Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A_2A receptor (A_2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A_2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A_2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A_2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A_2AR deletion; and (iv) selective A_2AR deletion in forebrain neurons. Notably, A_2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A_2AR antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A_2AR in the control of chronic stress-induced modifications and suggest A_2AR as candidate targets to alleviate the consequences of chronic stress on brain function.Repeated stress elicits neurochemical and morphological changes that negatively affect brain functioning (1, 2). Thus, repeated stress is a trigger or a risk factor for neuropsychiatric disorders, namely depression, in both humans and animal models (2, 3). Given the absence of effective therapeutic tools, novel strategies to manage the impact of chronic stress are needed, and analyzing particular lifestyles can provide important leads. Notably, caffeine consumption increases in stressful conditions (4) and correlates inversely with the incidence of depression (5, 6) and the risk of suicide (7, 8). However, the molecular targets operated by caffeine to afford these beneficial effects have not been defined.Caffeine is the most widely consumed psychoactive drug. The only molecular targets for caffeine at nontoxic doses are the main adenosine receptors in the brain, namely the inhibitory A₁ receptors (A₁R) and the facilitatory A_2A receptors (A_2AR) (9). A_2AR blockade affords robust protection against noxious brain conditions (10), an effect that might result from the ability of neuronal A_2AR to control aberrant plasticity (11, 12) and synaptotoxicity (13–15) or from A_2AR’s impact on astrocytes (16) or microglia (17). The protection provided by A_2AR blockade prompts the hypothesis that A_2AR antagonism may underlie the beneficial effects of caffeine on chronic stress, in accordance with the role of synaptic (18, 19) or glial dysfunction (20) in mood disorders. Thus, A_2AR antagonists prolonged escape behavior in two screening tests for antidepressant activity (21–23) and prevented maternal separation-induced long-term cognitive impact (12). We combined pharmacological and tissue-selective A_2AR transgenic mice (24, 25) to test if neuronal A_2AR controlled the modifications caused by chronic unpredictable stress (CUS).     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors

GABA_A-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABA_A receptors, and requires a newly described interaction with GABARAP, a GABA_A receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.Interneurons that release γ-aminobutyric acid (GABA) are a major source of inhibitory signaling in vertebrate nervous systems, and play important roles in cognition, mood, and behavior (1, 2). Many of these inhibitory interneurons release GABA, which binds to ionotropic ligand-gated GABA_A receptors located at GABAergic synapses and at extrasynaptic sites, and these GABA_A receptors are sites of action for benzodiazepine and barbiturates (3). GABA_A receptors are dynamic, with continuous exchange between synaptic and extrasynaptic sites in the plane of the membrane, as well as endocytic trafficking between the cell surface and intracellular compartments (3–6). GABA_A receptor cell surface expression is believed to be required for formation of GABAergic synapses based on studies with heterogeneously-expressed GABA_A receptors (7). However, the role of GABA_A receptors in preserving GABAergic synapses has not yet been described in a native neuronal environment.GABAergic synapses localize to both the axon initial segment (AIS) as well as somatodendritic sites of target neurons (2, 8, 9). In the cerebellum, basket and stellar interneurons project specific axon terminals to the AISs of Purkinje cells, forming GABAergic “pinceau” synapses (10). Formation of these pinceau synapses depends on a steep gradient of the cell adhesion molecule neurofascin, which is enriched at the AIS (11, 12). Both GABAergic pinceau synapses and the neurofascin gradient are missing in mice with cerebellar knockout out of the membrane adaptor ankyrin-G (11, 13). Ankyrin-G coordinates multiple proteins at AISs including voltage-gated sodium channels (VGSC), KCNQ2/3 channels, 186-kDa neurofascin, and beta-4 spectrin (14). A role of ankyrin-G in stabilizing GABAergic synapses outside of the the AIS of cerebellar neurons has not been explored.Assembly of AISs as well as their GABAergic synapses requires giant ankyrin-G, which contains a 7.8-kb alternatively spliced nervous system-specific exon found only in vertebrates (14). In addition to ANK repeats and a beta-spectrin-binding domain, giant ankyrin-G (480-kDa ankyrin-G) contains 2,600 residues configured as an extended fibrous polypeptide (14–17). Giant ankyrin-G has been assumed to be confined to AISs and nodes of Ranvier and a general role for ankyrin-G in GABAergic synapse stability at other cellular sites has not been entertained (14, 15, 18).Here we report that giant ankyrin-G is present in extrasynaptic microdomains on the somatodendritic surfaces of hippocampal and cortical neurons, and describe a giant ankyrin-G–based mechanism required for cell surface expression of GABA_A receptors and for maintaining somatodendritic GABAergic synapses. We find that somatodendritic giant ankyrin-G inhibits GABA_A receptor endocytosis through an interaction with the GABA_A receptor-associated protein (GABARAP). This previously unidentified role for giant ankyrin-G provides a newly resolved step in the formation of GABA_A-receptor-mediated circuitry in the cerebral cortex as well as a rationale for recent linkage of human mutations in the giant ankyrin exon with autism and severe cognitive dysfunction (19).     

Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice

Cannabinoid CB₂ receptors (CB₂Rs) have been recently reported to modulate brain dopamine (DA)-related behaviors; however, the cellular mechanisms underlying these actions are unclear. Here we report that CB₂Rs are expressed in ventral tegmental area (VTA) DA neurons and functionally modulate DA neuronal excitability and DA-related behavior. In situ hybridization and immunohistochemical assays detected CB₂ mRNA and CB₂R immunostaining in VTA DA neurons. Electrophysiological studies demonstrated that activation of CB₂Rs by JWH133 or other CB₂R agonists inhibited VTA DA neuronal firing in vivo and ex vivo, whereas microinjections of JWH133 into the VTA inhibited cocaine self-administration. Importantly, all of the above findings observed in WT or CB₁^−/− mice are blocked by CB₂R antagonist and absent in CB₂^−/− mice. These data suggest that CB₂R-mediated reduction of VTA DA neuronal activity may underlie JWH133''s modulation of DA-regulated behaviors.The presence of functional cannabinoid CB₂ receptors (CB₂Rs) in the brain has been controversial. When CB₂Rs were first cloned, in situ hybridization (ISH) failed to detect CB₂ mRNA in brain (1). Similarly, Northern blot and polymerase chain reaction (PCR) assays failed to detect CB₂ mRNA in brain (2–5). Therefore, CB₂Rs were considered “peripheral cannabinoid receptors” (1, 6).In contrast, other studies using ISH and radioligand binding assays detected CB₂ mRNA and receptor binding in rat retina (7), mouse cerebral cortex (8), and hippocampus and striatum of nonhuman primates (9). More recent studies using RT-PCR also detected CB₂ mRNA in the cortex, striatum, hippocampus, amygdala, and brainstem (9–14). Immunoblot and immunohistochemistry (IHC) assays detected CB₂R immunoreactivity or immunostaining in various brain regions (13, 15–20). The specificities of the detected CB₂R protein and CB₂-mRNA remain questionable, however, owing to a lack of controls using CB₁^−/− and CB₂^−/− mice in most previous studies (21). A currently accepted view is that brain CB₂Rs are expressed predominantly in activated microglia during neuroinflammation, whereas brain neurons, except for a very small number in the brainstem, lack CB₂R expression (21).On the other hand, we recently reported that brain CB₂Rs modulate cocaine self-administration and cocaine-induced increases in locomotion and extracellular dopamine (DA) in the nucleus accumbens in mice (22). This finding is supported by recent studies demonstrating that systemic administration of the CB₂R agonist O-1966 inhibited cocaine-induced conditioned place preference in WT mice, but not in CB₂^−/− mice (23), and that increased CB₂R expression in mouse brain attenuates cocaine self-administration and cocaine-enhanced locomotion (19). In addition, brain CB₂Rs may be involved in several DA-related CNS disorders, such as Parkinson’s disease (24), schizophrenia (25), anxiety (26), and depression (27). The cellular mechanisms underlying CB₂R modulation of DA-related behaviors and diseases are unclear, however. Given that midbrain DA neurons of the ventral tegmental area (VTA) play an important role in mediating the reinforcing and addictive effects of drugs of abuse (28, 29), we hypothesized that brain CB₂Rs, similar to other G protein-coupled receptors, are expressed in VTA DA neurons, where they modulate DA neuronal function and DA-related behaviors.In the present study, we tested this hypothesis using multiple approaches. We first assayed for CB₂ mRNA and protein expression in brain and in VTA DA neurons using quantitative RT-PCR (qRT-PCR), ISH, and double-label IHC techniques. We then examined the effects of the selective CB₂R agonist JWH133 and several other CB₂R agonists on VTA DA neuronal firing in both ex vivo and in vivo preparations using electrophysiological methods. Finally, we observed the effects of microinjections of JWH133 into the VTA on intravenous cocaine self-administration to study whether activation of VTA CB₂Rs modulates DA-dependent behavior. This multidisciplinary approach has provided evidence of functional CB₂Rs in VTA DA neurons. Importantly, all findings observed in WT or CB₁^−/− mice were blocked by a CB₂R antagonist and/or absent in CB₂^−/− mice, suggesting that CB₂Rs expressed in VTA DA neurons play an important role in modulating DA neuronal activity and DA-related functions.     

Differential vesicular sorting of AMPA and GABAA receptors

In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA_A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABA_A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABA_A receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA_A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABA_A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.In the mammalian central nervous system, neurons receive excitatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neurotransmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA_A receptors, which are predominantly heteropentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA_A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a “plasma membrane sorting model,” different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a “vesicle sorting model,” different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the exocytic pathways of AMPA and GABA_A receptors have similar but also distinct properties (1, 2).Increasing evidence has suggested roles for the SNARE protein family in vesicular trafficking of AMPA and GABA_A receptors (4–17). SNAREs are a large family of membrane-associated proteins critical for many intracellular membrane trafficking events. The family is subdivided into v-SNAREs (synaptobrevin/VAMP, vesicle-associated membrane proteins) and t-SNAREs (syntaxins and SNAP25, synaptosomal-associated protein of 25 kDa) based on their localization on trafficking vesicles or target membranes, respectively. To mediate vesicle fusion with target membranes, SNARE proteins form a four-helix bundle (SNARE complex) consisting of two coiled-coil domains from SNAP25, one coiled-coil domain from syntaxin, and a coiled-coil domain from VAMPs (18). Formation of the helical bundle can be disrupted by neurotoxins, which specifically cleave different SNARE proteins (19). Each SNARE subfamily is composed of genes with high homology but different tissue specificity and subcellular localization. It remains to be determined whether individual SNAREs play specific roles in regulating the membrane trafficking of individual proteins.To address how AMPA and GABA_A receptors are sorted in the exocytic pathway and what molecules are involved in regulating exocytosis of these receptors, we specifically studied constitutive exocytosis of AMPA and GABA_A receptor subunits using total internal reflection fluorescence microscopy (TIRFM) in combination with immunocytochemistry, electrophysiology, and electron microscopy methods. Together, we revealed that AMPA and GABA_A receptors are initially sorted into different vesicles in the Golgi apparatus and delivered to different domains at the plasma membrane and are regulated by specific Rab proteins and SNARE complexes. These results reveal fundamental mechanisms underlying the sorting of excitatory and inhibitory neurotransmitter receptors in neurons and uncover the specific trafficking machinery involved in the constitutive exocytosis of each receptor type.     

