Triple arginines as molecular determinants for pentameric assembly of the intracellular domain of 5-HT3A receptors

Abstract

Serotonin type 3 receptors (5-HT3Rs) are cation-conducting pentameric ligand-gated ion channels and members of the Cys-loop superfamily in eukaryotes. 5-HT3Rs are found in the peripheral and central nervous system, and they are targets for drugs used to treat anxiety, drug dependence, and schizophrenia, as well as chemotherapy-induced and postoperative 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 within the ICD of serotonin type 3A (5-HT3A) subunits for pentameric assembly by first identifying the segments contributing to pentamerization using deletion constructs of, and finally by making defined amino acid substitutions within, an isolated soluble ICD. Our work provides direct experimental evidence for the contribution of three intracellular arginines, previously implicated in governing the low conductance of 5-HT3ARs, in structural features such as pentameric assembly.

Evidence that ion conduction in 5-HT3A receptors proceeds through lateral portals in the cytosol

“…Another notable salt bridge is located between the MA-helix and the L1-loop: D312 at the center of L1 is straddled by the 0' and 4' arginine residues (mouse R420 and R424, human R436 and R440) from the MA-helix of the adjacent subunit. Amino acid substitutions introduced in the proximity of the five lateral portals, such as the QDA substitution, lead to the breakage of these salt bridges, which enhances flexibility of the MAbundle, increases single-channel conductance, and disrupts pentamerization of the ICD (21,33,35). Based on these observations, we hypothesize that partial release of intersubunit salt-bridges in the QDA background yields a more open and unclamped/flexible arrangement of the ICD, in particular the MAhelices.…”

“…A characteristic specific to the A subunit of 5-HT3 receptors is that the MA-helices are lined with positively and negatively charged residues that interact to form inter-and intrasubunit salt bridges (33,35). Another notable salt bridge is located between the MA-helix and the L1-loop: D312 at the center of L1 is straddled by the 0' and 4' arginine residues (mouse R420 and R424, human R436 and R440) from the MA-helix of the adjacent subunit.…”

“…Previously, we have shown that the ICD alone assembles into pentamers (34) and that disruption of saltbridges in conjunction with the QDA substitution also abolishes assembly of the ICD into pentamers (35). In the present study, we investigated the mobility of the lower end of the MA-helices (L402-R410, Fig 1), a segment of the MA stretch that remains largely unexplored.…”

Structural correlates of human muscle nicotinic acetylcholine receptor subunit assembly mediated by δ(+) interface residues

“…Given that pLGIC ICDs can assemble independently of the rest of the receptor [8] and that functional channels can be obtained without intracellular domains [56], it is unsurprising that our δδε chimeric subunit can still form functional pentamers. The ICD is also known to participate in interactions with cytoskeletal proteins and in the case of the δ subunit, is predicted to be mostly unfolded [57].…”

“…Studies in GlyRs [5,6], GABAARs [7], 5HT3Rs [8] and nAChRs [9][10][11][12][13][14][15][16] provide clues as to the individual amino acid residues responsible for governing receptor assembly, expression efficiency, and the underlying mechanisms for oligomerization. However, not all of the molecular determinants that control subunit order in the human nicotinic acetylcholine receptor have yet been found.…”