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The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

SUMMARYGenome-wide association analysis of electroencephalographic endophenotypes for alcohol use disorder has identified non-coding polymorphisms within theKCNJ6gene.KCNJ6encodes the GIRK2 protein, a subunit of a G-protein-coupled inwardly-rectifying potassium channel that regulates neuronal excitability. To elucidate how GIRK2 affects neuronal excitability and the response to ethanol exposure, we upregulatedKCNJ6in human glutamatergic neurons derived from induced pluripotent stem cells using two distinct strategies: CRISPRa induction and lentiviral expression. Multi-electrode-arrays, calcium imaging, patch-clamp electrophysiology, and mitochondrial stress tests collectively show that elevated GIRK2 acts in concert with 7-21 days of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate sensitivity, and to promote an increase intrinsic excitability. Furthermore, ethanol exposure did not alter basal nor activity-dependent mitochondrial respiration in elevated GIRK2 neurons. These data highlight a role for GIRK2 in mitigating the effects of ethanol on neuronal glutamatergic signaling and mitochondrial activity.Graphical Abstract

Understanding the signal transmission and processing within the central nervous system (CNS) is a grand challenge in neuroscience. The past decade has witnessed significant advances in the development of new tools to address this challenge. Development of these new tools draws diverse expertise from genetics, materials science, electrical engineering, photonics and other disciplines. Among these tools, nanomaterials have emerged as a unique class of neural interfaces due to their small size, remote coupling and conversion of different energy modalities, various delivery methods, and mitigated chronic immune responses. In this review, we will discuss recent advances in nanotransducers to modulate and interface with the neural system without physical wires. Nanotransducers work collectively to modulate brain activity through optogenetic, mechanical, thermal, electrical and chemical modalities. We will compare important parameters among these techniques including the invasiveness, spatiotemporal precision, cell-type specificity, brain penetration, and translation to large animals and humans. Important areas for future research include a better understanding of the nanomaterials-brain interface, integration of sensing capability for bidirectional closed-loop neuromodulation, and genetically engineered functional materials for cell-type specific neuromodulation.

G protein-gated inwardly rectifying potassium (GIRK) channels are essential regulators of cell excitability in the brain. While they are implicated in a variety of neurological diseases in both human and animal model studies, their therapeutic potential has been largely untapped. Here, we review recent advances in the development of small molecule compounds that specifically modulate GIRK channels and compare them with first-generation compounds that exhibit off-target activity. We describe the method of discovery of these small molecule modulators, their chemical features, and their effects in vivo. These studies provide a promising outlook on the future development of subunit-specific GIRK modulators to regulate neuronal excitability in a brain region-specific manner.

Remote and minimally-invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold-coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad-PC-Rad as a tool for the delivery of bioactive molecules into brain tissue. Near-infrared picosecond laser pulses activated the gold-coating on the surface of nanovesicles, creating nanomechanical stress and leading to near-complete vesicle cargo release in sub-seconds. Compared to natural phospholipid liposomes, the photo-release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

Neuropeptides are essential signaling molecules in the nervous system involved in modulating neural circuits and behavior. Although hypothesized to signal via volume transmission through G-protein coupled receptors (GPCR), remarkably little is known about their extrasynaptic diffusion. Here, we developed an all-optical approach to probe neuropeptide volume transmission in mouse neocortex. To control neuropeptide release, we engineered photosensitive nanovesicles with somatostatin-14 (SST) that is released with near-infrared light stimulation. To detect SST, we created a new cell-based neurotransmitter fluorescent engineered reporter (CNiFER) using the SST2 GPCR. Under two-photon imaging, we determined the time to activate SST2R at defined distances as well as the maximal distance and loss rate for SST volume transmission in neocortex. Importantly, we determined that SST transmission is significantly faster in neocortex with a chemically degraded extracellular matrix, a diseased condition indicated in neuroinflammation and Parkinson’s disease. These new neurotechnologies can reveal important biological signaling processes previously not possible, and provide new opportunities to investigate volume transmission in the brain.

