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Introduction Since the 1960s, countless studies have pointed to the participation of intrinsic inhibitory nerves in the relaxation of intestinal smooth muscle. These nerves make up the nonadrenergic, noncholinergic (NANC) [...], [gamma]-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adult central nervous system (CNS) synapses, but it excites immature CNS neurons as well as neurons in the myenteric plexus. The present work aimed to determine whether GABA-induced nonadrenergic, noncholinergic (NANC) neuronal-mediated relaxation of the rat duodenum is dependent on the activity of [Na.sup.+] [K.sup.+] [Cl.sup.-] cotransporters (NKCC) and requires calcium influx. In the presence of guanethidine (3 [micro]mol/L), atropine (3 [micro]mol/L), and indomethacin (1 [micro]mol/L), relaxations induced by GABA (100 [micro]mol/L), KCl (5-10 mmol/L) and electrical field stimulation (1-8 Hz, 2 ms, 60 V), but not those induced by bradykinin (10-100 nmol/L) were abolished by lidocaine (300 [micro]mol/L). However, only GABA-induced relaxations were reduced in a concentration-dependent manner by the NKCC1/2 inhibitors bumetanide (0.1-1 [micro]mol/L) and furosemide (1-10 [micro]mol/L). GABA-induced NANC neuronal relaxation was abolished by bicuculline (30 [micro]mol/L) and inhibited by N-nitroarginine methyl ester (L-NAME, 300 [micro]mol/L). The [omega]- conotoxin GVIA (1 pmol/L), which acts exclusively on neuronal CaV2 channels, but not on smooth muscle voltage-gated [Ca.sup.2+] [Ca.sub.V]1 channels, and nonselective blockers of these channels (verapamil 100 nmol/L and ruthenium red 10 [micro]mol/L), reduced GABA-induced relaxations. These results showed that the activation of [GABA.sub.A] receptors induces NANC nitrergic neuronal relaxations in the rat duodenum, which depend on NKCC activity and [CaV.sub.2] channel activation, suggesting that this phenomenon results from neuronal depolarization promoted by [Cl.sup.-] efflux through GABAa receptors, with subsequent [Ca.sup.2+] influx and nitric oxide release. Key words: GABA, NKCC cotransporters, NANC neurons, rat duodenum, nitric oxide and [Ca.sup.2+] Le GABA constitue le principal neurotransmetteur inhibiteur dans les synapses du SNC adulte, mais il joue un role excitateur dans les neurones du SNC immature, de meme que dans les neurones du plexus myenterique. Les presents travaux visaient a etablir si la relaxation du duodenum de rat mediee par le GABA dans les neurones non adrenergiques, non cholinergiques (NANC) est dependante de l'activite des cotransporteurs NKCC et si elle necessite un flux entrant de calcium. En presence de guanethidine (3 [micro]M), d'atropine (3 [micro]M) et d'indometacine (1 [micro]M), les relaxations obtenues avec le GABA (100 [micro]M), le KCl (5-10 mM) et la stimulation en champ electrique (SCE, 1-8 Hz, 2 ms, 60 V) etaient abolies avec la lidocaine (300 [micro]M), mais pas celles obtenues avec la bradykinine (10-100 [micro]M). Toutefois, uniquement les relaxations obtenues avec le GABA diminuaient de maniere proportionnelle a la concentration de bumetanide (0,1-1 [micro]M) et de furosemide (1-10 [micro]M), inhibiteurs des NKCC1/2. Les relaxations obtenues avec le GABA par les neurones NANC etaient abolies en presence de bicuculline (30 [micro]M) et inhibees en presence de l-NAME (300 [micro]M). L'[omega]-conotoxine-GVIA (1 [micro]M) (qui agit uniquement sur les canaux CaV2 neuronaux et pas sur les canaux [Ca.sup.2+] voltage-dependants CaV1 dans le muscle lisse) et des bloqueurs non selectifs de ces canaux (verapamil a 100 nM et rouge de ruthenium a 10 [micro]M) entrainaient une diminution des relaxations obtenues avec le GABA. Ces resultats montrent que dans le duodenum de rat, l'activation des recepteurs GABAA entraine des relaxations neuronales NANC nitrergiques, qui dependent de l'activite de NKCC et de l'activation des canaux CaV2, ce qui laisse entendre que ce phenomene proviendrait d'une depolarisation neuronale favorisee par le flux sortant d'ions [Cl.sup.-] par les recepteurs GABAA, avec ensuite un flux entrant d'ions [Ca.sup.2+] et la liberation d'oxyde nitrique. [Traduit par la Redaction] Mots-cles : GABA, cotransporteurs NKCC, neurones NANC, duodenum de rat, oxyde nitrique et ions [Ca.sup.2+].

Author(s): Ichia Chen [sup.1] , Shashank Pant [sup.2] [sup.3] [sup.4] , Qianyi Wu [sup.1] , Rosemary J. Cater [sup.1] [sup.7] , Meghna Sobti [sup.5] [sup.6] , Robert J. Vandenberg [sup.1] [...], Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity.sup.1. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism.sup.2-5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport.sup.6-8. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family. Glutamate transporters conduct chloride ions through an aqueous channel with hydrophobic gates that forms during the glutamate transport cycle.