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

The inositol 1,4,5-trisphosphate receptor (IP₃R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP₃Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP₃ at a distance. Defective allostery in IP₃R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP₃R type 1 (IP₃R1) and negatively regulates IP₃R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP₃R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.Ligand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ligand-gated ion channels that release calcium ions (Ca²⁺) from the endoplasmic reticulum (ER) (2, 3). IP₃Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3) are structurally divided into three domains: the IP₃-binding domain (IBD), the regulatory domain, and the channel domain (3–6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca²⁺ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP₃R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP₃R (IP₃R1) leads to perturbations in long-term potentiation/depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP₃R1 gene (13–15). Dysregulation of IP₃R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16–18) and Alzheimer’s disease (AD) (19–22). IP₃Rs also control fundamental cellular processes—for example, mitochondrial energy production (23, 24), autophagy regulation (24–27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophage inflammasomes (31). On the other hand, excessive IP₃R function promotes cell death processes including apoptosis by activating mitochondrial or calpain pathways (2, 17). Considering these versatile roles of IP₃Rs, appropriate IP₃R structure and function are essential for living systems, and aberrant regulation of IP₃R closely relates to various diseases.Several factors such as cytosolic molecules, interacting proteins, and posttranslational modifications control the IP₃-induced Ca²⁺ release (IICR) through allosteric sites in IP₃Rs. Cytosolic Ca²⁺ concentrations strictly control IICR in a biphasic manner with activation at low concentrations and inhibition at higher concentrations. The critical Ca²⁺ sensor for activation is conserved among the three isoforms of IP₃ and ryanodine receptors, and this sensor is located in the regulatory domain outside the IBD and the channel domain (32). A putative ATP regulatory region is deleted in opisthotonos mice, and IICR is also regulated by this mutation in the regulatory domain (33). Various interacting proteins, such as cytochrome c, Bcl-2-family proteins, ataxin-3, huntingtin (Htt) protein, Htt-associated protein 1A (HAP1A), and G-protein–coupled receptor kinase-interacting protein 1 (GIT1), target allosteric sites in the carboxyl-terminal tail (3–5). The regulatory domain and the carboxyl-terminal tail also undergo phosphorylation by the protein kinases A/G and B/Akt and contain the apoptotic cleavage sites for the protease caspase-3 (4, 5). These factors allosterically regulate IP₃R structure and function to control cellular fates; therefore, understanding the allosteric coupling of the IBD to channel gating will elucidate the regulatory mechanism of these factors.Transglutaminase (TG) catalyses protein cross-linking between a glutamine (Gln) residue and a lysine (Lys) residue via an N^ε-(γ-glutamyl)lysine isopeptide bond (34, 35). TG type 2 (TG2) is a Ca²⁺-dependent enzyme with widespread distribution and is highly inducible by various stimulations such as oxidative stress, cytokines, growth factors, and retinoic acid (RA) (34, 35). TG2 is considered a significant disease-modifying factor in neurodegenerative diseases including HD, AD, and Parkinson’s diseases (PD) (34, 36–45) because TG2 might enzymatically stabilize aberrant aggregates of proteins implicated in these diseases—that is, mutant Htt, β-amyloid, and α-synuclein; however, the causal role of TG2 in Ca²⁺ signaling in brain pathogenesis has been unclear. Ablation of TG2 in HD mouse models is associated with increased lifespan and improved motor function (46, 47). However, TG2 knockout mice do not show impaired Htt aggregation, suggesting that TG2 may play a causal role in these disorders rather than TG2-dependent cross-links in aberrant protein aggregates (47, 48).In this study, we discovered a new mode of chronic and irreversible allosteric regulation in IP₃R1 in which covalent modification of the receptor at Gln2746 is catalyzed by TG2. We demonstrate that up-regulation of TG2 modifies IP₃R1 structure and function in HD models and propose an etiologic role of this modification in the reduction of neuronal signaling and subsequent processes during the prodromal state of the neurodegenerative disease.     

Tobacco smoking interferes with GABAA receptor neuroadaptations during prolonged alcohol withdrawal

Understanding the effects of tobacco smoking on neuroadaptations in GABA_A receptor levels over alcohol withdrawal will provide critical insights for the treatment of comorbid alcohol and nicotine dependence. We conducted parallel studies in human subjects and nonhuman primates to investigate the differential effects of tobacco smoking and nicotine on changes in GABA_A receptor availability during acute and prolonged alcohol withdrawal. We report that alcohol withdrawal with or without concurrent tobacco smoking/nicotine consumption resulted in significant and robust elevations in GABA_A receptor levels over the first week of withdrawal. Over prolonged withdrawal, GABA_A receptors returned to control levels in alcohol-dependent nonsmokers, but alcohol-dependent smokers had significant and sustained elevations in GABA_A receptors that were associated with craving for alcohol and cigarettes. In nonhuman primates, GABA_A receptor levels normalized by 1 mo of abstinence in both groups—that is, those that consumed alcohol alone or the combination of alcohol and nicotine. These data suggest that constituents in tobacco smoke other than nicotine block the recovery of GABA_A receptor systems during sustained alcohol abstinence, contributing to alcohol relapse and the perpetuation of smoking.Alcohol dependence and tobacco smoking are highly comorbid (1). Alcohol-dependent smokers who quit drinking but continue smoking may have a reduced severity of alcohol withdrawal and relapse risk (2) compared with alcohol-dependent smokers who stop smoking and drinking at the same time (3–5). This has led to some complacency in the field about treating the addiction to nicotine in alcohol-dependent smokers, and few treatment settings provide any systematic tobacco treatment (6). However, a large part of the morbidity and mortality from alcohol dependence can be attributed to concurrent tobacco smoking (7), and a large number of alcohol-dependent individuals in treatment express a desire to quit smoking (8). Understanding the involvement of tobacco smoking in the neuroadaptations and behavioral changes that occur during alcohol withdrawal will provide critical insights to direct treatment strategies.Given the multiple molecular targets for alcohol in the brain and numerous constituents of tobacco smoke, it is likely that the neurobiology of this comorbidity is complex. However, the γ-aminobutyric acid (GABA) system may be an important point of convergence of the effects of tobacco smoke and alcohol in the brain. For example, nicotine reinforcement has been critically linked to activation of GABA neurons (9), and alcohol appears to both directly stimulate extrasynaptic GABA_A receptors with relatively high affinity (10) and to indirectly stimulate the release of GABA and neurosteroids (11), such as allopregnanolone, that also stimulate extrasynaptic GABA_A receptors (12, 13). Alcohol and neurosteroids can act at synaptic GABA_A receptors, but the affinity is low, the response is variable, and the dose of alcohol that would facilitate signaling at these synaptic receptors would induce a coma in humans (14, 15).Studies in both animals and humans have yielded a tentative model about the convergence of the codependency produced by smoking and alcohol consumption, as has been reviewed (16). In the absence of smoking, chronic alcohol administration produces an adaptive down-regulation of synaptic GABA_A receptor function by altering GABA_A receptor subunit composition and subtly shifting subpopulations of receptors from a relative predominance of low-affinity high Cl– conductance type to greater numbers of a high-affinity low Cl– conductance subtype, characteristic of extrasynaptic GABA_A receptors (15). In early recovery, there is a transitional phase, during which deficits in GABA_A receptor signaling are thought to contribute to withdrawal-related cortical hyperexcitability and low-affinity high-conductance receptors are recruited to reestablish the cortical balance of excitation and inhibition. The recruitment of the additional GABA_A receptors was demonstrated by a transient increase in ligand binding over the first week of alcohol withdrawal (17). In this same cross-sectional study (17), a subset of smokers did not show similar time-dependent alterations during early recovery. Moreover, GABA_A receptor availability was positively correlated with alcohol withdrawal symptoms in nonsmokers but not smokers, suggesting that smoking may have suppressed withdrawal symptoms by preventing alcohol-related neuroadaptations in GABA_A receptors.The goal of the current study was to systematically examine the effect of tobacco smoking on alcohol withdrawal-related neuroadaptations in GABA_A levels. The first study was designed to extend the previous cross-sectional findings to determine differences in GABA_A receptor levels in alcohol-dependent smokers versus nonsmokers at multiple times during early withdrawal and during extended abstinence. Additionally, tobacco smoke consists of over 4,000 chemicals. Many of these chemicals may play a role in influencing alcohol-related withdrawal symptoms; however, nicotine, the primary addictive chemical in tobacco smoke, has been the most widely studied tobacco constituent and has been associated with GABA system regulation (9, 18). Thus, a second parallel study was conducted in nonhuman primates that were randomized to self-administer alcohol with or without concurrent access to a nicotine solution rather than tobacco smoke to determine the role of nicotine per se on alcohol withdrawal-related neuroadaptations.     