Dopaminergic neurons are critical to movement, mood, addiction, and stress. Current techniques for generating dopaminergic neurons from human induced pluripotent stem cells (hiPSCs) yield heterogenous cell populations with variable purity and inconsistent reproducibility between donors, hiPSC clones, and experiments. Here, we report the rapid (5 weeks) and efficient (~90%) induction of induced dopaminergic neurons (iDANs) through transient overexpression of lineage-promoting transcription factors combined with stringent selection across five donors. We observe maturation-dependent increase in dopamine synthesis and electrophysiological properties consistent with midbrain dopaminergic neuron identity, such as slow-rising after- hyperpolarization potentials, an action potential duration of ~3 ms, tonic sub-threshold oscillatory activity, and spontaneous burst firing at a frequency of ~1.0-1.75 Hz. Transcriptome analysis reveals robust expression of genes involved in fetal midbrain dopaminergic neuron identity. Specifically expressed genes in iDANs, as well as those from isogenic induced GABAergic and glutamatergic neurons, were enriched in loci conferring heritability for cannabis use disorder, schizophrenia, and bipolar disorder; however, each neuronal subtype demonstrated subtype-specific heritability enrichments in biologically relevant pathways, and iDANs alone were uniquely enriched in autism spectrum disorder risk loci. Therefore, iDANs provide a critical tool for modeling midbrain dopaminergic neuron development and dysfunction in psychiatric disease.

The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABABreceptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+. Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENTDysregulation of GABABreceptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+. We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions.

Metabotropic GABA B receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABA B receptor complexes contain the principal subunits GABA B1 and GABA B2 , which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABA B receptors and modify the kinetics of GABA B receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABA B receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABA B2 receptor. A single GABA B2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABA B2 –KCTD16 interface disrupted both the biochemical association and functional modulation of GABA B receptors and G protein-activated inwardly rectifying K + channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABA B receptors. Defining the binding interface of GABA B receptor and KCTD reveals a potential regulatory site for modulating GABA B -receptor function in the brain.

In their pioneering study on dopamine release, Romo and Schultz speculated “…that the amount of dopamine released by unmodulated spontaneous impulse activity exerts a tonic, permissive influence on neuronal processes more actively engaged in preparation of self-initiated movements….”(1) Motivated by the suggestion of “spontaneous impulses,” as well as by the “ramp up” of dopaminergic neuronal activity that occurs when rodents navigate to a reward,(2–5) we asked two questions. First, are there spontaneous impulses of dopamine that are released in cortex? Using cell-based optical sensors of extrasynaptic dopamine, [DA](ex),(6) we found that spontaneous dopamine impulses in cortex of naive mice occur at a rate of ∼0.01 per second. Next, can mice be trained to change the amplitude and/or timing of dopamine events triggered by internal brain dynamics, much as they can change the amplitude and timing of dopamine impulses based on an external cue?(7–9) Using a reinforcement learning paradigm based solely on rewards that were gated by feedback from real-time measurements of [DA](ex), we found that mice can volitionally modulate their spontaneous [DA](ex). In particular, by only the second session of daily, hour-long training, mice increased the rate of impulses of [DA](ex), increased the amplitude of the impulses, and increased their tonic level of [DA](ex) for a reward. Critically, mice learned to reliably elicit [DA](ex) impulses prior to receiving a reward. These effects reversed when the reward was removed. We posit that spontaneous dopamine impulses may serve as a salient cognitive event in behavioral planning.

G protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability and have been implicated in a number of neurological diseases. In addition to G proteins, GIRK channels are also regulated by membrane cholesterol, which is elevated in the brain for neurodegenerative diseases like Alzheimer’s Dementia and Parkinson’s Disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and PIP2. The structures reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain. Our analysis suggests that CHS stabilizes PIP2 interaction with the channel and promotes the engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of residues in one CHS binding pocket occludes cholesterol-dependent potentiation. Elucidating the cholesterol-binding site and characterizing the structural mechanisms underlying modulation of neuronal GIRK channels could facilitate the development of novel therapeutics for treating human diseases.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen–Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient’s DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K+ selectivity and gained Ca2+ permeability. Notably, the inward current was inhibited by the Na+ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen–Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.

Summary Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (, Highlights • hiPSC-derived astrocyte populations generated from 42 NPC lines • Transcriptomic analysis shows hiPSC-astrocytes resemble primary human astrocytes • hiPSC-astrocyte transcription is consistent with a non-reactive state • hiPSC-astrocytes undergo inflammatory response and enhance microglial phagocytosis, Brennand, Goate, and colleagues report a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs) via a neural progenitor cell (NPC) intermediate. hiPSC-astrocytes resemble primary human fetal astrocytes, have a transcriptional signature consistent with a non-reactive state, respond to inflammatory stimulants, and enhance microglial phagocytosis.

