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.     

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

Adenosine A_2A receptor (A_2AR)-dopamine D₂ receptor (D₂R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A_2AR-D₂R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D₂R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A_2AR agonists, but also A_2AR antagonists, decrease the affinity and intrinsic efficacy of D₂R agonists and the affinity of D₂R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A_2AR-D₂R heteromers as heterotetramers, constituted by A_2AR and D₂R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A_2AR antagonists behaved as A_2AR agonists and decreased D₂R function in the brain.Most evidence indicates that G protein-coupled receptors (GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be viewed as multiples of dimers (1). It has been proposed that GPCR heteromers are constituted mainly by heteromers of homodimers (1, 2). Allosteric mechanisms determine a multiplicity of unique pharmacologic properties of GPCR homodimers and heteromers (1, 3). First, binding of a ligand to one of the receptors in the heteromer can modify the affinity of ligands for the other receptor (1, 3, 4). The most widely reproduced allosteric modulation of ligand-binding properties in a GPCR heteromer is the ability of adenosine A_2A receptor (A_2AR) agonists to decrease the affinity of dopamine D₂ receptor (D₂R) agonists in the A_2AR-D₂R heteromer (5). A_2AR-D₂R heteromers have been revealed both in transfected cells (6, 7), striatal neurons in culture (6, 8) and in situ, in mammalian striatum (9, 10), where they play an important role in the modulation of GABAergic striatopallidal neuronal function (9, 11).In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to the varying intrinsic efficacy of ligands for different signaling pathways (1–3). Intrinsic efficacy refers to the power of the agonist to induce a functional response, independent of its affinity for the receptor. Thus, allosteric modulation of an agonist can potentially involve changes in affinity and/or intrinsic efficacy (1, 3). This principle can be observed in the A_2AR-D₂R heteromer, where a decrease in D₂R agonist affinity cannot alone explain the ability of an A_2AR agonist to abolish the decreased excitability of GABAergic striatopallidal neurons induced by high concentration of a D₂R agonist (9), which should overcome the decrease in affinity. Furthermore, a differential effect of allosteric modulations of different agonist-mediated signaling responses (i.e., functional selectivity) can occur within GPCR heteromers (1, 2, 8). Again, the A_2AR-D₂R heteromer provides a valuable example. A recent study has shown that different levels of intracellular Ca²⁺ exert different modulations of A_2AR-D₂R heteromer signaling (8). This depends on the ability of low and high Ca²⁺ to promote a selective interaction of the heteromer with different Ca²⁺-binding proteins, which differentially modulate allosteric interactions in the heteromer (8).It has been hypothesized that the allosteric interactions between A_2AR and D₂R agonists within the A_2AR-D₂R heteromer provide a mechanism responsible not only for the depressant effects of A_2AR agonists, but also for the psychostimulant effects of adenosine A_2AR antagonists and the nonselective adenosine receptor antagonist caffeine (9, 11, 12), with implications for several neuropsychiatric disorders (13). In fact, the same mechanism has provided the rationale for the use of A_2AR antagonists in patients with Parkinson’s disease (13, 14). The initial aim of the present study was to study in detail the ability of caffeine to counteract allosteric modulations between A_2AR and D₂R agonists (affinity and intrinsic efficacy) within the A_2AR-D₂R heteromer. Unexpectedly, when performing control radioligand-binding experiments, not only an A_2AR agonist, but also caffeine, significantly decreased D₂R agonist binding. However, when coadministered, the A_2AR agonist and caffeine co-counteracted their ability to modulate D₂R agonist binding. By exploring the molecular mechanisms behind these apparent inconsistencies, the present study provides new insight into the quaternary structure and function of A_2AR-D₂R heteromers.     

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.     

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.     

