Proline substitution for glutamate at position 257 disrupted the m3 response. Also, single alanine insertions between residues 259 and 260 disrupted the m3 receptor-stimulated response while double alanine insertions at this site had no effect. Based on these results, we suggest that a region of the third cytoplasmic loop of the m3 receptor possesses an amphipathic alpha-helical conformation.
Duerson
K
K
Department of Pharmacology, Mayo Foundation, Rochester, MN 55905.
Carroll
R
R
Clapham
D
D
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D018118:Chloride Channels; D005260:Female; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D018118:Chloride Channels; D007473:Ion Channels; D008565:Membrane Proteins; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D002118:Calcium