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABA(B)R2 subunit, promotes internalization of the GABA(B) receptor (GABA(B)R) and leads to smaller GABA(B)R-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABA(B)R1 subunit, and that GABA(B)Rs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABA(B)R interaction can be regulated by intracellular Ca(2+). Finally, a peptide that potentially reduces recruitment of PP2A to GABA(B)Rs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABA(B)Rs may be a useful strategy to increase receptor signaling for treating diseases. SIGNIFICANCE STATEMENT Dysregulation of GABA(B) receptors (GABA(B)Rs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABA(B)Rs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABA(B)R1 subunit, and that this interaction is sensitive to intracellular Ca(2+). We demonstrate that a short peptide corresponding to the PP2A interaction site on GABA(B)R1 competes for PP2A binding, enhances phosphorylation GABA(B)R2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABA(B)Rs may provide a specific strategy to modulate GABA(B)R signaling in disease conditions.

The human GABA

SUMMARYTo study the molecular mechanisms driving the pathogenesis and identify novel therapeutic targets of late onset Alzheimer’s Disease (LOAD), we performed an integrative network analysis of whole-genome DNA and RNA sequencing profiling of four cortical areas, including the parahippocampal gyrus, across 364 donors spanning the full spectrum of LOAD-related cognitive and neuropathological disease severities. Our analyses revealed thousands of molecular changes and uncovered for the first-time multiple neuron specific gene subnetworks most dysregulated in LOAD. ATP6V1A, a critical subunit of vacuolar-type H+-ATPase (v-ATPase), was predicted to be a key regulator of one neuronal subnetwork and its role in disease-related processes was evaluated through CRISPR-based manipulation of human induced pluripotent stem cell derived neurons and RNAi-based knockdown in transgenic Drosophila models. This study advances our understanding of LOAD pathogenesis by providing the global landscape and detailed circuits of complex molecular interactions and regulations in several key brain regions affected by LOAD and the resulting network models provide a blueprint for developing next generation therapeutics against LOAD.

The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

Background Somatic cell reprogramming is routinely used to generate donor-specific human induced pluripotent stem cells (hiPSCs) to facilitate studies of disease in a human context. The directed differentiation of hiPSCs can generate large quantities of patient-derived cells; however, such methodologies frequently yield heterogeneous populations of neurons and glia that require extended timelines to achieve electrophysiological maturity. More recently, transcription factor-based induction protocols have been show to rapidly generate defined neuronal populations from hiPSCs. New method In a manner similar to our previous adaption of NGN2-glutamatergic neuronal induction from hiPSC-derived neural progenitor cells (NPCs), we now adapt an established protocol of lentiviral overexpression of ASCL1 and DLX2 to hiPSC-NPCs. Results We demonstrate induction of a robust and highly pure population of functional GABAergic neurons (iGANs). Importantly, we successfully applied this technique to hiPSC-NPCs derived from ten donors across two independent laboratories, finding it to be an efficient and highly reproducible approach to generate induced GABAergic neurons. Our results show that, like hiPSC-iGANs, NPC-iGANs exhibit increased GABAergic marker expression, electrophysiological maturity, and have distinct transcriptional profiles that distinguish them from other cell-types of the brain. Nonetheless, until donor-matched hiPSCs-iGANs and NPC-iGANs are directly compared, we cannot rule out the possibility that subtle differences in patterning or maturity may exist between these populations; one should always control for cell source in all iGAN experiments. Conclusions This methodology, relying upon an easily cultured starting population of hiPSC-NPCs, makes possible the generation of large-scale defined co-cultures of induced glutamatergic and GABAergic neurons for hiPSC-based disease models and precision drug screening.

Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABABreceptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABABR2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants.SIGNIFICANCE STATEMENTExposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABABreceptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

