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Neurotransmitter/[Na.sup.+] symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the [Na.sup.+] electrochemical gradient (1-6). NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine (1), as well as by antidepressants (7). The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two [Na.sup.+] are bound deep within the protein (8). This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold (9-22). Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan (23), and the [Na.sup.+]-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy (24). In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle (22). We investigated how substrate binding is coupled to structural transitions in LeuT during [Na.sup.+]-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ~10-fold faster than leucine (15,25), we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extra-cellular vestibule (15,20). Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 A away, in a manner that allows the [Na.sup.+] gradient to power the transport mechanism., The experiments were performed on LeuT engineered to contain a 15-amino-acid, carboxy-terminal biotinylation domain (26) and site-specifically labelled with the fluorophores Cy3 and Cy5 maleimide at residue position 7, after [...]

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His 250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation., Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) [1] transport saccharides across the cell membrane and phosphorylate them before their release into the cytosol (2-7). Phosphorylation of the incoming saccharide primes it for [...]

The TrkH/TrkG/KtrB proteins mediate [K.sup.+] uptake in bacteria and probably evolved from simple [K.sup.+] channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of [K.sup.+] channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of [K.sup.+] and [Rb.sup.+] over smaller ions such as [Na.sup.+] or [Li.sup.+]. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of [K.sup.+] flux. These results reveal the molecular basis of [K.sup.+] selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins., [K.sub.+] is highly concentrated in all living cells and plays diverse physiological roles such as setting membrane potential (1), regulating turgor pressure (2) and maintaining intracellular pH (3-5). Because [K.sup.+] [...]

Neurotransmitter: [NA.sup.+] symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the [NA.sup.+] gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport., The neurotransmitten [Na.sup.+] symporter (NSS) family stops cellular signalling by recapturing released neurotransmitter (1-3). These secondary active transporters enable the thermodynamically uphill transport of their substrates across the plasma membrane [...]

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the [sub.2] adrenergic receptor ([sub.2]AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G[sub.s] heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound [sub.2]ARG[sub.s] complexes are formed and the efficiency of nucleotide exchange leading to G[sub.s] activation. These data also reveal transient nucleotide-bound [sub.2]ARG[sub.s] species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism., Author(s): G. Glenn Gregorio [1]; Matthieu Masureel [2]; Daniel Hilger [2]; Daniel S. Terry [1]; Manuel Juette [1]; Hong Zhao [1]; Zhou Zhou [1]; Jose Manuel Perez-Aguilar [1]; Maria Hauge [...]

The binding site of the dopamine [D.sub.2] receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs), The high-resolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2). which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligand-binding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, 1184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs.

Background and Purpose: The cannabinoid CB2 receptor (CB2) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB2 desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β‐arrestin recruitment to CB2. Mutagenesis of several distal C‐terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB2. Experimental Approach In CB2‐expressing HEK 293 cells, β‐arrestin translocation was measured using real‐time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB2 in the presence of β‐arrestin 2. Key Results: Overexpression of GRK isoforms 1–6 failed to considerably improve translocation of either β‐arrestin 1 or β‐arrestin 2 to CB2. Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce β‐arrestin 2 translocation. Mutagenesis of C‐terminal aspartic acid residues resulted in attenuation of β‐arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation. Conclusion and Implications: Our findings suggest that CB2 does not adhere to the classical GPCR regulatory paradigm, entailing GRK‐mediated and β‐arrestin‐mediated desensitisation. Instead, C‐terminal aspartic acid residues may act as phospho‐mimics to induce β‐arrestin activation. This study provides novel insights into the regulatory mechanisms of CB2, which may aid in our understanding of drug tolerance and dependence. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB5 toxin, inhibits Gαi/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gαz. The role of Gαz signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gαi/o and Gαz G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein. A recently identified pertussis toxin-like AB5 toxin, OZITX, is found to inhibit Gαi/o and Gαz G proteins. In combination with directed mutations, it is a useful tool for interrogating Gαi/o/z G protein subunits individually. [ABSTRACT FROM AUTHOR]

Research has been conducted on the binding site of the dopamine D2 receptor contained within a water-accessible crevice formed among its transmembrane segments. The residues contributing to this binding-site crevice surface have been mapped via the substituted-cysteine-accessibility method and the results are discussed.

Research has been conducted on the mu, delta and kappa opioid receptor transmembrane domains (TMs). The use of methanethiosulfonate-insensitive mutants of mu and kappa opiod receptors and a wild-type delta receptor and application of the cystein accessibility method to identify amino acid residues within TMs are discussed.