title: Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.
abstract: Pentameric ligand-gated ion channels (pLGIC) are expressed in both excitable and non-excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo- or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC-3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5-HT3A ) that the interaction with RIC-3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC-3 attenuated serotonin-induced currents in 5-HT3A wild-type channels, but not in functional 5-HT3A glvM3M4 channels that have the 115-amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand-gated ion channel (GLIC) to contain the 5-HT3A -ICD confers sensitivity to RIC-3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC-3 protein expression in X. laevis oocytes. We purified RIC-3 to homogeneity after expression in Echericia coli. By using heterologously over-expressed and purified RIC-3 and the chimera consisting of the 5-HT3A -ICD and the extracellular and transmembrane domains of GLIC in pull-down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5-HT3 A R that mediates the interaction with RIC-3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5-HT3 A R-ICD and RIC-3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC-5-HT3A -ICD chimera interacts with RIC-3. We hypothesized that pentameric ligand-gated ion channels (pLGICs) associate directly with the chaperone protein RIC-3 (resistance to inhibitors of cholinesterase type 3), and that the interaction does not require other protein factors. We found that the two proteins indeed interact directly, that the pLGIC intracellular domain is required for the effect, and that pLGICs in their pentameric form associate with RIC-3. These results provide the basis for future studies aimed at investigating which motifs provide the interaction surfaces, and at delineating the mechanism(s) of RIC-3 modulation of functional pLGIC surface expression.
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.|The Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
authors.forename: Sita Nirupama
authors.initials: SN
authors.lastname: Nishtala
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.|The Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
authors.forename: Nelli
authors.initials: N
authors.lastname: Mnatsakanyan
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.|The Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
authors.forename: Akash
authors.initials: A
authors.lastname: Pandhare
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.|The Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.|Medical Student Summer Research Program, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
authors.forename: Chun
authors.initials: C
authors.lastname: Leung
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.|The Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
authors.forename: Michaela
authors.initials: M
authors.lastname: Jansen
mesh_terms: D000818:Animals; D003593:Cytoplasm / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D009865:Oocytes; D011485:Protein Binding / Q000502:physiology; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C509409:HTR3A protein, human; D047908:Intracellular Signaling Peptides and Proteins; C496701:RIC3 protein, human; D044406:Receptors, Serotonin, 5-HT3
keywords: Gloeobacter violaceus ligand-gated ion channel; intracellular domain; nell (nAChR); pentameric ligand-gated ion channels; resistance to inhibitors of cholinesterase; serotonin