The coupling of GABA(B) receptors to G-protein-gated inwardly rectifying potassium (GIRK) channels constitutes an important inhibitory pathway in the brain. Here, we examined the mechanism underlying desensitization of agonist-evoked currents carried by homomeric GIRK2 channels expressed in HEK-293T cells. The canonical GABA(B) receptor agonist baclofen produced GIRK2 currents that decayed by 57.3+/-1.4% after 60 s of stimulation, and then deactivated rapidly (time constant of 3.90+/-0.21 s) upon removal of agonist. Surface labeling studies revealed that GABA(B) receptors, in contrast to micro opioid receptors (MOR), did not internalize with a sustained stimulation for 10 min, excluding receptor redistribution as the primary mechanism for desensitization. Furthermore, heterologous desensitization was observed between GABA(B) receptors and MOR, implicating downstream proteins, such G-proteins or the GIRK channel. To investigate the G-protein turnover cycle, the non-hydrolyzable GTP analogue (GTPgammaS) was included in the intracellular solution and found to attenuate desensitization to 38.3+/-2.0%. The extent of desensitization was also reduced (45.3+/-1.3%) by coexpressing a mutant form of the Galphaq G-protein subunit that has been designed to sequester endogenous RGS proteins. Finally, reconstitution of GABA(B) receptors with Galphao G-proteins rendered insensitive to RGS resulted in significantly less desensitization (28.5+/-3.2%). Taken together, our results demonstrate that endogenous levels of RGS proteins effectively enhance GABA(B) receptor-dependent desensitization of GIRK currents.

G-protein-gated inwardly rectifying K+(GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation ofGirk1orGirk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.

Summary Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K + current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation.

G protein-gated inwardly rectifying potassium (GIRK) channels are regulators of neuronal excitability in the brain. Knockout mice lacking GIRK channels display altered behavioral responses to multiple addictive drugs, implicating GIRK channels in addictive behaviors. Here, we review the effects of GIRK subunit deletions on the behavioral response to psychostimulants, such as cocaine and methamphetamine. Additionally, exposure of mice to psychostimulants produces alterations in the surface expression of GIRK channels in multiple types of neurons within the reward system of the brain. Thus, we compare the subcellular mechanisms by which drug exposure appears to alter GIRK expression in multiple cell types and provide an outlook on future studies examining the role of GIRK channels in addiction. A greater understanding of how GIRK channels are regulated by addictive drugs may enable the development of therapies to prevent or treat drug abuse.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca(2+)-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred µm, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca(2+)-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred µm, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Although nearly half of today’s major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines.In vivoadministration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca2+and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the μ-selective (DAMGO) and δ-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba2+-sensitive basal K+currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine.

Recent work on G-protein coupled receptors (GPCRs) has highlighted the importance of homo- and heterodimerization in all areas of GPCR function, including trafficking, signaling and desensitization. Novel GPCR dimers and even high-order oligomers are constantly being discovered. Advances in techniques such as fluorescent microscopy have improved our ability to detect these interactions. As GPCRs represent the largest class of transmembrane signaling molecules in biology, these new insights into their function could vastly improve our understanding of the complex physiological role GPCRs play in cellular signaling. Utilizing a combination of classic biochemical approaches and newer techniques such as fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF), we recently demonstrated a novel interaction between M(2) muscarinic receptors and GABA(B) receptors. In this addendum, we address technical aspects of combining FRET and TIRF to study GPCR interactions and further discuss the physiological implications of the M(2)-GABA(B) heterodimer.

Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly, but the genetic code limits the building block to 20 common amino acids for protein manipulation in living cells. The genetic encoding of unnatural amino acids will remove this restriction and enable new chemical and physical properties to be precisely introduced into proteins. Here we present new strategies for generating orthogonal tRNA-synthetase pairs, which made possible the genetic encoding of diverse unnatural amino acids in different mammalian cells and primary neurons. Using this new methodology, we incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4, and found that the bulkiness of residues in the inactivation peptide is essential for fast channel inactivation, a finding that had not been possible using conventional mutagenesis. This technique will stimulate and facilitate new molecular studies using tailored unnatural amino acids for cell biology and neurobiology.

Many inhibitory neurotransmitters in the brain activate Kir3 channels by stimulating pertussis toxin (PTX)-sensitive G-protein-coupled receptors. Here, we investigated the regulation of native muscarinic receptors and Kir3 channels expressed in NGF-differentiated PC12 cells, which are similar to sympathetic neurons. Quantitative reverse transcription-PCR and immunocytochemistry revealed that NGF treatment significantly upregulated mRNA and protein for m(2) muscarinic receptors, PTX-sensitive Gα(o) G-proteins, and Kir3.2c channels. Surprisingly, these upregulated muscarinic receptor/Kir3 signaling complexes were functionally silent. Ectopic expression of m(2) muscarinic receptors or Kir3.2c channels was unable to produce muscarinic receptor-activated Kir3 currents with oxotremorine. Remarkably, pretreatment with muscarinic (m(2)/m(4)) receptor antagonists resulted in robust oxotremorine-activated Kir3 currents. Thus, sustained cholinergic stimulation of natively expressed m(2)/m(4) muscarinic receptors controlled cell surface expression and functional coupling of both receptors and Kir3 channels. This new pathway for controlling Kir3 signaling could help limit the potential harmful effects of excessive Kir3 activity in the brain.

Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein–protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 A of each other in the plasma membrane of living cells. GABAB R1 and R2 receptors, Kir3 channels, Gαo subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABAB R1 and R2 subunits (∼12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABAB R1 or R2 subunits (∼13% FRET), and between Gαo and GABAB R1 or R2 subunits (∼10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABAB R1 or R2 subunits (∼10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Gαo. These data are discussed in terms of a model in which GABAB receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

Steeply voltage-dependent block by intracellular polyamines underlies the strong inward rectification properties of Kir2.1 and other Kir channels. Mutagenesis studies have identified several negatively charged pore-lining residues (D172, E224, and E299, in Kir2.1) in the inner cavity and cytoplasmic domain as determinants of the properties of spermine block. Recent crystallographic determination of the structure of the cytoplasmic domains of Kir2.1 identified additional negatively charged residues (D255 and D259) that influence inward rectification. In this study, we have characterized the kinetic and steady-state properties of spermine block in WT Kir2.1 and in mutations of the D255 residue (D255E, A, K, R). Despite minimal effects on steady-state blockade by spermine, D255 mutations have profound effects on the blocking kinetics, with D255A marginally, and D255R dramatically, slowing the rate of block. In addition, these mutations result in the appearance of a sustained current (in the presence of spermine) at depolarized voltages. These features are reproduced with a kinetic model consisting of a single open state, two sequentially linked blocked states, and a slow spermine permeation step, with residue D255 influencing the spermine affinity and rate of entry into the shallow blocked state. The data highlight a “long-pore” effect in Kir channels, and emphasize the importance of considering blocker permeation when assessing the effects of mutations on apparent blocker affinity.

Since the discovery of somatic reprogramming, human induced pluripotent stem cells (hiPSCs) have been exploited to model a variety of neurological and psychiatric disorders. Because hiPSCs represent an almost limitless source of patient-derived neurons that retain the genetic variations thought to contribute to disease etiology, they have been heralded as a patient-specific platform for high throughput drug screening. However, the utility of current protocols for generating neurons from hiPSCs remains limited by protracted differentiation timelines and heterogeneity of the neuronal phenotypes produced. Neuronal induction via the forced expression of exogenous transcription factors rapidly induces defined populations of functional neurons from fibroblasts and hiPSCs. Here, we describe an adapted protocol that accelerates maturation of functional excitatory neurons from hiPSC-derived neural progenitor cells (NPCs) via lentiviral transduction of Neurogenin 2 (using both mNgn2 and hNGN2). This methodology, relying upon a robust and scalable starting population of hiPSC NPCs, should be readily amenable to scaling for hiPSC-based high-throughput drug screening.

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.

Background Repeated exposure to cocaine or social stress leads to lasting structural and functional synaptic alterations in medium spiny neurons (MSNs) of nucleus accumbens (NAc). Although cocaine-induced and stress-induced structural changes in dendritic spines have been well documented, few studies have investigated functional consequences of cocaine and stress at the level of single spines. Methods We exposed mice to chronic cocaine or chronic social defeat stress and used two-photon laser scanning microscopy with glutamate photo-uncaging and whole-cell recording to examine synaptic strength at individual spines on two distinct types of NAc MSNs in acute slices after 24 hours of cocaine withdrawal and after chronic social defeat stress. Results In animals treated with cocaine, average synaptic strength was reduced specifically at large mushroom spines of MSNs expressing dopamine receptor type 1 (D1-MSNs). In contrast, cocaine promoted a rightward shift in the distribution of synaptic weights toward larger synaptic responses in MSNs expressing dopamine receptor type 2 (D2-MSNs). After chronic social defeat stress, resilient animals displayed an upregulation of synaptic strength at large mushroom spines of D1-MSNs and a concomitant downregulation in D2-MSNs. Although susceptible mice did not exhibit a significant overall change in synaptic strength on D1-MSNs or D2-MSNs, we observed a slight leftward shift in cumulative distribution of large synaptic responses in both cell types. Conclusions This study provides the first functional cell type–specific and spine type–specific comparison of synaptic strength at a single spine level between cocaine-induced and stress-induced neuroadaptations and demonstrates that psychoactive drugs and stress trigger divergent changes in synaptic function in NAc.