Although radial oblique dendrites are a major synaptic input site in CA1 pyramidal neurons, little is known about their integrative properties. We have used multisite two-photon glutamate uncaging to deliver different spatiotemporal input patterns to single branches while simultaneously recording the uncaging-evoked excitatory postsynaptic potentials and local Ca2+ signals. Asynchronous input patterns sum linearly in spite of the spatial clustering and produce Ca2+ signals that are mediated by NMDA receptors (NMDARs). Appropriately timed and sized input patterns ( approximately 20 inputs within approximately 6 ms) produce a supralinear summation due to the initiation of a dendritic spike. The Ca2+ signals associated with synchronous input were larger and mediated by influx through both NMDARs and voltage-gated Ca2+ channels (VGCCs). The oblique spike is a fast Na+ spike whose duration is shaped by the coincident activation of NMDAR, VGCCs, and transient K+ currents. Our results suggest that individual branches can function as single integrative compartments.

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-gamma (PPARgamma, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct-specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg (flox/flox) mice. Deletion of collecting duct Pparg decreased renal Na(+) avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na(+) absorption and Scnn1g mRNA (encoding the epithelial Na(+) channel ENaCgamma) expression through a PPARgamma-dependent pathway. These studies identify Scnn1g as a PPARgamma target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

1. A modification of the methods of Miller and of Schimassek for the perfusion of the isolated rat liver, suitable for the study of gluconeogenesis, is described. 2. The main modifications concern the operative technique (reducing the period of anoxia during the operation to 3min.) and the use of aged (non-glycolysing) red cells in the semi-synthetic perfusion medium. 3. The performance of the perfused liver was tested by measuring the rate of gluconeogenesis, of urea synthesis and the stability of adenine nucleotides. Higher rates of gluconeogenesis (1mumole/min./g.) from excess of lactate and of urea synthesis from excess of ammonia (4mumoles/min./g. in the presence of ornithine) were observed than are likely to occur in vivo where rates are limited by the rate of supply of precursor. The concentrations of the three adenine nucleotides in the liver tissue were maintained within 15% over a perfusion period of 135min. 4. Ca(2+), Na(+), K(+), Mg(2+) and phosphate were found to be required at physiological concentrations for optimum gluconeogenesis but bicarbonate and carbon dioxide could be largely replaced by phosphate buffer without affecting the rate of gluconeogenesis. 5. Maximal gluconeogenesis did not decrease maximal urea synthesis in the presence of ornithine and ammonia and vice versa. This indicates that the energy requirements were not limiting the rates of gluconeogenesis or of urea synthesis. 6. Addition of lactate, and especially ammonium salts, increased the uptake of oxygen more than expected on the basis of the ATP requirements of the gluconeogenesis and urea synthesis.

We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g(Na)). At the level of a single channel, Pronase probably acts in an all-or-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces g(Na), possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.

The intracellular pH in animal cells in generally maintained at a higher level than would be expected if H+ were passively distributed across the plasma membrane. In a wide variety of cells including sea urchin eggs, skeletal muscle, renal and intestinal epithelial cells, and neuroblastoma cells, plasma membrane Na+-H+ exchangers mediate the uphill extrusion of H+ coupled to, and thus energized by, the downhill entry of Na+. Plasma membrane vesicles isolated from the luminal (microvillus, brush border) surface of renal proximal tubular cells possess a Na+-H+ exchanger that seems to be representative of the Na+-H+ exchangers found in other tissues. For example, the renal microvillus membrane Na+-H+ exchanger, like other Na+-H+ exchangers, mediates electroneutral cation exchange, is sensitive to inhibition by the diuretic drug amiloride, and has affinity for Li+ in addition to Na+ and H+ (refs 5, 9). Here we have examined the effect of internal H+ on the activity of the Na+-H+ exchanger in renal microvillus membrane vesicles. Our results suggest that internal H+, independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external Na+, has an important modifier role as an allosteric activator of the Na+-H+ exchanger. Allosteric behaviour with respect to internal H+ is a property that would enhance the ability of plasma membrane Na+-H+ exchangers to extrude intracellular acid loads and thereby contribute to the regulation of intracellular pH.