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K⁺ channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K⁺ channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K⁺ channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.Voltage-gated K⁺ channels are highly conserved among bilaterian metazoans and play a central role in the regulation of excitation in neurons and muscle. Understanding the functional evolution of these channels may therefore provide important insights into how neuromuscular excitation evolved within the Metazoa. Three major gene families, Shaker, KCNQ, and Ether-a-go-go (EAG) encode all voltage-gated K⁺ channels in bilaterians (1, 2). In this study, we examine the functional evolution and origins of the Shaker and KCNQ gene families. Shaker family channels can be definitively identified by a unique subunit structure that includes both a voltage-gated K⁺ channel core and a family-specific cytoplasmic domain within the N terminus known as the T1 domain. T1 mediates assembly of Shaker family subunits into functional tetrameric channels (3, 4). KCNQ channels are also tetrameric but lack a T1 domain and use a distinct coiled-coil assembly domain in the C terminus (5, 6). KCNQ channels can be identified by the presence of this family-specific assembly motif and high amino acid conservation within the K⁺ channel core. Both channel families are found in cnidarians (1, 7) and thus predate the divergence of cnidarians and bilaterians, but their ultimate evolutionary origins have not yet been defined.Shaker family K⁺ channels serve diverse roles in the regulation of neuronal firing and can be divided into four gene subfamilies based on function and sequence homology: Shaker, Shab, Shal, and Shaw (8, 9). The T1 assembly domain is only compatible between subunits from the same gene subfamily (4, 10) and thus serves to keep the subfamilies functionally segregated. Shaker subfamily channels activate rapidly near action potential threshold and range from rapidly inactivating to noninactivating. Multiple roles for Shaker channels in neurons and muscles have been described, but their most unique and fundamental role may be that of axonal action potential repolarization. Shaker channels are clustered to the axon initial segment and nodes of Ranvier in vertebrate neurons (11–13) and underlie the delayed rectifier in squid giant axons (14). The Shaker subfamily is diverse in cnidarians (15, 16), and the starlet sea anemone Nematostella vectensis has functional orthologs of most identified Shaker current types observed in bilaterians (16).The Shab and Shal gene subfamilies encode somatodendritic delayed rectifiers and A currents, respectively (17–20). Shab channels are important for maintaining sustained firing (21, 22), whereas the Kv4-based A current modulates spike threshold and frequency (17). Shab and Shal channels are present in cnidarians, but cnidarian Shab channels have not been functionally characterized, and the only cnidarian Shal channels expressed to date display atypical voltage dependence and kinetics compared with bilaterian channels (23). Shaw channels are rapid, high-threshold channels specialized for sustaining fast firing in vertebrates (24, 25) but have a low activation threshold and may contribute to resting potential in Drosophila (19, 26, 27). A Caenorhabditis elegans Shaw has slow kinetics but a high activation threshold (28), and a single expressed cnidarian Shaw channel has the opposite: a low activation threshold but relatively fast kinetics (29). Thus, the ancestral properties and function of Shaw channels is not yet understood. Further functional characterization of cnidarian Shab, Shal, and Shaw channels would provide a better understanding of the evolutionary status of the Shaker family in early parahoxozoans.KCNQ family channels underlie the M current in vertebrate neurons (30) that regulates subthreshold excitability (31). The M current provides a fundamental mechanism for regulation of firing threshold through the Gq G-protein pathway because KCNQ channels require phosphatidylinositol 4,5-bisphosphate (PIP₂) for activation (32, 33). PIP₂ hydrolysis and subsequent KCNQ channel closure initiated by Gq-coupled receptors produces slow excitatory postsynaptic potentials, during which the probability of firing is greatly increased (32, 33). The key functional adaptations of KCNQ channels for this physiological role that can be observed in vitro are (i) a requirement for PIP₂ to couple voltage-sensor activation to pore opening (34, 35), and (ii) a hyperpolarized voltage–activation curve that allows channels to open below typical action potential thresholds. Both key features are found in vertebrate (30, 34, 36–38), Drosophila (39), and C. elegans (40) KCNQ channels, suggesting they may have been present in KCNQ channels in a bilaterian ancestor. Evolution of the M current likely represented a major advance in the ability to modulate the activity of neuronal circuits, but it is not yet clear when PIP₂-dependent KCNQ channels first evolved.Here, we examine the origins and functional evolution of the Shaker and KCNQ gene families. If we assume the evolution of neuronal signaling provided a major selective pressure for the functional diversification of voltage-gated K⁺ channels, then we can hypothesize that the appearance of these gene families might accompany the emergence of the first nervous systems or a major event in nervous system evolution. Recent phylogenies that place the divergence of ctenophores near the root of the metazoan tree suggest that the first nervous systems, or at least the capacity to make neurons, may have been present in a basal metazoan ancestor (41–43) (Fig. S1). One hypothesis then is that much of the diversity of metazoan voltage-gated channels should be shared between ctenophores and parahoxozoans [cnidarians, bilaterians, and placozoans (44)]. However, genome analysis indicates that many “typical” neuronal genes are missing in ctenophores and the sponges lack a nervous system, leading to the suggestion that extant nervous systems may have evolved independently in ctenophores and parahoxozoans (42, 45). Thus, a second hypothesis is that important steps in voltage-gated K⁺ channel evolution might have occurred separately in ctenophores and parahoxozoans. We tested these hypotheses by carefully examining the phylogenetic distribution and functional evolution of Shaker and KCNQ family K⁺ channels. Our results support a model in which major innovations in neuromuscular excitability occurred specifically within the parahoxozoan lineage.     

