Running Title: Mobility of MA-helicesKeywords: pentameric ligand-gated ion channels, serotonin, intracellular domain (select 3 phrases and 3 techniques from a pre-determined list)
Statement of Significance: (120 words max)The intracellular domain (ICD) of pentameric ligand-gated ion channels (pLGICs) is the most diverse domain within receptors of the Cys-loop superfamily. Despite being the least understood domain of pLGICs, we know that it influences channel function in multiple ways and shapes the cytosolic exit pathway of the channel. X-ray and cryo-EM structures have captured the structured segments of the ICD of 5-HT3A receptors in different conformational states with lower resolution of the ICD as compared to the other domains.Here, we provide experimentally-derived evidence for the importance of the mobility of the MA-helices and functionally confirm ion-conduction through lateral portals as opposed to a vertical pathway for 5-HT3A receptors.
Abstract:Serotonin type 3A receptors (5-HT3ARs) are pentameric ligand-gated ion channels, pharmacologically targeted for the treatment of severe nausea and vomiting. The intracellular domain of 5-HT3ARs has been shown to be a crucial determinant for limiting conductance. Structurally, it consists of a short L1-loop following the third transmembrane segment M3, a short α-helical MX-segment, a large unstructured L2-loop, and the membrane-associated MA-helix that continues into the last transmembrane segment M4.During gating, conformational changes occur in all three domains. Extracellular and transmembrane domains rotate counterclockwise and clockwise, respectively. Within the intracellular domain the MA-M4 helix breaks at Gly430, leading to pore widening above and below the helix break, and the MX-helix moves outward and upward towards the membrane. At their bottom, the MA-helices come into close apposition to shape a narrow hydrophobic constriction below the cytosolic channel chalice that, in its upper section, is framed by lateral windows through which the L1-loops thread. In the present study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of the bottom segments of the MA-helices. Repeated agonist application or oxidation induced a current run down for channels with Cys pairs at I409C/R410C. For Cys pairs at L402C/L403C these conditions did not lead to altered currents. On the contrary, cross-linked subunits for both L402C/L403C and I409C/R410C were observed for both conditions using gel electrophoresis. Our results indicate that the proximity and orientation for Cys in both pairs is conducive for disulfide bond formation.While conformational changes associated with gating promote cross-linking for I409C/R410C that in turn inhibits gating, cross-linking of L402C/L403C is functionally silent. We infer that conformational changes associated with gating are more pronounced for the upper I409C/R410C pair in close proximity to the lateral windows as compared to the L402C/L403C pair at the apex of the inverted pen...
