title: The coupling interface and pore domain codetermine the single-channel activity of the α7 nicotinic receptor.
abstract: Ligand-gated ion channels play a role in mediating fast synaptic transmission for communication between neurons. However, the structural basis for the functional coupling of the binding and pore domains, resulting in channel opening, remains a topic of intense investigation. Here, a series of α7 nicotinic receptor mutants were constructed for expression in cultured mammalian cells, and their single-channel properties were examined using the patch-clamp technique combined with radio ligand binding and the fluorescence staining technique. We demonstrated that the replacement of the four pore-lining residues in the channel domain of the α7 nicotinic receptor with the hydrophilic residue serine prolongs the open-channel lifetime, although the conductance of these mutants decreases. At the coupling interface between the extracellular and transmembrane domains, when the VRW residues in the Cys-loop were substituted with the corresponding residues (i.e., IYN) in the 5-HT3A receptor, the single-channel activity elicited by acetylcholine is impaired. This effect occurred despite the expression of the mutant receptors on the cell surface and despite the fact that the apparent Kd values were much lower than those of the wild-type α7 receptor. When we further lowered the channel-gating barrier of this chimera to enhance the open-channel probability, the loss of function was rescued. Overall, we explored the microscopic mechanisms underlying the interplay between the channel domains and the coupling interface that affect the channel activity, and we generated an allosteric gating model for the α7 receptor. This model shows that the gating machinery and coupling assembly codetermine the single-channel gating kinetics. These results likely apply to all channels in the Cys-loop receptor family.
authors.affiliation: Department of Neurobiology, Beijing Institute of Brain Disorders, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders - State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Neurobiology, Beijing Institute of Brain Disorders, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders - State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China.
authors.forename: Na
authors.initials: N
authors.lastname: Pan
authors.affiliation: Medical Experiment and Test Center, Capital Medical University, Beijing 100069, China.
authors.forename: Fenqin
authors.initials: F
authors.lastname: Xue
authors.affiliation: Department of Neurobiology, Beijing Institute of Brain Disorders, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders - State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, Henan 453003, China.
authors.forename: Chaokun
authors.initials: C
authors.lastname: Li
authors.affiliation: Division of Neurobiology, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Department of Neurobiology, Beijing Institute of Brain Disorders, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders - State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Neurobiology, Beijing Institute of Brain Disorders, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders - State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China. Electronic address: huiyang@ccmu.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Neurobiology, Beijing Institute of Brain Disorders, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders - State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China. Electronic address: jlzhang@ccmu.edu.cn.
authors.forename: Jianliang
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009154:Mutation; D018408:Patch-Clamp Techniques; D011869:Radioligand Assay; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics; D012694:Serine / Q000235:genetics; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: D044406:Receptors, Serotonin, 5-HT3; D064569:alpha7 Nicotinic Acetylcholine Receptor; D012694:Serine; D000109:Acetylcholine
keywords: Channel coupling; Channel gating; Nicotinic receptor; Patch-clamp