Pentameric Structure of the Soluble Intracellular Domain of 5-HT3A Receptors

Abstract

The 115 amino acids spanning intracellular domain (ICD) links the third (M3) and fourth (M4) transmembrane segments within each subunit of the 5-HT3A receptor, a cationic homo-pentameric ligand-gated ion channel (pLGIC). The advent of increasingly sophisticated structural studies has unraveled, in part, the three-dimensional structure of eukaryotic as well as prokaryotic pLGICs at atomic resolution. Intriguingly, within all these structures, the ICD has commonly not been completely resolved. To illustrate the lack of completeness, a recent high-resolution 5-HT3A structure, albeit providing novel structural insights, is deprived of 61 amino acids from its ICD resulting in channel activity differing remarkably from wild-type channels. Prokaryotic pLGICs lack the ICD, and instead contain a very short M3-M4 linker. In eukaryotic pLGICs, the ICD exemplifies the diversity of domain architecture, unlike the other two domains - extracellular and transmembrane - which are highly-conserved. Given the expanding functional implications of ICDs in subunit folding, assembly, targeting, posttranslational modifications, reverse allosteric conductance, and protein-protein interactions, it is of great importance to resolve the structures of full-length ICDs in order to bridge this knowledge gap.To attain this set goal, we produced and purified a soluble chimera containing the ICD of the mouse 5-HT3A receptor. Molecular weight determination by both size exclusion chromatography and dynamic light scattering indicates stable pentameric assembly of the chimera in solution. This surprising finding was further corroborated when the chimera was cross-linked with glutaraldehyde vapors and then resolved either by SDS-PAGE or ‘on-chip electrophoresis’, revealing a ladder of discrete covalently-linked oligomers. The unexpected observation that the ICD alone assembles into stable pentamers in solution and its extreme diversity with regard to both length and amino acid-composition is further suggestive of its orphaned role in receptor oligomerization and potentially the specificity thereof.