Evidence that ion conduction in 5-HT3Areceptors proceeds through lateral portals in the cytosol

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. However, the specific ion conduction pathway through this domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short α-helical segment, a large unstructured loop, and finally the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a “closed vestibule”, with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In the present study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. While conformational changes associated with gating promote crosslinking for I409C/R410C, which in turn decreases channel currents, crosslinking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely. Significance 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, its impact on ion-channel function and contribution to the cytosolic exit pathway of the channel have been investigated. X-ray and cryo-EM structures have captured the structured segments of the ICD of 5-HT 3A 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 differential mobility of the cytosolic segment of the MA-helices, which supports the existence of lateral portals as opposed to a vertical pathway for 5-HT 3A receptors.