Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.

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Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligandinduced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute Ϸ1% of the cell membrane and to be sensitive to cholesterol depletion.ACP labeling ͉ G protein-coupled receptor (GPCR) oligomerization G protein-coupled receptors (GPCRs) were for a long time presumed to be distributed in the plasma membrane exclusively in a monomeric form (1, 2), but recent reports have unveiled a more complex behavior; in particular, dimeric structures have been found for several GPCRs using biochemical and biophysical methods (3-9). Dimerization can occur between receptors of the same subtype (homodimerization) or of different subtypes (heterodimerization). Some GPCRs remain dimeric all of the time, whereas others cycle between monomeric and dimeric states in a ligand-regulated process (7). Although GPCR homodimerization seems to be important for receptor ontology and trafficking, heterodimerization might result in altered ligand selectivity and distinctive coupling to signal transduction pathways, providing an additional possibility for the fine tuning of cellular signaling.In addition to dimerization, the lateral distribution of GPCRs in cell membranes has been extensively debated recently. Several reports based on biochemical (10), plasmon-resonance spectroscopy (11), single-molecule microscopy (12), and fluorescence recovery after photobleaching experiments (13) propose that GPCRs are localized in microdomains, but a clear demonstration of the existence and nature of such microdomains in living cells remains elusive, in particular because the interpretation of biochemical data can be rather equivocal (14-17). Compartmentalization in form of microdomains was proposed to explain the efficiency of signal transduction at the low physiological surface concentrations of the signaling partners by their enrichment inside specialized signaling platforms (10, 18).Bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) experiments have gained increasing interest to investigate these two central questions on GPCR signaling. (i) They can be p...

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