A ciliary synapse for "short-circuit" neuromodulation.

Nearly all neurons contain a primary cilium, but little is known about how this compartment contributes to neuromodulatory signaling. In a new study, Sheu et al. use cutting-edge electron microscopy and fluorescence imaging techniques to reveal a new type of synapse that enables chemical transmission between serotonergic axons and the primary cilia of hippocampal neurons.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Profiling the proximal proteome of the activated µ-opioid receptor.

The µ-opioid receptor (µOR) represents an important target of therapeutic and abused drugs. So far, most understanding of µOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet µOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated µOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the µOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new µOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin- dependent kinase II holoenzyme.

Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin-binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.

The GABAB (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly rectifying potassium channels (GIRKs)1,2. GABAB-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3-5. However, the mechanistic basis for KCTD modulation of GABAB signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABAB receptors and G-protein ßγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gßγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gßγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signalling by KCTD proteins.

Structural and Biophysical Mechanisms of Class C G Protein-Coupled Receptor Function.

Groundbreaking structural and spectroscopic studies of class A G protein-coupled receptors (GPCRs), such as rhodopsin and the ß2 adrenergic receptor, have provided a picture of how structural rearrangements between transmembrane helices control ligand binding, receptor activation, and effector coupling. However, the activation mechanism of other GPCR classes remains more elusive, in large part due to complexity in their domain assembly and quaternary structure. In this review, we focus on the class C GPCRs, which include metabotropic glutamate receptors (mGluRs) and gamma-aminobutyric acid B (GABAB) receptors (GABABRs) most prominently. We discuss the unique biophysical questions raised by the presence of large extracellular ligand-binding domains (LBDs) and constitutive homo/heterodimerization. Furthermore, we discuss how recent studies have begun to unravel how these fundamental class C GPCR features impact the processes of ligand binding, receptor activation, signal transduction, regulation by accessory proteins, and crosstalk with other GPCRs.

Deuteration as a General Strategy to Enhance Azobenzene-Based Photopharmacology.

Chemical photoswitches have become a widely used approach for the remote control of biological functions with spatiotemporal precision. Several molecular scaffolds have been implemented to improve photoswitch characteristics, ranging from the nature of the photoswitch itself (e.g. azobenzenes, dithienylethenes, hemithioindigo) to fine-tuning of aromatic units and substituents. Herein, we present deuterated azobenzene photoswitches as a general means of enhancing the performance of photopharmacological molecules. Deuteration can improve azobenzene performance in terms of light sensitivity (higher molar extinction coefficient), photoswitch efficiency (higher photoisomerization quantum yield), and photoswitch kinetics (faster macroscopic rate of photoisomerization) with minimal alteration to the underlying structure of the photopharmacological ligand. We report synthesized deuterated azobenzene-based ligands for the optimized optical control of ion channel and G protein-coupled receptor (GPCR) function in live cells, setting the stage for the straightforward, widespread adoption of this approach.

Sulfonated red and far-red rhodamines to visualize SNAP- and Halo-tagged cell surface proteins.

The (in)ability to permeate membranes is a key feature of chemical biology probes that defines their suitability for specific applications. Here we report sulfonated rhodamines that endow xanthene dyes with cellular impermeability for analysis of surface proteins. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to benzylguanine and choloralkane substrates for SNAP-tag and Halo-tag labelling. Sulfonated rhodamine-conjugated fluorophores maintain desirable photophysical properties, such as brightness and photostability. While transfected cells with a nuclear localized SNAP-tag remain unlabelled, extracellular exposed tags can be cleanly visualized. By multiplexing with a permeable rhodamine, we are able to differentiate extra- and intracellular SNAP- and Halo-tags, including those installed on the glucagon-like peptide-1 receptor, a prototypical class B G protein-coupled receptor. Sulfo549 and Sulfo646 also labelled transfected neurons derived from induced pluripotent stem cells (iPSCs), allowing STED nanoscopy of the axonal membrane. Together, this work provides a new avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags. We anticipate that Sulfo549, Sulfo646 and their congeners will be useful for a number of cell biology applications where labelling of intracellular sites interferes with accurate surface protein analysis.

Photoswitchable Serotonins for Optical Control of the 5-HT2A Receptor.

Serotonin receptors play central roles in neuromodulation and are critical drug targets for psychiatric disorders. Optical control of serotonin receptor subtypes has the potential to greatly enhance our understanding of the spatiotemporal dynamics of receptor function. While other neuromodulatory receptors have been successfully rendered photoswitchable, reversible photocontrol of serotonin receptors has not been achieved, representing a major gap in GPCR photopharmacology. Herein, we develop the first tools that allow for such control. Azo5HT-2 shows light-dependent 5-HT2A R agonism, with greater activity in the cis-form. Based on docking and test compound analysis, we also develop photoswitchable orthogonal, remotely-tethered ligands (PORTLs). These BG-Azo5HTs provide rapid, reversible, and repeatable optical control following conjugation to SNAP-tagged 5-HT2A R. Overall, this study provides a foundation for the broad extension of photopharmacology to the serotonin receptor family.