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere''s composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO₂-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO₂. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO₂. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.Rubisco, the most abundant enzyme in the biosphere (1), fixes CO₂ into organic carbon that supports nearly all life on Earth (2, 3). Over the past 3 billion y, the enzyme became a victim of its own success as it drew down the atmospheric CO₂ concentration to trace levels (4) and as the oxygen-producing reactions of photosynthesis filled our atmosphere with O₂ (4). In today’s atmosphere, O₂ competes with CO₂ at Rubisco''s catalytic site, producing the toxic compound phosphoglycolate (5). Phosphoglycolate must be metabolized at the expense of energy and loss of fixed carbon and nitrogen (6). To overcome Rubisco''s limitations, many photosynthetic organisms have evolved carbon-concentrating mechanisms (CCMs) (7, 8). CCMs increase the CO₂ concentration around Rubisco, decreasing O₂ competition and enhancing carbon fixation.At the heart of the CCM of eukaryotic algae is an organelle known as the pyrenoid (9). The pyrenoid is a spherical structure in the chloroplast stroma, discovered more than 130 y ago (10–12). Pyrenoids have been found in nearly all of the major oceanic eukaryotic primary producers and mediate ∼28–44% of global carbon fixation (SI Appendix, Table S1) (3, 13–17). A pyrenoid typically consists of a matrix surrounded by a starch sheath and traversed by membrane tubules continuous with the photosynthetic thylakoid membranes (18). This matrix is thought to consist primarily of tightly packed Rubisco and its chaperone, Rubisco activase (19). In higher plants and non–pyrenoid-containing photosynthetic eukaryotes, Rubisco is instead soluble throughout the chloroplast stroma. The molecular mechanism by which Rubisco aggregates to form the pyrenoid matrix remains enigmatic.Two mechanisms for Rubisco accumulation in the pyrenoid have been proposed: (i) Rubisco holoenzymes could bind each other directly through hydrophobic residues (20), or (ii) a linker protein may link Rubisco holoenzymes together (18, 20). The second model is based on analogy to the well-characterized prokaryotic carbon concentrating organelle, the β-carboxysome, where Rubisco aggregation is mediated by a linker protein consisting of repeats of a domain resembling the Rubisco small subunit (21). Here we find that Rubisco accumulation in the pyrenoid of the model alga Chlamydomonas reinhardtii is mediated by a disordered repeat protein, which we term Essential Pyrenoid Component 1 (EPYC1). Our findings suggest a mechanism for aggregation of Rubisco in the pyrenoid matrix, and highlight similarities and differences between the mechanisms of assembly of the eukaryotic and prokaryotic organelles.     

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.     

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.     