This review deals with physiological and biological mechanisms of neuropathic pain, that is, pain induced by injury or disease of the nervous system. Animal models of neuropathic pain mostly use injury to a peripheral nerve, therefore, our focus is on results from nerve injury models. To make sure that the nerve injury models are related to pain, the behavior was assessed of animals following nerve injury, i.e. partial/total nerve transection/ligation or chronic nerve constriction. The following behaviors observed in such animals are considered to indicate pain: (a) autotomy, i.e. self-attack, assessed by counting the number of wounds implied, (b) hyperalgesia, i.e. strong withdrawal responses to a moderate heat stimulus, (c) allodynia, i.e. withdrawal in response to non-noxious tactile or cold stimuli. These behavioral parameters have been exploited to study the pharmacology and modulation of neuropathic pain. Nerve fibers develop abnormal ectopic excitability at or near the site of nerve injury. The mechanisms include unusual distributions of Na(+) channels, as well as abnormal responses to endogenous pain producing substances and cytokines such as tumor necrosis factor alpha (TNF-alpha). Persistent abnormal excitability of sensory nerve endings in a neuroma is considered a mechanism of stump pain after amputation. Any local nerve injury tends to spread to distant parts of the peripheral and central nervous system. This includes erratic mechano-sensitivity along the injured nerve including the cell bodies in the dorsal root ganglion (DRG) as well as ongoing activity in the dorsal horn. The spread of pathophysiology includes upregulation of nitric oxide synthase (NOS) in axotomized neurons, deafferentation hypersensitivity of spinal neurons following afferent cell death, long-term potentiation (LTP) of spinal synaptic transmission and attenuation of central pain inhibitory mechanisms. In particular, the efficacy of opioids at the spinal level is much decreased following nerve injury. Repeated or prolonged noxious stimulation and the persistent abnormal input following nerve injury activate a number of intracellular second messenger systems, implying phosphorylation by protein kinases, particularly protein kinase C (PKC). Intracellular signal cascades result in immediate early gene (IEG) induction which is considered as the overture of a widespread change in protein synthesis, a general basis for nervous system plasticity. Although these processes of increasing nervous system excitability may be considered as a strategy to compensate functional deficits following nerve injury, its by-product is widespread nervous system sensitization resulting in pain and hyperalgesia. An important sequela of nerve injury and other nervous system diseases such as virus attack is apoptosis of neurons in the peripheral and central nervous system. Apoptosis seems to induce neuronal sensitization and loss of inhibitory systems, and these irreversible processes might be in common to nervous system damage by brain trauma or ischemia as well as neuropathic pain. The cellular pathobiology including apoptosis suggests future strategies against neuropathic pain that emphasize preventive aspects.

Although epithelia, which often are in intimate contact with lymphoid cells, may bear receptors for various cytokines, it is unclear whether cytokines directly effect epithelial function. We examine the effects of the cytokine interferon (IFN) on barrier function of cultured monolayers of the T84 human intestinal epithelial cell line. Gamma IFN, in concentrations and exposures required to show its other biological effects, directly affects such monolayers. Monolayer resistance is substantially diminished by gamma IFN. Such effects were not due to cytotoxicity as judged morphologically and by LDH assays. Solute fluxes and dual Na+-mannitol flux analysis indicate that the resistance decrease is due to an effect of gamma IFN on tight junction permeability. The effects of gamma IFN on monolayer barrier function were not duplicated by the cytokines interleukin 1, interleukin 2, or tumor necrosis factor. We speculate that such products of activation of lymphoid cells might influence barrier function of intestinal, and perhaps other epithelia in disease states.