Conformational dynamics of a class C G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in eukaryotes. Crystal structures have provided insight into GPCR interactions with ligands and G proteins, but our understanding of the conformational dynamics of activation is incomplete. Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders. A 'clamshell' ligand-binding domain (LBD), which contains the ligand-binding site, is coupled to the transmembrane domain via a cysteine-rich domain, and LBD closure seems to be the first step in activation. Crystal structures of isolated mGluR LBD dimers led to the suggestion that activation also involves a reorientation of the dimer interface from a 'relaxed' to an 'active' state, but the relationship between ligand binding, LBD closure and dimer interface rearrangement in activation remains unclear. Here we use single-molecule fluorescence resonance energy transfer to probe the activation mechanism of full-length mammalian group II mGluRs. We show that the LBDs interconvert between three conformations: resting, activated and a short-lived intermediate state. Orthosteric agonists induce transitions between these conformational states, with efficacy determined by occupancy of the active conformation. Unlike mGluR2, mGluR3 displays basal dynamics, which are Ca(2+)-dependent and lead to basal protein activation. Our results support a general mechanism for the activation of mGluRs in which agonist binding induces closure of the LBDs, followed by dimer interface reorientation. Our experimental strategy should be widely applicable to study conformational dynamics in GPCRs and other membrane proteins.

Dual optical control and mechanistic insights into photoswitchable group II and III metabotropic glutamate receptors.

G protein-coupled receptor (GPCR) signaling occurs in complex spatiotemporal patterns that are difficult to probe using standard pharmacological and genetic approaches. A powerful approach for dissecting GPCRs is to use light-controlled pharmacological agents that are tethered covalently and specifically to genetically engineered receptors. However, deficits in our understanding of the mechanism of such photoswitches have limited application of this approach and its extension to other GPCRs. In this study, we have harnessed the power of bioorthogonal tethering to SNAP and CLIP protein tags to create a family of light-gated metabotropic glutamate receptors (mGluRs). We define the mechanistic determinants of photoswitch efficacy, including labeling efficiency, dependence on photoswitch structure, length dependence of the linker between the protein tag and the glutamate ligand, effective local concentration of the glutamate moiety, and affinity of the receptor for the ligand. We improve the scheme for photoswitch synthesis as well as photoswitch efficiency, and generate seven light-gated group II/III mGluRs, including variants of mGluR2, 3, 6, 7, and 8. Members of this family of light-controlled receptors can be used singly or in specifically labeled, independently light-controlled pairs for multiplexed control of receptor populations.

Genetically Targeted Optical Control of an Endogenous G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in biology. However, our understanding of their function in complex living systems is limited because we lack tools that can target individual receptors with sufficient precision. State-of-the-art approaches, including DREADDs, optoXRs, and PORTL gated-receptors, control GPCR signaling with molecular, cell type, and temporal specificity. Nonetheless, these tools are based on engineered non-native proteins that may (i) express at nonphysiological levels, (ii) localize and turnover incorrectly, and/or (iii) fail to interact with endogenous partners. Alternatively, membrane-anchored ligands (t-toxins, DARTs) target endogenous receptors with molecular and cell type specificity but cannot be turned on and off. In this study, we used a combination of chemistry, biology, and light to control endogenous metabotropic glutamate receptor 2 (mGluR2), a Family C GPCR, in primary cortical neurons. mGluR2 was rapidly, reversibly, and selectively activated with photoswitchable glutamate tethered to a genetically targeted-plasma membrane anchor (membrane anchored Photoswitchable Orthogonal Remotely Tethered Ligand; maPORTL). Photoactivation was tuned by adjusting the length of the PORTL as well as the expression level and geometry of the membrane anchor. Our findings provide a template for controlling endogenous GPCRs with cell type specificity and high spatiotemporal precision.

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics.

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist.

Family A G protein-coupled receptors (GPCRs) control diverse biological processes and are of great clinical relevance. Their archetype rhodopsin becomes naturally light sensitive by binding covalently to the photoswitchable tethered ligand (PTL) retinal. Other GPCRs, however, neither bind covalently to ligands nor are light sensitive. We sought to impart the logic of rhodopsin to light-insensitive Family A GPCRs in order to enable their remote control in a receptor-specific, cell-type-specific, and spatiotemporally precise manner. Dopamine receptors (DARs) are of particular interest for their roles in motor coordination, appetitive, and aversive behavior, as well as neuropsychiatric disorders such as Parkinson's disease, schizophrenia, mood disorders, and addiction. Using an azobenzene derivative of the well-known DAR ligand 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), we were able to rapidly, reversibly, and selectively block dopamine D1 and D2 receptors (D1R and D2R) when the PTL was conjugated to an engineered cysteine near the dopamine binding site. Depending on the site of tethering, the ligand behaved as either a photoswitchable tethered neutral antagonist or inverse agonist. Our results indicate that DARs can be chemically engineered for selective remote control by light and provide a template for precision control of Family A GPCRs.

Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid.

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.

Emerging modes of regulation of neuromodulatory G protein-coupled receptors.

In the nervous system, G protein-coupled receptors (GPCRs) control neuronal excitability, synaptic transmission, synaptic plasticity, and, ultimately, behavior through spatiotemporally precise initiation of a variety of signaling pathways. However, despite their critical importance, there is incomplete understanding of how these receptors are regulated to tune their signaling to specific neurophysiological contexts. A deeper mechanistic picture of neuromodulatory GPCR function is needed to fully decipher their biological roles and effectively harness them for the treatment of neurological and psychiatric disorders. In this review, we highlight recent progress in identifying novel modes of regulation of neuromodulatory GPCRs, including G protein- and receptor-targeting mechanisms, receptor-receptor crosstalk, and unique features that emerge in the context of chemical synapses. These emerging principles of neuromodulatory GPCR tuning raise critical questions to be tackled at the molecular, cellular, synaptic, and neural circuit levels in the future.

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP.

It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαßγ) regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gßγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs, yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.