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H₂O₂ sensitive and that they can detect strong UV light via sensing light-induced production of H₂O₂. We found that ectopic expression of these H₂O₂-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H₂O₂-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H₂O₂-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H₂O₂-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H₂O₂ production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H₂O₂. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae.Light is an important sensory cue that has a wide-ranging influence on animal physiology and behavior. In addition to its role in vision, light detection also contributes to circadian rhythm regulation, sleep, phototaxis, and even mood control (1–5). Animals of different species have evolved diverse light sensors and light-detection cells to regulate various light-dependent physiological processes. For example, whereas rods and cones in mammals are critical for detecting and relaying image-forming visual information to the visual cortex, the intrinsically light-sensitive melanopsin-expressing retinal ganglion cells relay nonimage-forming visual information primarily to the suprachiasmatic nucleus to regulate circadian rhythm (6, 7).Similar to the diversity of cell types that sense light, the molecular mechanisms for light detection also vary. For example, in mammalian rods and cones, light activates a Rhodopsin-dependent phototransduction pathway that hyperpolarizes the cells via closing a cyclic nucleotide-gated channel (5). In the intrinsically light-sensitive retinal ganglion cells, however, light activates a melanopsin-dependent pathway that depolarizes the cells via opening TRP channels (6, 8). Further, in Drosophila PDF neurons, light has been shown to activate cryptochrome signaling to depolarize the cells (9). And in the ASJ sensory neuron in Caenorhabditis elegans, UV and blue light have been shown to activate the cell via signaling a specific GPCR known as LITE-1 (high energy light unresponsive protein 1) (10, 11).It has been shown that short wavelength UV and blue light can trigger reactive oxygen species (ROS) and H₂O₂ production in cultured cells (12), and H₂O₂ can modulate ion channel activities directly or indirectly (13–15). We therefore reasoned that sensing H₂O₂ might constitute another light-sensing mechanism. Indeed, a recent finding has suggested that two specific GPCRs—GUR-3 and LITE-1—both of which play important roles in light sensing in C. elegans, may also be H₂O₂ sensitive (16). Moreover, TRPA1, an evolutionarily conserved TRP channel known for its role in sensing many chemical irritants, has been shown to sense H₂O₂ (17–24), thus TRPA1 may be activated by light via sensing light-induced H₂O₂ production. Some recent evidence has demonstrated a role for TRPA1 in light sensing. First, in human melanocyte, TRPA1 has been shown to act to increase melanin production in response to UV (25). Moreover, in Drosophila, TRPA1 has been shown to increase neuronal activities of a specific group of larval somatosensory neurons (also known as the C4da neurons) in response to UV (26). However, in both cases, GPCRs—rhodopsin in the case of human melanocyte and Gr28b in the case of C4da neurons—are thought to mediate light-dependent activation of TRPA1 (25, 26); thus, it is not clear whether TRPA1 can be activated by light through H₂O₂ production.Here, we show that two specific isoforms of Drosophila dTRPA1 are H₂O₂ sensitive and that their H₂O₂ sensitivity allows them to detect UV without relying on conventional phototransduction molecules. We found that ectopic expression of these H₂O₂-sensitive dTRPA1 isoforms conferred UV sensitivity to light-insensitive cultured HEK293 cells and a few types of light-insensitive Drosophila neurons. The light and H₂O₂ sensitivities are specific to certain dTRPA1 isoforms, consistent with previous findings that different dTRPA1 isoforms exhibit distinct thermal sensitivities (20, 21). Strikingly, the H₂O₂-sensitive dTRPA1 was as effective as channelrhodopsin-2 (ChR2) in triggering light-produced motor responses when expressed in a specific group of motor neurons that control proboscis extension in adult Drosophila. We further discovered that the corpus cardiacum (CC) cells, a group of adipokinetic hormone (AKH)-producing cells that reside in the Drosophila larval ring gland, expressed the H₂O₂-sensitive dTRPA1 isoforms endogenously, and that these cells were UV and H₂O₂ sensitive. Their sensitivity required dTRPA1 and H₂O₂ production: Reducing dual oxidase (DUOX) or increasing catalase (cat) expression in them reduced their UV sensitivity significantly. In contrast, reducing PLC or GR28b in CC cells had little impact. Our results suggest specific H₂O₂-sensitive TRPA1 isoforms can be activated by UV via a photochemical transduction cascade and that these isoforms may be exploited as a UV-dependent optogenetic tool for controlling neuronal activities.     