High-temperature in situ crystallographic observation of reversible gas sorption in impermeable organic cages

Crystallographic observation of adsorbed gas molecules is a highly difficult task due to their rapid motion. Here, we report the in situ single-crystal and synchrotron powder X-ray observations of reversible CO₂ sorption processes in an apparently nonporous organic crystal under varying pressures at high temperatures. The host material is formed by hydrogen bond network between 1,3,5-tris-(4-carboxyphenyl)benzene (H₃BTB) and N,N-dimethylformamide (DMF) and by π–π stacking between the H₃BTB moieties. The material can be viewed as a well-ordered array of cages, which are tight packed with each other so that the cages are inaccessible from outside. Thus, the host is practically nonporous. Despite the absence of permanent pathways connecting the empty cages, they are permeable to CO₂ at high temperatures due to thermally activated molecular gating, and the weakly confined CO₂ molecules in the cages allow direct detection by in situ single-crystal X-ray diffraction at 323 K. Variable-temperature in situ synchrotron powder X-ray diffraction studies also show that the CO₂ sorption is reversible and driven by temperature increase. Solid-state magic angle spinning NMR defines the interactions of CO₂ with the organic framework and dynamic motion of CO₂ in cages. The reversible sorption is attributed to the dynamic motion of the DMF molecules combined with the axial motions/angular fluctuations of CO₂ (a series of transient opening/closing of compartments enabling CO₂ molecule passage), as revealed from NMR and simulations. This temperature-driven transient molecular gating can store gaseous molecules in ordered arrays toward unique collective properties and release them for ready use.Guest capture, storage, and removal in porous materials have been interesting topics in chemistry (1–7). The porosity of solids is one of the important factors determining their potential applications in guest storage. Because the storage generally uses void spaces interconnected through large open channels between the voids inside the crystal, nonporous or seemingly nonporous materials have received less attention. Over the past decades, nevertheless, due to the potential for selective guest capture and release in a controlled manner there have been attempts to study such materials which include calixarenes (8–17), 4-phenoxyphenol (18), biconcave molecules (19), tris(5-acetyl-3-thienyl)methane (20), metallocyclic complex (21), clarithromycin (22), and metal-organic frameworks (23, 24).Single-crystal X-ray diffraction can provide the crucial information on the binding interactions or structural changes of guest molecules within pores (25–27). However, the crystallographic observation of adsorbed gas adsorbents generally has not been possible due to the poor crystalline order of adsorbents upon removing residual solvent/guest molecules and the high mobility of gases even at low temperatures. Only a limited number of gas-adsorbed single-crystal structures have been determined at low temperatures (16, 28–36).If the permanent channels large enough for the guest diffusion between voids are not present, such a material is nonporous and impermeable to the guest molecules even if there are cages available for the guest storage. If transient pathways between voids can be made with molecular gates in special conditions, the cages can be used for storing special molecules in well-ordered arrays. Furthermore, if they confine the gas molecules in cages, it would be possible to observe the gas molecule using single-crystal X-ray crystallography at high temperatures, even for the relatively weak interactions between the frameworks and gas molecules. Based on this assumption, we studied the reversible CO₂ sorption in a seemingly nonporous organic crystalline cage material composed of 1,3,5-tris-(4-carboxyphenyl)benzene (H₃BTB) and N,N-dimethylformamide (DMF) using in situ single-crystal and synchrotron powder X-ray diffraction at 323 K. The dynamical motion of CO₂ is investigated using solid-state NMR as well as density functional calculations of transition pathways and molecular dynamics (MD) simulations.     

Hydrogen sulphide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia

Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H₂S), through multiple steps involving cystathionine β-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H₂S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1–100 µM) or l-cysteine (0.1–100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide–6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine β-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H₂S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A₂ production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A₂, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H₂S. Finally, phosphorylated–phospholipase A₂ expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H₂S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.Hyperhomocysteinemia (HHcy) is a risk factor for neurovascular and cardiovascular disease associated with endothelial dysfunction and accelerated atherosclerosis (1–3). Many clinical and epidemiological studies have demonstrated a positive correlation between homocysteine (Hcy) plasma levels and cardiovascular disorders (4, 5), leading to the general conclusion that Hcy is a prothrombotic factor (6–8). However, the mechanism(s) through which elevated circulating levels of Hcy promote vascular disease and thrombosis is still unclear (9). Hcy has two primary fates: conversion through a reaction catalyzed by 5,10 methylenetetrahydrofolate reductase (MTHFR) into l-methionine or conversion to l-cysteine (l-Cys) via a transulfuration pathway (10, 11). The transulfuration pathway relies upon cystathionine β-synthase (CBS) to transform Hcy in cysthathionine, which is converted by cystathionine γ-lyase (CSE) into l-Cys. Thereafter, both enzymes convert l-Cys to generate hydrogen sulphide (H₂S) (12, 13). H₂S has been recognized as the third member of the family of gaseous transmitters (14), and it is present in human blood at micromolar concentrations (10–100 μM) (15). It rapidly travels through cell membranes without using any specific receptor/transporter or intracellular signaling proteins. CBS and CSE are differentially expressed in cardiovascular as well as in several other body districts (13). The physiological functions of H₂S are mediated by a variety of molecular targets, including ion channels and signaling proteins (16–18). Alterations in H₂S metabolism contribute to an array of cardiovascular disorders such as hypertension, atherosclerosis, heart failure, and diabetes (19). Nevertheless, the influence of H₂S on platelet function and, in turn, on blood clotting has been poorly explored. We hypothesized that H₂S could be involved in the thrombotic events associated with HHcy. To address this issue, we used human platelets harvested either from healthy volunteers or from patients with a C677T polymorphism of the MTHFR gene (MTHFR++) that is linked to HHcy (20–22).     

Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm

Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca_v3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca²⁺ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca²⁺ channels in the medial septal GABAergic projection neurons in this behavior.When confronted with an unfamiliar environment, or physical or social objects, animals often exhibit behavior patterns that can broadly be termed exploration, such as moving around the environment, touching or sniffing novel objects, and interacting with social stimuli (1). Social exploration involves complex processes that differ from those involved in the nonsocial exploration (2). Several distinctions were proposed to categorize the different forms of nonsocial exploratory behaviors from a motivational perspective (3). Behaviorally, two types of nonsocial exploration are observed in rodents and humans (3–5): object exploration and spatial or environmental exploration in the absence of objects. Object exploration is the behavior to explore discrete novel objects. This activity is elicited and sustained by the physical presence of an object. Several types of preference or “novelty” tests have been developed to investigate object exploration in rodents (3, 5–7). Environmental or spatial exploration in the absence of objects refers to the inquisitive activity of an animal in a new space, where the eliciting and sustaining stimulus is the “place” itself. Various forms of open-field tests have been used to investigate environmental or spatial exploration in rodents (3, 5, 8). Experimentally, however, the distinction can be less obvious because both can occur together (4, 7–9). Spatial exploration is suggested to be hippocampal-dependent (10)—although that is controversial (11)—whereas object exploration is suggested to be hippocampal-independent (12). Thus, it is still a matter of debate whether animal exploration belongs to a unitary category or not (9). To resolve this issue, neural definitions of these two previously proposed exploratory behaviors are needed.Interestingly, the medial septum (MS), where Ca_v3.1 T-type Ca²⁺ channels are highly expressed (13), is suggested to be critical for exploratory behaviors (5, 14–16). Moreover, the MS is also the nodal point for ascending afferent systems involved in the generation of hippocampal theta rhythms, the largest synchronous oscillatory signals in the mammalian brain, which are implicated in diverse brain functions (17, 18). Although the heterogeneity of hippocampal theta rhythms has long been under debate (19), recent studies based on genetic mutations in mice and optogenetics provide strong support for theta rhythm heterogeneity (20–22). However, their exact behavioral correlates are still debated. Ca_v3.1 Ca²⁺ channels play an important role in diverse behaviors, as well as the generation of physiologic and pathophysiologic brain rhythms (23). Notably, T-type, low-threshold Ca²⁺ currents are assumed to be a candidate ionic mechanism of theta rhythm genesis (24), analogous to the role of T-type channels in the generation of oscillations in the reticular nucleus of the thalamus (25). Nevertheless the involvement of T-type Ca²⁺ channels in hippocampal theta rhythms or exploratory behavior has not been examined. Here, we analyzed global KO mice and mice with MS-specific inactivation of the Ca_v3.1 gene encoding T-type Ca²⁺ channels, focusing on finding the neural mechanism that control the exploratory behaviors. Using a combination of tools, we provide evidence that object and open field exploratory behaviors are processed differently in the brain. Furthermore, Ca_v3.1 T-type Ca²⁺ channels in the septo-hippocampal GABAergic projection neurons are critically involved in controlling object exploration through modulating hippocampal type 2 theta rhythm.     

From the Cover: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca²⁺ leak at rest, and depressed force production due to impaired SR Ca²⁺ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca²⁺-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.It is increasingly clear that regular physical exercise plays a key role in the general well-being, disease prevention, and longevity of humans. Impaired muscle function manifesting as muscle weakness and premature fatigue development are major health problems associated with the normal aging process as well as with numerous common diseases (1). Physical exercise has a fundamental role in preventing and/or reversing these muscle problems, and training also improves the general health status in numerous diseases (2–4). On the other side of the spectrum, excessive muscle use can induce prolonged force depressions, which may set the limit on training tolerance and performance of top athletes (5, 6).Recent studies imply a key role of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor 1 (RyR1), in the reduced muscle strength observed in numerous physiological conditions, such as after strenuous endurance training (6), in situations with prolonged stress (7), and in normal aging (8, 9). Defective RyR1 function is also implied in several pathological states, including generalized inflammatory disorders (10), heart failure (11), and inherited conditions such as malignant hyperthermia (12) and Duchenne muscular dystrophy (13). In many of the above conditions, there is a link between the impaired RyR1 function and modifications induced by reactive oxygen/nitrogen species (ROS) (6, 8, 10, 12, 13). Conversely, altered RyR1 function may also be beneficial by increasing the cytosolic free [Ca²⁺] ([Ca²⁺]_i) at rest, which can stimulate mitochondrial biogenesis and thereby increase fatigue resistance (14–16). Intriguingly, effective antioxidant treatment hampers beneficial adaptations triggered by endurance training (17–19), and this effect might be due to antioxidants preventing ROS-induced modifications of RyR1 (20).A high-intensity interval training (HIIT) session typically consists of a series of brief bursts of vigorous physical exercise separated by periods of rest or low-intensity exercise. A major asset of HIIT is that beneficial adaptations can be obtained with much shorter exercise duration than with traditional endurance training (21–25). HIIT has been shown to effectively stimulate mitochondrial biogenesis in skeletal muscle and increase endurance in untrained and recreationally active healthy subjects (22, 26), whereas positive effects in elite endurance athletes are less clear (21, 27, 28). Moreover, HIIT improves health and physical performance in various pathological conditions, including cardiovascular disease, obesity, and type 2 diabetes (29, 30). Thus, short bouts of vigorous physical exercise trigger intracellular signaling of large enough magnitude and duration to induce extensive beneficial adaptations in skeletal muscle. The initial signaling that triggers these adaptations is not known.In this study, we tested the hypothesis that a single session of HIIT induces ROS-dependent RyR1 modifications. These modifications might cause prolonged force depression due to impaired SR Ca²⁺ release during contractions. Conversely, they may also initiate beneficial muscular adaptations due to increased SR Ca²⁺ leak at rest.     

