G protein ß4 as a structural determinant of enhanced nucleotide exchange in the A2AAR-Gs complex.

Adenosine A2A receptors (A2AAR) evoke pleiotropic intracellular signaling events via activation of the stimulatory heterotrimeric G protein, Gs. Here, we used cryoEM to solve the agonist-bound structure of A2AAR in a complex with full-length Gs α and Gß4γ2 (A2AAR-Gs α:ß4γ2). The orthosteric binding site of A2AAR-Gs α:ß4γ2 was similar to other structures of agonist-bound A2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gß4 versus the closest analog, A2AAR-miniGs α:ß1γ2. Consequently, there are fewer interactions between the switch II in Gs α and the Gß4 torus. In reconstitution experiments Gß4γ2 displayed a ten-fold higher efficiency over Gß1γ2 in catalyzing A2AAR dependent GTPγS binding to Gs α. We propose that the less constrained switch II in A2AAR-Gs α:ß4γ2 accounts for this increased efficiency. These results suggest that Gß4 functions as a positive allosteric enhancer versus Gß1.

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry.

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, "crossmember" helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.

A model for how Gßγ couples Gα to GPCR.

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gßγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine-proline-phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR-G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ-rhodopsin interactions may facilitate GPCR dimer transactivation.

Binding of ß4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry.

Characterization of G protein ßγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native ßγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and ßγ labeled with heavy isotopes purified. Heavy ßγ was combined with either recombinant ßγ purified from Sf9 cells, ßγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched ßγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing ß and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three ß isoforms, ß(1), ß(2), and ß(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. ß(1) and γ(5) were most abundant in the enriched ßγ fraction, and this ßγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more ß(4) and γ(5) compared to the enriched ßγ fraction; also, more ß(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched ßγ fraction. These results suggest that preferences for particular ßγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.

A Steric "Ball-and-Chain" Mechanism for pH-Mediated Regulation of Gap Junction Channels.

Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric "ball-and-chain" gating particle.

Glycosylation of CaV3.2 Channels Contributes to the Hyperalgesia in Peripheral Neuropathy of Type 1 Diabetes.

Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.

Structural determinants involved in the formation and activation of G protein betagamma dimers.

Heterotrimeric G proteins, composed of an alpha, beta and gamma subunit, represent one of the most important and dynamic families of signaling proteins. As a testament to the significance of G protein signaling, the hundreds of seven-transmembrane-spanning receptors that interact with G proteins are estimated to occupy 1-2% of the human genome. This broad diversity of receptors is echoed in the number of potential heterotrimer combinations that can arise from the 23 alpha subunit, 7 beta subunit and 12 gamma subunit isoforms that have been identified. The potential for such vast complexity implies that the receptor G protein interface is the site of much regulation. The historical model for the activation of a G protein holds that activated receptor catalyzes the exchange of GDP for GTP on the alpha subunit, inducing a conformational change that substantially lowers the affinity of alpha for betagamma. This decreased affinity enables dissociation of betagamma from alpha and receptor. The free form of betagamma is thought to activate effectors, until the hydrolysis of GTP by G alpha (aided by RGS proteins) allows the subunits to re-associate, effectively deactivating the G protein until another interaction with activated receptor.

Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

Ca2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K+ channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K+ channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells.

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

Reconstitution of G protein-coupled receptors with recombinant G protein alpha and beta gamma subunits.

The methods outlined in this article describe experiments that can probe the first steps in receptor:G protein interaction using defined, recombinant receptors and G proteins. The protocols have the advantages that the receptors are inserted properly in a cell membrane and that the investigator has complete control of the proteins reconstituted with the receptor. Specific mutations in the receptors or G proteins are studied easily and the protocols allow precise examination of the stoichiometry of the receptor:alpha:beta gamma interaction.

Function and dynamics of macromolecular complexes explored by integrative structural and computational biology.

Three vignettes exemplify the potential of combining EM and X-ray crystallographic data with molecular dynamics (MD) simulation to explore the architecture, dynamics and functional properties of multicomponent, macromolecular complexes. The first two describe how EM and X-ray crystallography were used to solve structures of the ribosome and the Arp2/3-actin complex, which enabled MD simulations that elucidated functional dynamics. The third describes how EM, X-ray crystallography, and microsecond MD simulations of a GPCR:G protein complex were used to explore transmembrane signaling by the ß-adrenergic receptor. Recent technical advancements in EM, X-ray crystallography and computational simulation create unprecedented synergies for integrative structural biology to reveal new insights into heretofore intractable biological systems.

Reversal of neuropathic pain in diabetes by targeting glycosylation of Ca(V)3.2 T-type calcium channels.