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

H⁺-transporting F₁F₀ ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F₀ and F₁. H⁺ transport at the subunit a–c interface in transmembranous F₀ drives rotation of a cylindrical c₁₀ oligomer within the membrane, which is coupled to rotation of subunit γ within the α₃β₃ sector of F₁ to mechanically drive ATP synthesis. F₁F₀ functions in a reversible manner, with ATP hydrolysis driving H⁺ transport. ATP-driven H⁺ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag⁺ and/or Cd⁺² with the substituted sulfhydryl groups. The H⁺ transport pathway mapped via these Ag⁺(Cd⁺²)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H⁺ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd⁺² directly inhibits passive H⁺ transport mediated by F₀ reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H⁺ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H⁺ transport likely interact in a single structural domain, which then functions in gating H⁺ release to the cytoplasm.The F₁F₀-ATP synthase of oxidative phosphorylation uses the energy of a transmembrane electrochemical gradient of H⁺ or Na⁺ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F₁ and F₀ sectors of the enzyme (1). H⁺ transport through the transmembrane F₀ sector is coupled to ATP synthesis or hydrolysis in the F₁ sector at the surface of the membrane. Homologous ATP synthases are found in mitochondria, chloroplasts, and many bacteria. In Escherichia coli and other eubacteria, F₁ consists of five subunits in an α₃β₃γδε stoichiometry. F₀ is composed of three subunits in a likely ratio of a₁b₂c₁₀ in E. coli and Bacillus PS3 (2, 3) or a₁b₂c₁₁ in the Na⁺ translocating Ilyobacter tartaricus ATP synthase (1, 4) and may contain as many as 15 c subunits in other bacterial species (5). Subunit c spans the membrane as a hairpin of two α-helices, with the first transmembrane helix (TMH) on the inside and the second TMH on the outside of the c ring (1, 4). The binding of Na⁺ or H⁺ occurs at an essential, membrane-embedded Glu or Asp on cTMH2. High-resolution X-ray structures of both Na⁺- and H⁺-binding c-rings have revealed the details and variations in the cation binding sites (4–8). In the H⁺-translocating E. coli enzyme, Asp-61 at the center of cTMH2 is thought to undergo protonation and deprotonation, as each subunit of the c ring moves past the stationary subunit a. In the functioning enzyme, the rotation of the c ring is thought to be driven by H⁺ transport at the subunit a/c interface. Subunit γ physically binds to the cytoplasmic surface of the c-ring, which results in the coupling of c-ring rotation with rotation of subunit γ within the α₃β₃ hexamer of F₁ to mechanically drive ATP synthesis (1).E. coli subunit a folds in the membrane with five TMHs and is thought to provide aqueous access channels to the H⁺-binding cAsp-61 residue (9, 10). Interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation–protonation cycle of cAsp-61 (1, 11, 12). At this time, very limited biophysical or crystallographic information is available on the 3D arrangement of the TMHs in subunit a. TMHs 2–5 of subunit a pack in a four-helix bundle, which was initially defined by cross-linking (13), but now, such a bundle, packing at the periphery of the c-ring, has been viewed directly by high-resolution cryoelectron microscopy in the I. tartaricus enzyme (14). Previously published cross-linking experiments support the identification of aTMH4 and aTMH5 packing at the periphery of the c-ring and the identification of aTHM2 and aTMH3 as the other components of the four-helix bundle seen in these images (13, 15, 16). More recently, published cross-linking experiments identify the N-terminal α-helices of two b subunits, one of which packs at one surface of aTMH2 with close enough proximity to the c-ring to permit cross-linking (17). The other subunit b N-terminal helix packs on the opposite peripheral surface of aTMH2 in a position where it can also be cross-linked to aTMH3 (17). The last helix density shown in Hakulinen and colleagues (14) packs at the periphery of the c-ring next to aTMH5 and is very likely to be aTMH1.The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag⁺ and other thiolate-reactive agents (18–20). Two regions of aqueous access were found with distinctly different properties. One region in TMH4, extending from Asn-214 and Arg-210 at the center of the membrane to the cytoplasmic surface, contains Cys substitutions that are sensitive to inhibition by both N-ethylmaleimide (NEM) and Ag⁺ (18–20; Fig. 1). These NEM- and Ag⁺-sensitive residues in TMH4 pack at or near the peripheral face and cytoplasmic side of the modeled four-helix bundle (11, 13). A second set of Ag⁺-sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18, 19), and Ag⁺-sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19, 20). The Ag⁺-sensitive substitutions on the periplasmic side of TMHs 2–5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11, 13, 19, 20). We have proposed that the movement of H⁺ from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a–c subunit interface (16, 21–24). This gating is thought to be coupled with ionization of a protonated cAsp61 in the adjacent subunit of the c-ring and with release of the H⁺ into the cytoplasmic half-channel at the subunit a–c interface. The route of aqueous access to the cytoplasmic side of the c subunit packing at the a–c interface has also been mapped by the chemical probing of Cys substitutions and, more recently, by molecular dynamics simulations (22, 25, 26).Open in a separate windowFig. 1.The predicted topology of subunit a in the E. coli inner membrane. The location of the most Ag⁺-sensitive Cys substitutions are highlighted in red (>85% inhibition) or orange (66–85% inhibition). The five proposed TMHs are shown in boxes, each with a span of 21 amino acids, which is the minimum length required to span the hydrophobic core of a lipid bilayer. The α-helical segments shown in loops 1–2 and 3–4 are consistent with the predictions of TALOS, based on backbone chemical shifts seen by NMR (29). Others have also predicted extensive α-helical regions in these loops (12, 30), but the possible positions remain largely speculative. aArg210 is highlighted in green. Figure is modified from those shown previously (21, 23, 24, 27).We have also reported Ag⁺-sensitive Cys substitutions in two cytoplasmic loops of subunit a (27) and, more recently, in the cytoplasmic loop of subunit c (28). The mechanism by which Ag⁺ inhibited F₁F₀-mediated H⁺ transport was uncertain. Several of these substitutions were also sensitive to inhibition by Cd⁺², and these substitutions provided a means of testing whether Cd⁺² directly inhibits passive H⁺ transport through F₀ (28). In the case of two subunit c loop substitutions, Cd⁺² was shown to directly inhibit passive F₀-mediated transport activity. In this study, we have extended the survey to Cd⁺²-sensitive Cys substitutions in cytoplasmic loops of subunit a. We report four loop substitutions in which Cd⁺² inhibits passive F₀-mediated H⁺ transport. Further, in two cases, we show cross-linking between pairs of Cys substitutions, which lie in subunits a and c, respectively, and which individually mediate passive H⁺ transport activity. These results suggest that the a and c loops, which gate H⁺ release to the cytoplasm, fold into a single domain at the surface of F₀.     

