The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABA A receptors containing a1 and b3 subunits could be expressed with specific activities of 29-34 pmol/mg corresponding to 140-170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT 3A ) receptors were 49-63 pmol/mg and 245-315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3-3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one Abbreviations: 5-HT 3 AR, 5-hydroxytryptamine-3A receptor; CMC, critical micelle concentration; DDM, n-dodecyl-b-D-maltopyranoside; EPR, electron paramagnetic resonance; GABA A a1b3R, gamma-aminobutyric acid type A receptor with a1 and b3 subunits; GPCR, G protein-coupled receptor; GlyR, glycine receptor; HEK293, human embryonic kidney cells; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; NMR, nuclear magnetic resonance; PEG, poly(ethylene glycol).Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2010 The Protein Society step after solubilization using ANTI-FLAG affinity chromatography with yields of 30-40%. Purified receptors were functional. Binding of the agonist [ 3 H]muscimol to the purified GABA A R was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles.

No abstract

The nicotinic acetylcholine receptor (nAChR) is a member of the important Cys-loop ligand-gated ion channel superfamily that modulates neuronal excitability. After responding to their agonists, their actions are terminated either by removal of ligand or by fast and slow desensitization, processes that play an important role in modulating the duration of conducting states and hence of integrated neuronal behavior. We monitored structural changes occurring during fast and slow desensitization in the transmembrane domain of the Torpedo nAChR using time-resolved photolabeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID). After channel opening, TID photolabels a residue on the δ-subunit's M2-M3 loop and a cluster of four residues on δM1 and δM2, defining an open state pocket [Arevalo, E. et al. (2005) J. Biol. Chem. 280, 13631-13640]. We now find that photolabeling of this pocket persists during the transition to the fast desensitized state, decreasing only with the transition to the slow desensitized state. In contrast, photoincorporation in the channel lumen at the conserved 9′ leucines on the second transmembrane helix (M2-9′) decreased successively during the resting to open and open to fast desensitized state transitions, implying that the local conformation is different in each state, a conclusion consistent with the hypothesis that there are separate gates for channel opening and desensitization. Thus, although during fast desensitization there is a conformation change in the channel lumen at the level of M2-9′, there is none in the regions of the δ-subunit's M2-M3 loop and the interior of its M1-M4 helix bundle until slow desensitization occurs.The Cys-loop ligand-gated ion channel superfamily, which includes nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine type 3 (5-HT 3 ) receptors, γ-aminobutyric acid type A (GABA A ) receptors, and glycine receptors, has been studied intensively. These channels modulate synaptic and extrasynaptic neuronal excitability in response to their agonists and their actions are terminated either by removal of ligand or by desensitization, a process that plays an important role in modulating the duration of ligand-gated conducting states and hence of integrated neuronal behavior (1-3). A primary challenge is not only to understand how agonist binding in the extracellular ligand-binding domain (LBD) triggers a conformation *To whom correspondence may be addressed: Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 32 Fruit St., Boston. MA 02114. Tel.: 617-726-8985, Fax: 617-724-8644, k_miller@helix.mgh.harvard.edu. IHY and DCC contributed equally to this work. Supporting InformationSupporting information is available showing HPLC purification of labeled peptides and a molecular model. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 January 12. Published in final ...

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by Plasmodium falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the Plasmodium falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP119 and 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1.