Dual role of CO in the stability of subnano Pt clusters at the Fe3O4(001) surface

Interactions between catalytically active metal particles and reactant gases depend strongly on the particle size, particularly in the subnanometer regime where the addition of just one atom can induce substantial changes in stability, morphology, and reactivity. Here, time-lapse scanning tunneling microscopy (STM) and density functional theory (DFT)-based calculations are used to study how CO exposure affects the stability of Pt adatoms and subnano clusters at the Fe₃O₄(001) surface, a model CO oxidation catalyst. The results reveal that CO plays a dual role: first, it induces mobility among otherwise stable Pt adatoms through the formation of Pt carbonyls (Pt₁–CO), leading to agglomeration into subnano clusters. Second, the presence of the CO stabilizes the smallest clusters against decay at room temperature, significantly modifying the growth kinetics. At elevated temperatures, CO desorption results in a partial redispersion and recovery of the Pt adatom phase.Subnanometer metal particles exhibit a range of interesting electronic or catalytic properties that can vary substantially with the removal or addition of a single atom (1–6). Understanding the mechanistic details underlying the rearrangement of the active phase is important because changes in cluster size and shape are known to be commonplace under the conditions used in heterogeneous catalysis (7, 8), and because such processes are associated with deactivation phenomena such as sintering. Although sintering is usually regarded as a thermally activated process, there is increasing evidence that adsorbates influence sintering rates in a reactive environment by formation of mobile metal-molecule intermediates (2, 8–30). Indeed, in a previous study we demonstrated that the formation of highly mobile Pd₁–CO species led to enhanced sintering in the Pd/Fe₃O₄(001) system (31). Here, we turn our attention to Pt. Mobility is induced in the form of Pt₁–CO. In addition, CO stabilizes the smallest clusters. When it desorbs, Pt dimers break up into single atoms; thus, the CO is necessary for preserving nuclei that act as seeds for further growth. Using room-temperature scanning tunneling microscopy (STM), complemented by X-ray photoelectron spectroscopy (XPS) and density functional theory with an on-site Hubbard U (DFT+U), we follow the CO-induced diffusion and coalescence of Pt atom-by-atom, creating catalytically active (32) subnano clusters with a well-defined size distribution. On heating, desorption of CO leads to significant redispersion of Pt into the adatom phase.     

From the Cover: PNAS Plus: Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P₂ and probably PI(3,4,5)P₃ as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P₃ is 55-fold slower than 5-phosphatase activity against PI(4,5)P₂; thus, PI(4,5)P₂ generated more slowly from dephosphorylating PI(3,4,5)P₃ might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P₂ in parallel to PI(3,4,5)P₃ dephosphorylation, emphasizing that VSPs can cleave the 3-phosphate of PI(3,4,5)P₃.This paper concerns the substrate specificity and voltage dependence of a unique voltage-sensitive phosphoinositide (PI) phosphatase in intact live cells. Bioelectricity, caused by ion channels and differences in ion concentrations between the inside and outside of a cell, regulates essential biological activities like generation, propagation, and processing of neuronal signals; muscle contraction; and secretion of hormones. Voltage-gated ion channels were the first protein family identified that possessed bioelectric voltage-sensing domains (VSDs) and participated in these signaling activities. Recently, a quite unanticipated voltage-sensing enzyme with a VSD was cloned from the sea squirt Ciona intestinalis (1). Biochemical and electrophysiological examination revealed a voltage-dependent phosphatase activity toward polyphospho-PIs that was given the name C. intestinalis voltage-sensing phosphatase (Ci-VSP). Since then, homologs have been discovered in other vertebrates, for example, Danio rerio (zebrafish; Dr-VSP), African frog (Xi-VSP and Xt-VSP), chicken (Gg-VSP), and salamander (Hn-VSP and Cp-VSP) (2–5). In addition, two mammalian homologs have been discovered in human Hs-transmembrane phosphatase with tensin homology (TPTE) and mouse (Mm-VSP) tissues (6, 7). Although knowledge about the physiological function of these two proteins is still lacking, a recent study showed that mouse VSP seems to be localized to intracellular membranes of neuronal cells, suggesting a different role for mammalian VSPs than for plasma membrane-localized Ci-VSP or Dr-VSP (7). As in voltage-gated ion channels, the VSD of VSPs consists of four transmembrane segments, S1–S4, with the charged voltage-sensing S4 segment being moved by the intense electric fields across the plasma membrane upon depolarization (8). However, VSPs are monomeric and have a cytosolic catalytic domain instead of the pore-forming domain (S5–S6) of ion channels. This enzyme domain is homologous to tumor suppressor phosphatase and tensin homolog (PTEN), a phosphoinositide 3-phosphatase that dephosphorylates both phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (1). Three distinctive regions of PTEN, an N-terminal phospholipid-binding motif that anchors the protein at the plasma membrane, a phosphatase domain that has the enzymatic site, and a C-terminal lipid-interacting C2 domain (9, 10), are well conserved in sequence and structure in the VSPs (11–14).Propagation of the depolarization-induced conformational changes of the VSD to the cytosolic catalytic domain activates the unique voltage-activated phosphoinositide phosphatase activity (12, 14, 15). Unlike its analog PTEN, which has only 3-phosphatase activity (9, 16), Ci-VSP was seen initially to cleave the 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ in response to membrane depolarization, generating phosphatidylinositol 4-phosphate [PI(4)P] and PI(3,4)P₂, respectively (1, 17–19). Subsequently, VSPs were reported to cleave 3-phosphate from PI(3,4)P₂, generating PI(4)P upon larger depolarization (20), and, very recently, Ci-VSP was indicated to cleave 3-phosphate from PI(3,4,5)P₃, generating PI(4,5)P₂ (21, 22). Because different substrate reactions are best seen at different voltages, several authors have suggested that changing electric fields can drive VSPs successively through several catalytically active states that favor one set of substrates or reactions over another (20–22). The active site of VSPs is well conserved among species (12) and shows only a single amino acid difference from the active site of PTEN (1). When this residue in Ci-VSP was mutated to the corresponding amino acid of the PTEN active site, the mutated Ci-VSP still showed both 5- and 3-phosphatase activities toward polyphosphoinositides (14). Therefore, the observed substrate specificity and the voltage-dependent dual phosphatase activity of VSPs might be determined by the environment surrounding the active site rather than only by the active site itself (14). When the entire enzymatic domain of Ci-VSP was replaced by PTEN, the resulting chimera, called VSPTEN, had the enzymatic properties of a voltage-dependent pure 3-phosphatase (23).VSPs are found in remarkably diverse tissues, including the testis and brain of mice (7, 24, 25); testis, brain, and stomach of humans (6); testis and neuronal complex of the ascidian (1); and testis, ovary, kidney, and liver of the African frog (26). Surprisingly, the physiological functions of this widespread enzyme remain a puzzle. Recent work suggests that VSPs might have roles in egg fertilization (26) and in neuronal signaling in the brain (7). To understand its physiological enzymology more completely, we screened Dr-VSP–induced phosphoinositide changes in living cells by engineered Förster resonance energy transfer (FRET) probes that specifically report the cellular dynamics of PI(4)P, PI(3,4)P₂, PI(4,5)P₂, and PI(3,4,5)P₃. Our results show that when exogenous expression of PI(4)P 5-kinase type Iγ (PIPKIγ) was used selectively to counteract the 5-phosphatase activity of Dr-VSP, PI(4,5)P₂ accumulated, revealing an intrinsic 3-phosphatase activity of VSP toward PI(3,4,5)P₃. The voltage dependence and substrate specificity of 3- and 5-phosphatase subreactions of Dr-VSP were then extracted quantitatively by a comprehensive kinetic systems analysis. Together, our data demonstrate that Dr-VSP possesses both 3- and 5-phosphatase activities toward PI(3,4,5)P₃, with the same voltage dependence but with quite different absolute catalytic rates. These results should help clarify the roles of VSPs in fertilization, neural computations, and other signaling events that involve voltage changes.     

Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K⁺ channel α-subunits to form I_Kslow (I_Ks) channels in the heart and ear. The number of E1 subunits in I_Ks channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface I_Ks channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, I_Ks channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce I_Ks channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.Voltage-gated potassium (K_V) channels include four α-subunits that form a single, central ion conduction pathway with four peripheral voltage sensors (1–3). Incorporation of accessory β-subunits modifies the function of K_V channels to suit the diverse requirements of different tissues. KCNE genes encode minK-related peptides (MiRPs) (4–6), β-subunits with a single transmembrane span that assemble with a wide array of K_V α-subunits (7, 8) to control surface expression, voltage dependence, and kinetics of gating transitions, unitary conductance, ion selectivity, and pharmacology of the resultant channel complexes (4, 9–15). I_Kslow (I_Ks) channels in the heart and inner ear are formed by the α-subunit encoded by KCNQ1 (called Q1, K_VLQT1, K_V7.1, or KCNQ1) and the β-subunit encoded by KCNE1 (called E1, mink, or KCNE1) (16, 17). Inherited mutations in Q1 and E1 are associated with cardiac arrhythmia and deafness.The number of E1 subunits in I_Ks channels has been a longstanding matter of disagreement. We first argued for two E1 subunits per channel based on the suppression of current by an E1 mutant (18). Subsequently, we reached the same conclusion by determining the total number of channels using radiolabeled charybdotoxin (CTX), a scorpion toxin that blocks channels when one molecule binds in the external conduction pore vestibule, and an antibody-based luminescence assay to tally E1 subunits (19). Morin and Kobertz (20) used iterative chemical linkage between CTX in the pore and E1, and they also assigned two accessory subunits to >95% of I_Ks channels without gathering evidence for variation in subunit valence. Furthermore, when we formed I_Ks channels from separate E1 and Q1 subunits and compared them with channels enforced via genetic encoding to contain two or four E1 subunits (19), we observed the natural I_Ks channels to have the same gating attributes, small-molecule pharmacology, and CTX on and off rates (a reflection of pore vestibule structure) as channels encoded with two E1 subunits but not those with four. These findings support the conclusion that two E1 subunits are necessary, sufficient, and the normal number in I_Ks channels.In contrast, others have argued that I_Ks channels have variable stoichiometry with one to four E1 subunits, or even more (21–24). Recently, Nakajo et al. (25) applied single-particle spectroscopy to the question; this powerful “gold-standard” tool has been a valuable strategy to assess the subunit composition of ion channels (26–28) and should be expected to improve on prior investigations conducted on populations of I_Ks channels and subject, therefore, to the simplifying assumptions that attend macroscopic studies (29). Nakajo et al. (25) reported a variable number of E1 subunits, from one to four, in I_Ks channels studied in Xenopus laevis oocytes. The impact of this result has been striking because it has engendered new models of cardiac physiology, altered models of I_Ks channel biosynthesis and function, stimulated the use of transgenic animals artificially enforced to express I_Ks channels with four E1 subunits (by expression of a fused E1–Q1 subunit), and prompted cardiac drug design based on the assumption that I_Ks channels can form with one E1 subunit (23, 30–32).We were concerned that the conclusions of Nakajo et al. (25) were in error because they appraised only a limited fraction of particles that were immobile in the oocyte membrane; counted E1 and Q1 asynchronously rather than simultaneously (increasing the risk that particles moved into or out of the field of view); and studied Q1 and E1 appended not only with the fluorescent proteins (FP) required to count subunits by photobleaching but also with a common trafficking motif that suppressed channel mobility by interacting with an overexpressed anchoring protein, thereby risking nonnatural aggregation of subunits.Here, to resolve mobility problems and obviate the need for modification of subunits with targeting motifs, we describe and perform single-fluorescent-particle photobleaching at the surface of live mammalian cells, demonstrating three spectroscopic counting approaches: standard, asynchronous subunit counting; simultaneous, two-color subunit counting; and toxin-directed, simultaneous, two-color photobleaching. To analyze the data, we use two statistical approaches—one to assess the degree of colocalization of objects in dual-color images (33) and the other to infer stoichiometry from single-molecule photobleaching (34). These methods also allow determination of the surface density of assemblies of defined subunit composition and are therefore useful to assess the formation and life cycle of membrane protein complexes.We report that single I_Ks channels at the surface of mammalian cells contain two E1 subunits—no more and no less. This finding refutes the single-particle studies of Nakajo et al. (25) in oocytes and macroscopic studies (21–24, 30–32), arguing that forcing cells to express excess E1 produces I_Ks channels containing more than two E1 subunits and that low levels of E1 yields I_Ks channels with less than two E1 subunits. Not once did we observe an I_Ks channel with three or four E1 subunits. Moreover, simultaneous, two-color subunit counting revealed that low amounts of E1 relative to Q1 [ratios like those reported in human cardiac ventricle (35, 36)] produced two types of channels on the cell surface: I_Ks channels (with two E1 subunits) and Q1 channels (with no E1 subunits). Finally, E1 was shown to increase in I_Ks channel surface expression threefold, as we predicted based on assessment of I_Ks channel unitary conductance (11), whereas few E1 subunits were on the surface outside of I_Ks channels, even when E1 was expressed alone. This finding indicates that E1 does not travel to the surface readily on its own, that two E1 subunits facilitate I_Ks channel trafficking to the surface (or enhance surface residence time compared with Q1 channels), and that I_Ks channels with only one E1 subunit do not form, do not reach the surface, or are rapidly recycled.     

