title: Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.
abstract: Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Nelli
authors: N
authors: Mnatsakanyan
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Sita Nirupama
authors: SN
authors: Nishtala
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Akash
authors: A
authors: Pandhare
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Mariana C
authors: MC
authors: Fiori
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Raman
authors: R
authors: Goyal
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Biotechnology and Genomics, Texas Tech University, Lubbock, Texas 79430, United States.
authors: Jonathan E
authors: JE
authors: Pauwels
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Medical Student Summer Research Program, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Andrew F
authors: AF
authors: Navetta
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Undergraduate Science Education Program of the Howard Hughes Medical Institute, Texas Tech University, Lubbock, Texas 79430, United States.
authors: Afzal
authors: A
authors: Ahrorov
authors: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.|Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.
authors: Michaela
authors: M
authors: Jansen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D016974:Dickeya chrysanthemi / Q000737:chemistry* / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D014101:Torpedo; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029941:Fish Proteins; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor