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

ABSTRACT Serotonin type 3A receptors (5-HT 3A Rs) are cation-conducting homo-pentameric ligand-gated ion channels (pLGICs) also known as the Cys-loop superfamily in eukaryotes. 5-HT 3 Rs are found in the peripheral and central nervous system, and they are targets for drugs used to treat anxiety, drug dependence, schizophrenia, as well as chemotherapy-induced and post-operative nausea and emesis. Decades of research of Cys-loop receptors have identified motifs in both the extracellular and transmembrane domains that mediate pentameric assembly. Those efforts have largely ignored the most diverse domain of these channels, the intracellular domain (ICD). Here we identify molecular determinants inside the ICD for pentameric assembly by first identifying the segments contributing to pentamerization using deletion constructs, and remarkably by making a small number of defined amino acid substitutions. Our work provides direct experimental evidence for the contribution of three arginines, previously implicated in governing the low conductance of 5-HT 3A Rs, in structural features such as pentameric assembly.