title: Engineering a prokaryotic Cys-loop receptor with a third functional domain.
abstract: Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
authors.forename: Raman
authors.initials: R
authors.lastname: Goyal
authors.forename: Ahmed Abdullah
authors.initials: AA
authors.lastname: Salahudeen
authors.forename: Michaela
authors.initials: M
authors.lastname: Jansen
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000458:Cyanobacteria / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; D007477:Ions; D008024:Ligands; D008051:Lipid Bilayers; D044406:Receptors, Serotonin, 5-HT3; D003545:Cysteine