Improving recombinant Rubisco biogenesis,plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO₂-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco’s biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob^AtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob^AtL-R1 plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob^AtL-R1 and Arabidopsis leaves at 10–15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L₈^AS₈^t Rubisco [comprising AtL and tobacco small (S) subunits] in tob^AtL-R1 leaves compared with tob^AtL, despite >threefold lower steady-state Rubisco mRNA levels in tob^AtL-R1. Accompanying twofold increases in photosynthetic CO₂-assimilation rate and plant growth were measured for tob^AtL-R1 lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.The increasing global demands for food supply, bioenergy production, and CO₂-sequestration have placed a high need on improving agriculture yields and resource use (1, 2). It is now widely recognized that yield increases are possible by enhancing the light harvesting and CO₂-fixation processes of photosynthesis (3–5). A major target for improvement is the enzyme Rubisco [ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase] whose deficiencies in CO₂-fixing speed and efficiency pose a key limitation to photosynthetic CO₂ capture (6, 7). In plants, the complex, multistep catalytic mechanism of Rubisco to bind its 5-carbon substrate RuBP, orient its C-2 for carboxylation, and then process the 6-carbon product into two 3-phosphoglycerate (3PGA) products, limits its throughput to one to four catalytic cycles per second (8). The mechanism also makes Rubisco prone to competitive inhibition by O₂ that produces only one 3PGA and 2-phosphoglycolate (2PG). Metabolic recycling of 2PG by photorespiration requires energy and results in most plants losing 30% of their fixed CO₂ (5). To compensate for these catalytic limitations, plants like rice and wheat invest up to 50% of the leaf protein into Rubisco, which accounts for ∼25% of their leaf nitrogen (9).Natural diversity in Rubisco catalysis demonstrates that plant Rubisco is not the pinnacle of evolution (6, 7). Better-performing versions in some red algae have the potential to raise the yield of crops like rice and wheat by as much as 30% (10). Bioengineering Rubisco in leaves therefore faces two key challenges: identifying the structural changes that promote performance and identifying ways to efficiently transplant these changes into Rubisco within a target plant. A significant hurdle to both challenges is the complex biogenesis requirements of Rubisco in plant chloroplasts (7, 11). A number of ancillary proteins are required to correctly process and assemble the chloroplast made Rubisco large (L) subunit (coded by the plastome rbcL gene) and cytosol made small (S) subunits (coded by multiple RbcS genes in the nucleus) into L₈S₈ complexes in the chloroplast stroma. The complicated assembly requirements of Rubisco in chloroplasts prevent their functional testing in Escherichia coli and conversely impedes, sometimes prevents, the biogenesis of Rubisco from other higher plants, cyanobacteria, and algae (12–14). For example, the L-subunits from sunflower and varying Flaveria sp. showed fivefold differences in their capacity to form hybrid L₈S₈ Rubisco (that comprise tobacco S-subunits) in tobacco chloroplasts despite each rbcL transgene sharing the same genetic regulatory sequences and showing >92% amino acid identity (13, 14). Evidently, evolution of Rubisco function may have been constrained to maintain compatibility with the molecular chaperones required for its biogenesis (7, 15).The necessity of chloroplast chaperonin (CPN) complexes for Rubisco biogenesis has been known for some time (16). Upon release from the hetero-oligomeric CPN ring structures in chloroplasts (17) the folded L-subunits are thought to sequentially assemble into dimers (L₂) then octamers (L₂)₄ before S-subunit binding (18). The molecular details of this process remain unclear. The maize Photosynthetic Mutant Library has provided useful insight by identifying three chaperones with roles associated with Rubisco synthesis, assembly, and stability: Rubisco accumulation factors-1 (RAF1) (19) and-2 (RAF2; a Pterin-4a-Carbinolamine Dehydratase-like protein) (20) and Bundle Sheath Defective-2 (BSDII; a DnaJ-like protein) (21). Results of chemical cross-linking experiments in maize leaves suggest all three proteins might associate with the S-subunit during Rubisco biogenesis (20). Other studies, however, suggest RAF1 interacts with post-CPN folded L-subunits to assist in L₂ then (L₂)₄ formation (19, 22). This function mirrors that shown for RbcX, a Rubisco chaperone that acts as a “molecular staple” to assemble folded L-subunits into L₂ units for (L₂)₄ assembly before S-subunit binding to displace the RbcX and trigger catalytic potential (18). Although the function of RbcX in L₈S₈ Rubisco biogenesis has been resolved in exquisite molecular detail in vitro and in E. coli, its functional role in cyanobacteria and in leaf chloroplasts remain unresolved. Comparable molecular details on RAF1, RAF2, and BSDII structure and function remain incomplete, making it difficult to reliably assign their roles and interactions with Rubisco in chloroplasts.Targeted transformation of the chloroplast genome (plastome) provides a reliable but time-consuming tool for engineering Rubisco (23). This technology is best developed in tobacco with the ^cmtrL genotype specifically made for bioengineering Rubisco and testing its effects on leaf photosynthesis and growth (6, 7, 13, 14). Here we use chloroplast transformation in ^cmtrL to examine the function of RAF1 from Arabidopsis (AtRAF1) in Rubisco biogenesis. We show that AtRAF1 forms a stable dimer that, when coexpressed with its cognate Arabidopsis Rubisco L-subunits (AtL), enhances hybrid L₈^AS₈^t Rubisco (containing Arabidopsis L- and tobacco S-subunits) assembly in tobacco chloroplasts and concomitantly improves leaf photosynthesis and plant growth by more than twofold.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.