Anticonvulsant activity of artificial sweeteners: a structural link between sweet-taste receptor T1R3 and brain glutamate receptors

A virtual screening campaign based on application of a topological discriminant function capable of identifying novel anticonvulsant agents indicated several widely-used artificial sweeteners as potential anticonvulsant candidates. Acesulfame potassium, cyclamate and saccharin were tested in the Maximal Electroshock Seizure model (mice, ip), showing moderate anticonvulsant activity. We hypothesized a probable structural link between the receptor responsible of sweet taste and anticonvulsant molecular targets. Bioinformatic tools confirmed a highly significant sequence-similarity between taste-related protein T1R3 and several metabotropic glutamate receptors from different species, including glutamate receptors upregulated in epileptogenesis and certain types of epilepsy.     

Homer蛋白介导谷氨酸受体信号转导

Homer蛋白是一类联系突触内细胞骨架蛋白、信号蛋白的重要物质。Homer家族蛋白可和mGluRI、IP3R、Shank、RyR中富含脯氨酸的序列结合。Homer蛋白可以自我交联形成同聚或异聚体 ,此多聚体通过与多种蛋白、受体形成复合体并相互作用 ,在信号转导、突触形成、受体在细胞定位起重要作用。     

Homer and the ryanodine receptor

Homer proteins have recently been identified as novel high-affinity ligands that modulate ryanodine receptor (RyR) Ca²⁺ release channels in heart and skeletal muscle, through an EVH1 domain which binds to proline-rich regions in target proteins. Many Homer proteins can also self-associate through a coiled-coil domain that allows their multimerisation. In other tissues, especially neurons, Homer anchors proteins embedded in the surface membrane to the Ca²⁺ release channel in the endoplasmic reticulum and can anchor membrane or cytosolic proteins to the cytoskeleton. Although this anchoring aspect of Homer function has not been extensively investigated in muscle, there are consensus sequences for Homer binding in the RyR and on many of the proteins that it interacts with in the massive RyR ion channel complex. In this review we explore the potential of Homer to contribute to a variety of cell processes in muscle and neurons that also involve RyR channels.     

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: modulation by Homer

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.     

Remote control of neuronal activity with a light-gated glutamate receptor

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.     

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.     

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG-TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.     

Osteoblastic glutamate receptor function regulates bone formation and resorption

Previous studies showed that a variety of bone cells express protein components necessary for neuronal-like glutamatergic signaling and implicated glutamate as having a role in mechanically induced bone remodeling. Initial functional studies concentrated on the role of glutamate signaling in bone resorption and provided compelling evidence to suggest that glutamate signaling through functional NMDA type ionotropic glutamate receptors (iGluRs) is a prerequisite for in vitro osteoclastogenesis. Originally, effects of iGluR antagonists seen in co-cultures were attributed to antagonists acting directly on osteoclast precursors. However, in the light of recent osteoblast studies it now seems likely that the observed effects on osteoclastogenesis are an indirect effect of modulating the function of pre-osteoblast present within these cultures. The presence of iGluRs in osteoblasts suggests a role for them in bone formation and this paper reviews and discusses the emerging data relating to the role of glutamate signaling in osteoblasts. A number of recently published studies have shown that osteoblasts not only express a wide number of 'pre-synaptic' glutamatergic proteins but also possess the ability to both regulate glutamate release and actively recycle extracellular glutamate. The functionality of osteoblastic 'post-synaptic' glutamatergic components has also been shown as both primary and clonal osteoblasts express electrophysiologically active iGluRs, metabotropic type glutamate receptors (mGluRs) along with a variety of glutamate receptor associated signaling proteins. There is, however, little published data regarding the actual role of glutamatergic signaling in osteoblastic bone formation. In vivo and in vitro studies performed provide evidence that glutamatergic signaling is a necessity for normal osteoblast function. In a number of different models of in vitro bone formation, the addition of non-competitive antagonists of iGluRs prevents the formation of mineralized bone, moreover antagonizing some sub-types of iGluR mediates the differentiation of pre-osteoblasts. iGluR antagonists modulate osteoblast function in a manner that correlates with the previously reported data regarding in vitro osteoclastogenesis. Interestingly iGluR mediated glutamate signaling appears to function differently in osteoblasts derived from flat and long bones. This implies the components of osteoblastic glutamatergic signaling may be adapted in vivo possibly to reflect the differential function of osteoblasts in those regions of the skeleton.     

