Ion channels gated by acetylcholine and serotonin: structures, biology, and drug discovery.

The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 receptors (5-HT3Rs) are cation-selective members of the pentameric ligand-gated ion channels (pLGICs), which are oligomeric protein assemblies that convert a chemical signal into an ion flux through postsynaptic membrane. They are critical components for synaptic transmission in the nervous system, and their dysfunction contributes to many neurological disorders. The diverse subunit compositions of pLGICs give rise to complex mechanisms of ligand recognition, channel gating, and ion-selective permeability, which have been demonstrated in numerous electrophysiological and molecular biological studies, and unraveled by progress in studying the structural biology of this protein family. In this review, we discuss recent insights into the structural and functional basis of two cation-selective pLGICs, the nAChR and the 5-HT3R, including their subunit compositions, ligand binding, and channel gating mechanisms. We also discuss their relevant pharmacology and drug discovery for treating various neurological disorders. Finally, we review a model of two alternative ion conducting pathways based on the latest 5-HT3A crystal structure.

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

AIM: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. METHODS: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. RESULTS: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. CONCLUSION: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.

High yield and efficient expression and purification of the human 5-HT3A receptor.

AIM: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. METHODS: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). RESULTS: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. CONCLUSION: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor.