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-HT3AglvM3M4 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 the present 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 overexpressed 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-HT3AR that mediates the interaction with RIC-3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5-HT3AR-ICD and RIC-3 does not require other proteins. Additionally, we demonstrate that the pentameric assembly of the GLIC-5-HT3A-ICD chimera interacts with RIC-3.

Delineating the site of interaction on the intracellular domain of 5-HT3A receptors with the chaperone protein RIC-3

“…In summary, from previous studies, it is known that the 5-HT 3A -ICD is required and sufficient for interaction of the 5-HT 3A R and RIC-3. Furthermore, this direct interaction between the ICD and RIC-3 does not require the presence of other host cell proteins [21][22][23][24][25]. In the current study, we aimed to identify the segment of the ICD responsible for this interaction with the hypothesis that the interaction site lies within an independent segment of the ICD.…”

“…Additionally, we demonstrated that the interaction between GLIC-5-HT 3A -ICD and RIC-3 is direct and does not require the presence of additional proteins. Both proteins were overexpressed in and purified from Escherichia coli (E. coli) and were subjected to a pull-down assay through which we observed an interaction between the two proteins [24]. Subsequently, in a separate study we examined if the 5-HT 3A -ICD alone is sufficient to retain an interaction with RIC-3.…”

“…fused to the C-terminus of maltose binding protein (MBP) in pMALX vector (New England Biolabs), MBP-5-HT 3A -ICD-pMALX, was used as the template to design constructs for the present study [25,29]. The fusion constructs were generated by deletion of amino acids from [24,31,32]. The MBP-RIC-3-His 6 -pMAL-c2x and hRIC-3-pHEMH19 were used in pull-down and in-vivo interaction assays, respectively.…”

“…We have characterized and identified a potential oligomerization motif related to the conductance-limiting arginines within the MA-helix and an aspartate within L1 (bioRxiv, doi: https://doi.org/10.1101/561282, under review). It is important to mention that subsequent deletions of amino acids in designing further constructs did not cause any significant changes in pI of the proteins, and therefore, the same expression and purification conditions were used for all MBP-5-HT 3A -ICD constructs.We additionally overexpressed the MBP-hRIC-3-His 6 chimera[24] in E. coli and optimized the lysis and purification of this membrane protein to obtain a homogenous and stable protein with appropriate yield for our direct protein-protein interaction studies. RIC-3 was purified using two sequential affinity purification steps with amylose and Talon super-flow resins and additionally SEC.…”

Triple Arginines as Molecular Determinants for Pentameric Assembly of the Intracellular Domain of 5-HT3A Receptors

“…The ICD is involved in determining ion conductance (23, 35) and modulation by drugs (36). Additionally, it interacts with various entities of the intracellular apparatus, which facilitate receptor sorting, assembly, trafficking, and anchorage (37, 38). Studies of subunit assembly and oligomerization have investigated the individual contributions of ECD (39, 40) as well as TMD (30, 31) at the molecular level.…”