The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB ( 5-HT3 ͉ ligand-gated ion channel ͉ polymorphism ͉ serotonin ͉ receptor kinetics S erotonin [5-hydroxytryptamine (5-HT)] is a major neurotransmitter in both the CNS and the peripheral nervous system (PNS), where it plays a key role in basic functions such as mood, sleep, appetite regulation, and libido (1). Serotonergic signaling is mediated via a plethora of G protein-coupled receptors, whereas only one serotonin-gated ion channel has been identified: the 5-HT 3 receptor (2).5-HT 3 receptors are expressed in both the CNS and the PNS. Presynaptic 5-HT 3 receptors are known to modulate the synaptic release of various neurotransmitters (3-5), whereas postsynaptic 5-HT 3 receptors are responsible for the fast excitatory response to serotonin (6). The role of the 5-HT 3 receptor in general physiology and pathophysiology is not well established. However, the 5-HT 3 receptor is known to mediate the nausea and emesis caused by radio/chemotherapy and anesthetics (7). Furthermore, 5-HT 3 antagonists have proven effective in the treatment of irritable bowel disease (7). Finally, the 5-HT 3 receptor has been suggested to be involved in anxiety, depression, pain, alcohol dependence, and eating disorders (7,8).The 5-HT 3 receptor is a nonselective cation channel belonging to the superfamily of Cys-loop ligand-gated ion channels that includes receptors for the neurotransmitters ACh, ␥-aminobutyric acid, and glycine. These receptors are pentameric assemblies, where the five subunits form a central ion channel path (9). To date, five 5-HT 3 subunits have been cloned: 5-HT 3A -5-HT 3E (10-12). Transcripts of 5-HT 3A , 5-HT 3B , and 5-HT 3C subunits have been demonstrated in both the CNS and the PNS (11-13), whereas 5-HT 3D and 5-HT 3E transcripts have been detected only in peripheral tissues (12). By using 5-HT 3B -specific antibodies, the existence of the 5-HT 3B subunit in the human hippocampus (14) as well as in the CNS in rodents (15) has been confirmed. Whereas homomeric 5-HT 3A receptors are functional, the other subunits only form functional receptors when coexpressed with 5-HT 3A (11,16). The homomeric 5-HT 3A and the heteromeric 5-HT 3AB receptors are the best characterized 5-HT 3 receptors, whereas the physiological relevance of subunits 5-HT 3c -5-HT 3E has just started to be unraveled. In heterologous expression systems, the 5-HT 3A :5-HT 3B stoichiometry an...

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.

The conductance of the BK channel was evaluated in reconstituted bilayers made of POPE/POPS (3.3:1), or POPE/POPS with an added 20% of either SPM (3.3:1:1), CER (3.3:1:1), or CHL (3.3:1:1). The presence of SPM, which is known to increase bilayer thickness, significantly reduced the conductance of the BK channel. To directly test the role of membrane thickness, the conductance of the BK channel was measured in bilayers formed from PCs with acyl chains of increasing length (C14:1-C24:1), all in the absence of SPM. Slope conductance was maximal at a chain length of (C18:1) and much reduced for both thinner (C14:1) and thicker (C24:1) bilayers, indicating that membrane thickness alone can modify slope conductance. Further, in a simplified binary mixture of DOPE/SPM that forms a confined, phase-separated bilayer, the measured conductance of BK channels shows a clear bimodal distribution. In contrast, the addition of CER, which has an acyl chain structure similar to SPM but without its bulky polar head group to POPE/POPS, was without effect, as was the addition of CHL. The surface structure of membranes made from these same lipid mixtures was examined with AFM. Incorporation of both SPM and CER resulted in the formation of microdomains in POPE/POPS monolayers, but only SPM promoted a substantial increase in the amount of the high phase observed for the corresponding bilayers. The addition of CHL to POPE/POPS eliminated the phase separation observed in the POPE/POPS bilayer. The decrease in channel conductance observed with the incorporation of SPM into POPE/POPS membranes was, therefore, attributed to larger SPM-rich domains that appear thicker than the neighboring bilayer.