The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na(+) channels (ENaC), the Na(+)-K(+)-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na(+) feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl(-) secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl(-) channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl(-) secretion and enhanced Na(+) absorption in the colon of cystic fibrosis (CF) patients. Ca(2+)- and cAMP-activated basolateral K(+) channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.

Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel Na(V)1.1 causes Dravet's syndrome, a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit and autism-spectrum behaviours. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviours in Dravet's syndrome are poorly understood. Here we report that mice with Scn1a haploinsufficiency exhibit hyperactivity, stereotyped behaviours, social interaction deficits and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odours and social odours are aversive to Scn1a(+/-) mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of Na(V)1.1 channels in forebrain interneurons is sufficient to cause these behavioural and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABA(A) receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model of Dravet's syndrome, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for Na(V)1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviours in Dravet's syndrome.

The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.

Influenza A virus causes seasonal epidemics, sporadic pandemics and is a significant global health burden. Influenza virus is an enveloped virus that contains a segmented negative strand RNA genome. Assembly and budding of progeny influenza virions is a complex, multi-step process that occurs in lipid raft domains on the apical membrane of infected cells. The viral proteins hemagglutinin (HA) and neuraminidase (NA) are targeted to lipid rafts, causing the coalescence and enlargement of the raft domains. This clustering of HA and NA may cause a deformation of the membrane and the initiation of the virus budding event. M1 is then thought to bind to the cytoplasmic tails of HA and NA where it can then polymerize and form the interior structure of the emerging virion. M1, bound to the cytoplasmic tails of HA and NA, additionally serves as a docking site for the recruitment of the viral RNPs and may mediate the recruitment of M2 to the site of virus budding. M2 initially stabilizes the site of budding, possibly enabling the polymerization of the matrix protein and the formation of filamentous virions. Subsequently, M2 is able to alter membrane curvature at the neck of the budding virus, causing membrane scission and the release of the progeny virion. This review investigates the latest research on influenza virus budding in an attempt to provide a step-by-step analysis of the assembly and budding processes for influenza viruses.

We examine the role of synaptic activity in the development of identified Drosophila embryonic motorneurons. Synaptic activity was blocked by both pan-neuronal expression of tetanus toxin light chain (TeTxLC) and by reduction of acetylcholine (ACh) using a temperature-sensitive allele of choline acetyltransferase (Cha(ts2)). In the absence of synaptic activity, aCC and RP2 motorneurons develop with an apparently normal morphology and retain their capacity to form synapses. However, blockade of synaptic transmission results in significant changes in the electrical phenotype of these neurons. Specifically, increases are seen in both voltage-gated inward Na(+) and voltage-gated outward K(+) currents. Voltage-gated Ca(2+) currents do not change. The changes in conductances appear to promote neuron excitability. In the absence of synaptic activity, the number of action potentials fired by a depolarizing ramp (-60 to +60 mV) is increased and, in addition, the amplitude of the initial action potential fired is also significantly larger. Silencing synaptic input to just aCC, without affecting inputs to other neurons, demonstrates that the capability to respond to changing levels of synaptic excitation is intrinsic to these neurons. The alteration to electrical properties are not permanent, being reversed by restoration of normal synaptic function. Whereas our data suggest that synaptic activity makes little or no contribution to the initial formation of embryonic neural circuits, the electrical development of neurons that constitute these circuits seems to depend on a process that requires synaptic activity.

Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene SCN9A, which encodes hNa(v)1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Na(v)1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.

Crop performance is severely affected by high salt concentrations in soils. To engineer more salt-tolerant plants it is crucial to unravel the key components of the plant salt-tolerance network. Here we review our understanding of the core salt-tolerance mechanisms in plants. Recent studies have shown that stress sensing and signaling components can play important roles in regulating the plant salinity stress response. We also review key Na+ transport and detoxification pathways and the impact of epigenetic chromatin modifications on salinity tolerance. In addition, we discuss the progress that has been made towards engineering salt tolerance in crops, including marker-assisted selection and gene stacking techniques. We also identify key open questions that remain to be addressed in the future.