It has been established that Ca(V)3.2 T-type voltage-gated calcium channels (T-channels) play a key role in the sensitized (hyperexcitable) state of nociceptive sensory neurons (nociceptors) in response to hyperglycemia associated with diabetes, which in turn can be a basis for painful symptoms of peripheral diabetic neuropathy (PDN). Unfortunately, current treatment for painful PDN has been limited by nonspecific systemic drugs with significant side effects or potential for abuse. We studied in vitro and in vivo mechanisms of plasticity of Ca(V)3.2 T-channel in a leptin-deficient (ob/ob) mouse model of PDN. We demonstrate that posttranslational glycosylation of specific extracellular asparagine residues in Ca(V)3.2 channels accelerates current kinetics, increases current density, and augments channel membrane expression. Importantly, deglycosylation treatment with neuraminidase inhibits native T-currents in nociceptors and in so doing completely and selectively reverses hyperalgesia in diabetic ob/ob mice without altering baseline pain responses in healthy mice. Our study describes a new mechanism for the regulation of Ca(V)3.2 activity and suggests that modulating the glycosylation state of T-channels in nociceptors may provide a way to suppress peripheral sensitization. Understanding the details of this regulatory pathway could facilitate the development of novel specific therapies for the treatment of painful PDN.

Protein kinase A activity controls the regulation of T-type CaV3.2 channels by Gbetagamma dimers.

Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gbetagamma dimers, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gbeta(2)gammax dimers to effect voltage-independent inhibition of Ca(v)3.2 channels. Inhibition requires phosphorylation of Ser(1107), a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gbeta(2)gamma(2) dimers but does not disrupt dimer binding nor change its specificity. Gbetagamma dimers released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Ca(v)3.2 whole cell current is precluded by Gbetagamma-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, D(1) or D(2) agonists do not elicit channel inhibition yet together synergize to inhibit Ca(v)3.2 channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Ca(v)3.2 channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides.

We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.

Differential sensitivity of P-Rex1 to isoforms of G protein betagamma dimers.

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.

Influence of differential stability of G protein ßγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-ß.

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.

Inhibition of a background potassium channel by Gq protein alpha-subunits.

Two-pore-domain K(+) channels provide neuronal background currents that establish resting membrane potential and input resistance; their modulation provides a prevalent mechanism for regulating cellular excitability. The so-called TASK channel subunits (TASK-1 and TASK-3) are widely expressed, and they are robustly inhibited by receptors that signal through Galphaq family proteins. Here, we manipulated G protein expression and membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels in intact and cell-free systems to provide electrophysiological and biochemical evidence that inhibition of TASK channels by Galphaq-linked receptors proceeds unabated in the absence of phospholipase C (PLC) activity, and instead involves association of activated Galphaq subunits with the channels. Receptor-mediated inhibition of TASK channels was faster and less sensitive to a PLCbeta1-ct minigene construct than inhibition of PIP(2)-sensitive Kir3.4(S143T) homomeric channels that is known to be dependent on PLC. TASK channels were strongly inhibited by constitutively active Galphaq, even by a mutated version that is deficient in PLC activation. Receptor-mediated TASK channel inhibition required exogenous Galphaq expression in fibroblasts derived from Galphaq/11 knockout mice, but proceeded unabated in a cell line in which PIP(2) levels were reduced by regulated overexpression of a lipid phosphatase. Direct application of activated Galphaq, but not other G protein subunits, inhibited TASK channels in excised patches, and constitutively active Galphaq subunits were selectively coimmunoprecipitated with TASK channels. These data indicate that receptor-mediated TASK channel inhibition is independent of PIP(2) depletion, and they suggest a mechanism whereby channel modulation by Galphaq occurs through direct interaction with the ion channel or a closely associated intermediary.

Inhibition of mammalian Gq protein function by local anesthetics.

BACKGROUND: Local anesthetics have been shown to selectively inhibit functioning of Xenopus laevis Gq proteins. It is not known whether a similar interaction exists with mammalian G proteins. The goal of this study was to determine whether mammalian Gq protein is inhibited by local anesthetics. METHODS: In Xenopus oocytes, the authors replaced endogenous Gq protein with mouse Gq (expressed in Sf9 cells using baculovirus vectors). Cells endogenously expressing lysophosphatidic acid or recombinantly expressing muscarinic m3 receptors were injected with phosphorothioate DNA antisense (or sense as control) oligonucleotides against Xenopus Gq. Forty-eight hours later, oocytes were injected with purified mouse Gq (5 x 10(-8) M) or solvent as control. Two hours later, the authors injected either lidocaine, its permanently charged analog QX314 (at IC50, 50 nl), or solvent (KCl 150 mM) as control and measured Ca-activated Cl currents in response to lysophosphatidic acid or methylcholine (one tenth of EC50). RESULTS: Injection of anti-Gq reduced the mean response size elicited by lysophosphatidic acid to 33 +/- 7% of the corresponding control response. In contrast, responses were unchanged (131 +/- 29% of control) in cells in addition injected with mouse Gq protein. Injection of mouse Gq protein "rescued" the inhibitory effect of intracellularly injected QX314: whereas QX314 was without effect on Gq-depleted oocytes, responses to lysophosphatidic acid after QX314 injection were inhibited to 44 +/- 10% of control response in cells in addition injected with mouse Gq protein (5 x 10(-8) M). Similar results were obtained for m3 signaling and intracellularly injected lidocaine. CONCLUSION: Inhibition of Gq function by local anesthetics is not restricted to Xenopus G proteins. Therefore, Gq should be considered as one additional intracellular target site for local anesthetics, especially relevant for those effects not explainable by sodium channel blockade (e.g., antiinflammatory effects).