Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.
Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.
Rayes
Diego
D
Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, 8000 Bahía Blanca, Argentina.
De Rosa
María José
MJ
Sine
Steven M
SM
Bouzat
Cecilia
C
D000109:Acetylcholine / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics; D000596:Amino Acids / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D055592:Biophysical Phenomena / Q000187:drug effects / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism; D002461:Cell Line, Transformed; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D018722:Nicotinic Agonists / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods; D064569:alpha7 Nicotinic Acetylcholine Receptor
D000596:Amino Acids; D002038:Bungarotoxins; C579167:Chrna7 protein, human; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3; D064569:alpha7 Nicotinic Acetylcholine Receptor; D003545:Cysteine; D000109:Acetylcholine