1. The acetylcholine-sensitive ionic channels at the neuromuscular junction were studied in voltage-clamped single muscle fibres from a monolayer preparation of the cutaneous pectoris muscle from Rana pipiens. The experimental observations were of three types: (a) reversal potential as a function of external Na and Ca concentrations, (b) the single channel conductance (gamma) from noise analysis as a function of these same concentrations, and (c) gamma as a function of membrane potential. 2. The reversal potential in normal Na Ringer was -3.8 +/- 0.5 mV (+/- S.E. of mean, n = 22) and decreased approximately linearly as the logarithm of the outside Na activity as this activity decreased to 10% of normal. 3. The single channel conductance in normal Na Ringer was 27.5 +/- 0.7 pS (n = 28) and reached a limiting value close to 10 pS as Na was replaced with sucrose. 4. Increasing [Ca]o from 2 to 10 mM made the reversal potential more positive and decreased the single channel conductance. Mg caused similar effects. 5. Various theories that have been used to describe the mechanism of ion permeation through e.p.c. channels were tested. Constant field theory (eqns. (3), (4) and (5)), a modified Takeuchi approach (eqn. (6)), and a single barrier theory (eqns. (8), (9) and (10)) could not account for all of the experimental observations. 6. In particular, constant field theory, with no surface charge density, could account for the following: (a) the reversal potential measurements for solutions containing 2 mM-Ca (with PK/PNa = 1.2 and PCa/PNa = 1.02), (b) the single channel conductance values for solutions containing 2 mM-Ca and Na concentrations down to 20% of normal, (c) that gamma has little voltage dependence. 7. However, constant field theory, with no assumed surface charge density, could not account for the following: (a) the reversal potential observed for Ringer containing 80 mM-Ca, (b) the gamma values observed for very low Na concentrations, (c) the observation that increasing Ca from 2 to 10 mM in a solution containing 75% normal Na results in a decrease in gamma. 8. From the failure of the Takeuchi approach (eqn. (6)), it is argued that ion interactions must occur at e.p.c. channels because ion flux independence is the only asumption in the derivation of eqn. (6) without experimental verification. 9. The ion interactions at e.p.c. channels probably include both surface charge effects and competition for a binding site.

Antidepressants targeting Na(+)/Cl(-)-coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses.

Although there has been impressive progress in the elucidation of the genetic and molecular basis of cystic fibrosis (CF), the pathogenesis of CF lung disease remains obscure. The elucidation of the pathogenesis of CF lung disease requires both a full description of normal innate airway defence and how absent function of the cystic fibrosis transmembrane regulator protein (CFTR) adversely perturbs this activity. Recent data have linked the abnormal ion transport properties of CF airway epithelia to depleted airway surface liquid (ASL) volume, reflecting the combined defects of accelerated Na+ transport and the failure to secrete Cl-. Depletion of a specific compartment of the ASL, i.e. the periciliary liquid (PCL), appears to abrogate both cilia-dependent and cough clearance. Subsequent to PCL depletion, mucus adheres to airway surfaces and persistent mucin secretion generates the formation of "thickened" mucus plaques and plugs, which become the nidus for bacterial infection. The paucity of liquid in these plaques/plugs, and the hypoxia in this environment, appear to promote biofilm bacterial infection. Therapeutic agents that restore airway surface liquid volume, i.e. blockers of Na+ transport, initiators of Cl- transport and osmolytes, are reviewed, as are strategies that may be required to use volume-restoring agents safely in patients with cystic fibrosis.

