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-HT_3A) 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-HT_3ARs, in structural features such as pentameric assembly.

Figure 1.

Structural representation and alignments of constructs. (A) Cartoon representation of the pentameric mouse 5-HT_3A receptor x-ray structure (PDB ID: 4PIR; Hassaine et al., 2014) viewed parallel to the plane of the membrane. ECD and TMD in green, MA and MX helices in blue and cyan. (B) A single subunit of the 5-HT_3A receptor viewed parallel to the membrane. The 62 residues not resolved in L2 are depicted as a dashed line. (C) Cartoon representation of MBP–5-HT_3A–ICD–WT. All structural models of the fusion proteins are hypothetical models. The cytoplasmic domain of 5-HT_3AR is fused to the C-terminus of MBP and serves as the template for the chimeras. (D) The chimera MBP-ΔMA has the entire MA helix removed. (E) MBP-MA has the MA helix attached to the C-terminus of MBP. (F) MBP-Δ44 contains a 44-amino acid deletion in L2. (G) MBP-QDA contains a triple mutation in its MA helix and no deletions. (H) Multiple sequence alignment of 5-HT_3AR and the ICD chimeras highlighting deletions (dashed lines) or mutations (red).

Figure 2.

Affinity column purification of the ICD chimeras. The chimeras were purified by amylose resin column chromatography. The soluble protein was loaded onto columns (L, loading material; FT, flow-through), followed by washes (W), and eluted. SDS-PAGE indicates the quality of purified protein and identifies peak fractions.

Figure 3.

Weight determination of ICD chimeras by SDS-PAGE and SEC. (A) The weight of the ICD chimeras in monomeric state was determined by resolving SEC-purified protein on SDS-gel electrophoresis. Molecular weight standards are shown on top, and ICD (53 kD), ΔMA (49 kD), MA (45 kD), Δ44 (48 kD), and QDA (52 kD) are shown below. (B) The complete SDS-gel electrophoresis image from which the analysis in section A is drawn. A standard protein ladder is shown next to each protein for increased accuracy of the analysis. Lane compositions are as follows: lane 2, ICD; lane 4, ΔMA; lane 6, MA; lane 8, Δ44; and lane 10, QDA. (C) SEC yielded a weight of 267 kD for ICD, 261 kD for Δ44, 180 kD for ΔMA, 141 kD for QDA, and 73 kD for MA. Chromatogram of standard proteins (T, thyroglobulin; F, ferritin; A, aldolase; C, conalbumin; O, ovalbumin) in gray, and chimeras in their respective colors are shown above. A280, UV absorbance at 280 nm.

Figure 4.

SEC-MALS determination of oligomeric assembly. (A) Both the ICD (dotted black) and the Δ44 construct (purple line) maintain pentameric assembly, whereas deletions in the ΔMA (pink line) and the MA (orange line) constructs abolish pentamerization of the ICD. (B) The QDA substitutions (red line) disrupt the pentameric assembly of the ICD. A representative SEC-MALS profile showing the Rayleigh ratio. Note that different flow rates were used during the monitoring period for A (0.5 ml/min) and B (0.3 ml/min).