Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.     

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.     

Positive and Negative Feedbacks and Free-Scale Pattern Distribution in Rural-Population Dynamics

Depopulation of rural areas is a widespread phenomenon that has occurred in most industrialized countries, and has contributed significantly to a reduction in the productivity of agro-ecological resources. In this study, we identified the main trends in the dynamics of rural populations in the Central Pyrenees in the 20^th C and early 21^st C, and used density independent and density dependent models and identified the main factors that have influenced the dynamics. In addition, we investigated the change in the power law distribution of population size in those periods. Populations exhibited density-dependent positive feedback between 1960 and 2010, and a long-term positive correlation between agricultural activity and population size, which has resulted in a free-scale population distribution that has been disrupted by the collapse of the traditional agricultural society and by emigration to the industrialized cities. We concluded that complex socio-ecological systems that have strong feedback mechanisms can contribute to disruptive population collapses, which can be identified by changes in the pattern of population distribution.     

Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys⁵⁴ in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys⁵⁴ α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit.     

Synthesis of new thiophene and benzo[b]thiophene hydrazide derivatives as human NPY Y(5) antagonists

Neuropeptide Y is one of the most potent appetite stimulating hormones known. Novel thiophene and benzo[b]thiophene hydrazide derivatives were synthetized and evaluated biologically as NPY Y(1) and Y(5) receptor subtype antagonists. They were found to have nanomolar binding affinities for human NPY Y(5) receptor, obtaining the lead compound, trans-N-4-[N'-(thiophene-2-carbonyl)hydrazinocarbonyl]cyclohexylmethyl-4-bromobenzenesulfonamide, which binds with a 7.70 nM IC(50) to the hY(5) receptor.     

Early detection of graft incompatibility in apricot (Prunus armeniaca) using in vitro techniques

Graft compatibility has been studied in vitro using callus tissues of apricot ( Prunus armeniaca) and different Prunus rootstocks to form scion/rootstock combinations with different degrees of graft compatibility. In these species, incompatibility is manifested by a breakdown of the trees at the union area that can occur some years after grafting. Here, the possibility of obtaining an early detection method to determine graft incompatibility is explored by callus fusion in vitro. The adhesion of the two callus partners, the development of the cells at the contact surface (cell arrangement, intensity of cell-wall staining), and the presence of lipid and phenolic compounds have been studied during the first 3 weeks after grafting in both compatible and incompatible combinations. Differences were observed at the second and the third week of callus co-culture in most of the characters determined, although these differences were present as early as the first week in the case of phenolic compounds. The behaviour of the grafts grown in vitro was correlated to that of the same combinations in the field, suggesting that callus fusion in vitro could be a possible and reliable method for an early detection of graft incompatibility in different Prunus combinations.     

Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation

Background

Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage.

Methods/Principal Findings

To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage.

Conclusion

The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.