Introduction Mounting evidence supports the paradigm of specific nucleoporins as noncanonical epigenomic regulators of differentiation and disease (Capelson and Hetzer 2009; Ibarra et al. 2016). Alternative to their role as [...], Nucleoporins are a specialized subset of nuclear proteins that comprise the nuclear pore complex and regulate nucleocytoplasmic transport. Recent demonstrations of roles for individual nucleoporins in multiple paradigms of differentiation via mechanisms independent of nuclear trafficking represent conceptual advances in understanding the contributions of nucleoporins to cellular development. Among these, a functional role for nucleoporins in reproductive fitness and gametogenesis has been identified, supported by robust models and clinical studies that leverage the power of next generation sequencing technology to identify reproductive-disease-associated mutations in specific nucleoporins. Proper nucleoporin function manifests in different ways during oogenesis and spermatogenesis. However, nonhuman models of gametogenesis may not recapitulate human mechanisms, which may confound translational interpretation and relevance. To circumvent these limitations, identification of reproductive pathologies in patients, combined with next generation sequencing approaches and advanced in silico tools, offers a powerful approach to investigate the potential function of nucleoporins in human reproduction. Ultimately, elucidating the role of nucleoporins in reproductive biology will provide opportunities for predictive, diagnostic, and therapeutic strategies to address reproductive disorders. Key words: nucleoporins, oogenesis, spermatogenesis, nuclear pore complex, reproduction. Les nucleoporines constituent un sous-ensemble specialise de proteines nucleaires, qui comprennent le complexe des pores nucleaires et participent a la regulation du transport nucleo-cytoplasmique. De recentes demonstrations des roles de nucleoporines donnees dans de multiples paradigmes de differentiation par l'intermediaire de modes d'action independants des echanges nucleaires correspondent a des avancees conceptuelles dans la comprehension des contributions de nucleoporines au developpement de la cellule. Parmi celles-la, on a etabli que les nucleoporines jouent un role fonctionnel dans la sante de la reproduction et la gametogenese, observations appuyees par des modeles et des etudes cliniques robustes qui tirent profit de la puissance de nouvelles generations de technologies de sequencage en vue de deceler des mutations de nucleoporines specifiques associees a des maladies de la reproduction. Le bon fonctionnement des nucleoporines se manifeste de differentes manieres au cours de l'ovogenese et de la spermatogenese. Cependant, les modeles de gametogenese non humains pourraient ne pas reprendre les modes d'action chez l'humain, ce qui pourrait contrecarrer l'interpretation et la pertinence translationnelles. En vue de contourner ces limites, relever des pathologies reproductives chez les patients, en association avec de nouvelles generations d'approches de sequencage et des outils informatiques evolues, offre une approche puissante pour l'etude d'eventuelles fonctions des nucleoporines dans la reproduction humaine. Ultimement, elucider le role des nucleoporines dans la biologie de la reproduction fournira l'occasion d'etablir des strategies predictives, diagnostiques et therapeutiques en vue de s'attaquer aux troubles de la reproduction. [Traduit par la Redaction] Mots-cles : nucleoporines, ovogenese, spermatogenese, complexe des pores nucleaires, reproduction.

Author(s): Sho Morioka [sup.1] [sup.2] , Justin S. A. Perry [sup.1] [sup.2] , Michael H. Raymond [sup.1] [sup.3] , Christopher B. Medina [sup.1] [sup.2] , Yunlu Zhu [sup.4] , Liyang [...], Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis.sup.1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood.sup.1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis.sup.2--that is, 'smell' ('find-me' signals or sensing factors released by apoptotic cells), 'taste' (phagocyte-apoptotic cell contact) and 'ingestion' (corpse internalization)--activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis.sup.3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells.Distinct transcriptional programs are activated during different stages of apoptotic cell engulfment, including a unique program of genes coding for solute carrier proteins and enzymes in the glycolytic pathway.

Author(s): Ambre M. Bertholet [sup.1] , Edward T. Chouchani [sup.2] , Lawrence Kazak [sup.2] , Alessia Angelin [sup.3] , Andriy Fedorenko [sup.1] , Jonathan Z. Long [sup.2] , Sara Vidoni [...], The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H.sup.+ transport. The AAC-mediated H.sup.+ current requires free fatty acids and resembles the H.sup.+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H.sup.+ leak, but does not completely inhibit it. This suggests that the H.sup.+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H.sup.+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria. The mitochondrial ADP/ATP carrier mediates the proton leak in mitochondria from all tissues that lack UCP1, thereby linking coupled (ATP production) and uncoupled (thermogenesis) energy conversion.

Cellular uptake of glucose is a fundamental process for metabolism, growth and homeostasis (1). The SLC2 family glucose transporters (GLUTs) catalyse facilitative diffusion of glucose and other monosaccharides across biomembranes [...], The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 A in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.