Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition.

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. In this study, the role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the human A2A adenosine receptor was examined. To this end, cysteine-to-alanine mutations were conducted to replace individual and both cysteines in three disulfide bonds present in the first two extracellular loops. Although none of the disulfide bonds were essential for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity. These results indicate that in contrast to many class A GPCRs, the extracellular disulfide bonds of the A2A receptor are not essential, but can modulate the ligand-binding activity, by either changing the conformation of the extracellular loops or perturbing the interactions of the transmembrane domains.

Structural model of the open-closed-inactivated cycle of prokaryotic voltage-gated sodium channels.

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.

Differential lipid dependence of the function of bacterial sodium channels.

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative (22)Na(+) uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different "guest" glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.

Molecular dynamics of ion transport through the open conformation of a bacterial voltage-gated sodium channel.

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.

Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing.

Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing.

Simplified bacterial "pore" channel provides insight into the assembly, stability, and structure of sodium channels.

Eukaryotic sodium channels are important membrane proteins involved in ion permeation, homeostasis, and electrical signaling. They are long, multidomain proteins that do not express well in heterologous systems, and hence, structure/function and biochemical studies on purified sodium channel proteins have been limited. Bacteria produce smaller, homologous tetrameric single domain channels specific for the conductance of sodium ions. They consist of N-terminal voltage sensor and C-terminal pore subdomains. We designed a functional pore-only channel consisting of the final two transmembrane helices, the intervening P-region, and the C-terminal extramembranous region of the sodium channel from the marine bacterium Silicibacter pomeroyi. This sodium "pore" channel forms a tetrameric, folded structure that is capable of supporting sodium flux in phospholipid vesicles. The pore-only channel is more thermally stable than its full-length counterpart, suggesting that the voltage sensor subdomain may destabilize the full-length channel. The pore subdomains can assemble, fold, and function independently from the voltage sensor and exhibit similar ligand-blocking characteristics as the intact channel. The availability of this simple pore-only construct should enable high-level expression for the testing of potential new ligands and enhance our understanding of the structural features that govern sodium selectivity and permeability.

Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking.

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A(2)a (hA(2)aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA(2)aR, as expected, and partially cleaved for hA(2)bR, hCCR5R, and hD(2L)R. Ligand-binding assays conducted on the adenosine family (hA(1)R, hA(2)aR, hA(2)bR, and hA(3)R) of receptors show that hA(2)aR and hA(2)bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.

Heterologous GPCR expression: a bottleneck to obtaining crystal structures.

G protein-coupled receptors (GPCRs) are an important, medically relevant class of integral membrane proteins. Laboratories throughout all disciplines of science devote time and energy into developing practical methods for the discovery, isolation, and characterization of these proteins. Since the crystal structure of rhodopsin was solved 6 years ago, the race to determine high-resolution structures of more GPCRs has gained momentum. Since certain GPCRs are currently produced at sufficient levels for X-ray crystallography trials, it is speculated that heterologous expression of GPCRs may no longer be a bottleneck in obtaining crystal structures. This Review focuses on the current approaches in heterologous expression of GPCRs and explores the problems associated with obtaining crystal structures from GPCRs expressed in different systems. Although milligram amounts of certain GPCRs are attainable, the majority of GPCRs are still either produced at very low levels or not at all. Developing reliable expression techniques for GPCRs is still a major priority for the structural characterization of GPCRs.