Hyperpolarization-activated,cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K⁺ and Na⁺ ions, and is selectively blocked by Cs⁺ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (I_h) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances I_h and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning.     

Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy

Protein palmitoylation regulates many aspects of cell function and is carried out by acyl transferases that contain zf-DHHC motifs. The in vivo physiological function of protein palmitoylation is largely unknown. Here we generated mice deficient in the acyl transferase Aph2 (Ablphilin 2 or zf-DHHC16) and demonstrated an essential role for Aph2 in embryonic/postnatal survival, eye development, and heart development. Aph2^−/− embryos and pups showed cardiomyopathy and cardiac defects including bradycardia. We identified phospholamban, a protein often associated with human cardiomyopathy, as an interacting partner and a substrate of Aph2. Aph2-mediated palmitoylation of phospholamban on cysteine 36 differentially alters its interaction with PKA and protein phosphatase 1 α, augmenting serine 16 phosphorylation, and regulates phospholamban pentamer formation. Aph2 deficiency results in phospholamban hypophosphorylation, a hyperinhibitory form. Ablation of phospholamban in Aph2^−/− mice histologically and functionally alleviated the heart defects. These findings establish Aph2 as a critical in vivo regulator of cardiac function and reveal roles for protein palmitoylation in the development of other organs including eyes.Protein S-acylation on cysteine residues by palmitate regulates substrate protein localization, trafficking, and protein–protein interactions (1–3) and could potentially play important roles in vivo (4–9). Palmitoylation is the only reversible lipid modification that can be removed by protein palmitoyl thioesterases. Recently, several palmitoyl acyltransferases (PATs) containing a unique zinc finger domain called zf-DHHC have been identified (10). Comparative genome searches have uncovered 23 PAT-like proteins in mammals (11). However, the physiological function of these PATs remains poorly understood (4).Heart disease is a leading cause of morbidity and mortality worldwide (12). Recent studies have established a pivotal role for defects of calcium cycling in the onset of heart disease (12–16), with a particularly critical role for the regulation of the sarcoplasmic reticulum (SR) calcium ATPase (SERCA). SERCA2a activity is controlled by phospholamban (PLN), an abundant SR protein. Upon β-adrenergic signaling, PKA can phosphorylate serine 16 of PLN, leading to PLN pentamer formation and loss of ability to inhibit SERCA2a (13, 14, 17). Ser16 phosphorylation is removed by phosphatases such as protein phosphatase 1 α (PP1α) (18, 19).It has been reported that some cardiac proteins are palmitoylated, including Na pump regulatory subunit phospholemman and β1-adrenergic receptor (20–22). However, there is a lack of genetic evidence that protein palmitoylation plays a role in heart development and function. Here we have taken a reverse genetic approach to study the physiological function of Aph2 (Ablphilin 2), first identified as an interacting protein of nonreceptor tyrosine kinase c-Abl (23), and provide direct evidence that Aph2 is a PAT, with PLN as one substrate, and that Aph2 is essential for embryonic/postnatal survival, eye development, proper embryonic heart development, and in vivo cardiac function. Some of the heart phenotypes can be rescued by PLN deficiency. Thus, Aph2-deficient mice represent an animal model for cardiomyopathy, whose pathogenesis involves defective PLN palmitoylation.     

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.