The structure and function of glutamate receptor ion channels

As in the case of many ligand-gated ion channels, the biochemical and electrophysiological properties of the ionotropic glutamate receptors have been studied extensively. Nevertheless, we still do not understand the molecular mechanisms that harness the free energy of agonist binding, first to drive channel opening, and then to allow the channel to close (desensitize) even though agonist remains bound. Recent crystallographic analyses of the ligand-binding domains of these receptors have identified conformational changes associated with agonist binding, yielding a working hypothesis of channel function. This opens the way to determining how the domains and subunits are assembled into an oligomeric channel, how the domains are connected, how the channel is formed, and where it is located relative to the ligand-binding domains, all of which govern the processes of channel activation and desensitization.     

The zinc sensing receptor, a link between zinc and cell signaling

Zinc is essential for cell growth. For many years it has been used to treat various epithelial disorders, ranging from wound healing to diarrhea and ulcerative colon disease. The physiological/molecular mechanisms linking zinc and cell growth, however, are not well understood. In recent years, Zn2+ has emerged as an important signaling molecule, activating intracellular pathways and regulating cell fate. We have functionally identified an extracellular zinc sensing receptor, called zinc sensing receptor (ZnR), that is specifically activated by extracellular Zn2+ at physiological concentrations. The putative ZnR is pharmacologically coupled to a Gq-protein which triggers release of Ca2+ from intracellular stores via the Inositol 1,4,5-trisphosphate (IP3) pathway. This, in turn results in downstream signaling via the MAP and phosphatidylinositol 3-kinase (PI3 kinase) pathways that are linked to cell proliferation. In some cell types, e.g., colonocytes, ZnR activity also upregulates Na+/H+ exchange, mediated by Na+/H+ exchanger isoform 1 (NHE1), which is involved in cellular ion homeostasis in addition to cell proliferation. Our overall hypothesis, as discussed below, is that a ZnR, found in organs where dynamic zinc homeostasis is observed, enables extracellular Zn2+ to trigger intracellular signaling pathways regulating key cell functions. These include cell proliferation and survival, vectorial ion transport and hormone secretion. Finally, we suggest that ZnR activity found in colonocytes is well positioned to attenuate erosion of the epithelial lining of the colon, thereby preventing or ameliorating diarrhea, but, by signaling through the same pathways, a ZnR may enhance tumor progression in neoplastic disease.     

FANCE: the link between Fanconi anaemia complex assembly and activity

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.     

Mutual regulation between metabotropic glutamate type 1alpha receptor and caveolin proteins: from traffick to constitutive activity

In this paper, a molecular and functional interaction between metabotropic glutamate receptor type 1alpha (mGlu1alpha receptor) and caveolin-1 or caveolin-2beta is described. An overlapping pattern of staining for mGlu1alpha receptor with caveolin-1 and caveolin-2 by confocal laser microscopy in transiently transfected HEK-293 cells is observed. The presence of mGlu1alpha receptor in caveolin-enriched membrane fractions was demonstrated by flotation gradient analysis in the absence of detergents and the interaction between mGlu1alpha receptor with caveolin-1 and with caveolin-2beta was demonstrated by coimmunoprecipitation experiments. In HEK-293 cells, caveolin-2beta accumulates surrounding lipid droplets when single expressed but coexpression with mGlu1alpha receptor changed dramatically the subcellular localization of caveolin-2beta, directing it from lipid droplets to the cell surface. At the membrane level, the interaction between caveolin-1 and mGlu1alpha receptor could abrogate the constitutive activity exhibited by mGlu1alpha receptor. Overall, these results show that mGlu1alpha receptor interacts with caveolins and that this interaction is physiologically relevant for receptor function. Interestingly, we provide evidence that caveolin-1 is not just acting as a scaffolding protein for the mGlu1alpha receptor but that also regulates mGlu1alpha receptor constitutive activity.     

Dysregulated Src upregulation of NMDA receptor activity: a common link in chronic pain and schizophrenia

Upregulation of N-methyl-D-aspartate (NMDA) receptor function by the nonreceptor protein tyrosine kinase Src has been implicated in physiological plasticity at glutamatergic synapses. Here, we highlight recent findings suggesting that aberrant Src upregulation of NMDA receptors may also be key in pathophysiological conditions. Within the nociceptive processing network in the dorsal horn of the spinal cord, pathologically increased Src upregulation of NMDA receptors is critical for pain hypersensitivity in models of chronic inflammatory and neuropathic pain. On the other hand, in the hippocampus and prefrontal cortex, the physiological upregulation of NMDA receptors by Src is blocked by neuregulin 1-ErbB4 signaling, a pathway that is genetically implicated in the positive symptoms of schizophrenia. Thus, either over-upregulation or under-upregulation of NMDA receptors by Src may lead to pathological conditions in the central nervous system. Therefore, normalizing Src upregulation of NMDA receptors may be a novel therapeutic approach for central nervous system disorders, without the deleterious consequences of directly blocking NMDA receptors.     

Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity

Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.