The SLC7 family is divided into two subgroups, the cationic amino acid transporters (the CAT family, SLC7A1-4) and the glycoprotein-associated amino acid transporters (the gpaAT family, SLC7A5-11), also called light chains or catalytic chains of the hetero(di)meric amino acid transporters (HAT). The associated glycoproteins (heavy chains) 4F2hc (CD98) or rBAT (D2, NBAT) form the SLC3 family. Members of the CAT family transport essentially cationic amino acids by facilitated diffusion with differential trans-stimulation by intracellular substrates. In some cells, they may regulate the rate of NO synthesis by controlling the uptake of l-arginine as the substrate for nitric oxide synthase (NOS). The heterodimeric amino acid transporters are, in contrast, quite diverse in terms of substrate selectivity and function (mostly) as obligatory exchangers. Their selectivity ranges from large neutral amino acids (system L) to small neutral amino acids (ala, ser, cys-preferring, system asc), negatively charged amino acid (system x(c)(-)) and cationic amino acids plus neutral amino acids (system y(+)L and b(0,+)-like). Cotransport of Na(+) is observed only for the y(+)L transporters when they carry neutral amino acids. Mutations in b(0,+)-like and y(+)L transporters lead to the hereditary diseases cystinuria and lysinuric protein intolerance (LPI), respectively.

Nociception is essential for survival whereas pathological pain is maladaptive and often unresponsive to pharmacotherapy. Voltage-gated sodium channels, Na(v)1.1-Na(v)1.9, are essential for generation and conduction of electrical impulses in excitable cells. Human and animal studies have identified several channels as pivotal for signal transmission along the pain axis, including Na(v)1.3, Na(v)1.7, Na(v)1.8, and Na(v)1.9, with the latter three preferentially expressed in peripheral sensory neurons and Na(v)1.3 being upregulated along pain-signaling pathways after nervous system injuries. Na(v)1.7 is of special interest because it has been linked to a spectrum of inherited human pain disorders. Here we review the contribution of these sodium channel isoforms to pain.

We present the intramolecular G-quadruplex structure of human telomeric DNA in physiologically relevant K(+) solution. This G-quadruplex, whose (3 + 1) topology differs from folds reported previously in Na(+) solution and in a K(+)-containing crystal, involves the following: one anti.syn.syn.syn and two syn.anti.anti.anti G-tetrads; one double-chain reversal and two edgewise loops; three G-tracts oriented in one direction and the fourth in the opposite direction. The topological characteristics of this (3 + 1) G-quadruplex scaffold should provide a unique platform for structure-based anticancer drug design targeted to human telomeric DNA.

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Na(v)1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Na(v)1.6-deficient mouse mutant strain med, whose nodes were negative for Na(v)1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Na(v)1.6 is the predominant channel type in this structure. In the central nervous system, Na(v)1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Na(v)1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

Five experiments examined the effects of destruction of the dorsal noradrenergic bundle (DNAB), arising in the locus coeruleus, both on brightness and spatial visual discrimination, and selective attention. An analogue o Leonard's 5-choice serial reaction task for human subjects was used. Hungry rats were trained to detect brief (0.5 sec) flashes of light presented randomly in one of 5 locations with a fixed intertrial interval of 5 sec, paced by the rat. Correct responses were rewarded with food and incorrect responses punished by time-out (darkness + delay). Following training to high levels of accuracy (80%, with less than 20% errors of omission), rats received either 6-OHDA (4 micrograms/2 microliters) injected bilaterally into the trajectory of the dorsal bundle, or injection of vehicle (0.1% ascorbic acid in 0.9% saline). The 6-OHDA lesion was sufficient to reduce cortical NA by 84%. Performance on both the spatial discrimination and brightness (produced by graded reductions in the brightness of the stimuli) discrimination was unaffected by DNAB lesions. However, the DNAB lesion produced significant decreases in accuracy and increases in omissions when the stimuli were presented at faster, unpredictable rates. In addition, although intense white noise failed to produce differential impairments when presented simultaneously with the visual discriminanda, the DNAB lesion significantly impaired accuracy when the noise was presented immediately prior to, but not overlapping, the onset of the visual stimuli. The implications of this pattern of deficits in performance found following DNAB lesions is discussed in terms of disruptive effects of cortical NA depletion upon mechanisms of selective attention and arousal.