Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood-brain barrier (BBB), are enriched in two proteins, aquaporin-4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1(G93A) rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole-cell patch-clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir-specific Cs(+)- and Ba(2+)-sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death.

The acid-sensitive ion channel 1 (ASIC1␣ or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that has the broadest distribution in the nervous system. Hallmarks of ASIC1␣ are gating by external protons and rapid desensitization. In sensory neurons ASIC1 may constitute a nociceptor for pain induced by local acidification, whereas in central neurons it may modulate synaptic activity. To gain insight into the functional roles of ASIC1, we cloned and examined the properties of the evolutionarily distant species toadfish (Opsanus tau), ϳ420-million year divergent from mammals. Analysis of the protein sequence from fish ASIC1 revealed 76% amino acid identity with the rat orthologue. The regions of highest conservation are the second transmembrane domain and the ectodomain, whereas the amino and carboxyl termini and first transmembrane domain are poorly conserved. At the functional level, fish ASIC1 is gated by external protons with a half-maximal activation at pH o 5.6 and a halfmaximal inactivation at pH o 7.30. The fish differs from the rat channel on having a 25-fold faster rate of desensitization. Functional studies of chimeras made from rat and fish ASIC1 indicate that the extracellular domain specifically, a cluster of three residues, confers the faster desensitization rate to the fish ASIC1.The acid-sensitive ion channels (ASICs) 1 constitute a subfamily of the large epithelial sodium channel (ENaC)/DEG family of ion channels (17, 27). The ASIC1␣ protein (or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that is expressed in most neurons of the central and peripheral nervous systems (1, 2). The mammalian ASIC1, ASIC2, and ASIC3 are all activated by protons but the degree and rate of desensitization markedly differ in each type of channel. For instance, rat ASIC1 and ASIC3 exhibit rapid and complete desensitization at pH o 5.0, whereas ASIC2 has a slow and incomplete desensitization, leaving a substantial component of persistent current in the continual presence of protons (29). Indeed, most of the functional differences in the currents generated by the ASICs can be attributed to differences in desensitization rate, suggesting that this property may be important in conferring specificity to the response of the various ASICs in different regions of the nervous system. Numerous functions have been proposed for ASIC1 including a role in sensory transduction, specifically in nociception (pain induced by ischemia and inflammation) (7,9,15,20,21,24), and as modulators of synaptic transmission and long term plasticity (2, 28). For many of these functions, in particular nociception, proton sensitivity plays a fundamental role in the physiology of these channels.In order to gain more insight in the structure-function of these channels, we cloned and examined the functional properties of ASIC1 from an evolutionarily distant species, the fish Opsanus tau. Comparison of the properties of the fish and mammalian ASIC1 revealed significant differences, pri...

The serum‐ and glucocorticoid‐induced kinase‐1 (sgk1) increases the activity of a number of epithelial ion channels and transporters. The present study examines the distribution and subcellular localization of sgk1 protein in the rat kidney and the regulation of levels of expression induced by steroids. The results indicate that the kidney expresses predominantly the sgk1 isoform with a distribution restricted to the thick ascending limb of Henle, distal convoluted, connecting and cortical collecting tubules. Within cells, sgk1 strongly associates with the microsomal fraction of homogenates and it colocalizes with the Na+,K+‐ATPase to the basolateral membrane. Analysis of the levels of expression of sgk1 by Western blotting and immunohistochemistry indicates constitutive high expression under basal conditions. Approximately half of the basal level is maintained by glucocorticoids whereas physiological fluctuations of aldosterone produce minor changes in sgk1 abundance in adrenal‐intact animals. These results do not support the notion that physiological changes of aldosterone concentration turn the expression of sgk1 ‘on and off’ in the mammalian kidney. Additionally, localization of sgk1 to the basolateral membrane indicates that the effects mediated by sgk1 do not require a direct interaction with the ion channels and transporters whose activity is modulated, since most of these proteins are located in the apical membrane of renal epithelial cells.

Two newly synthesized 4-hydroxycoumarin bidentate ligands (L1 and L2) and their palladium(II) complexes (C1 and C2) were screened for their biological activities, in vitro and in vivo. Structures of new compounds were established based on elemental analysis, 1H NMR, 13C NMR, and IR spectroscopic techniques. The obtained compounds were tested for their antioxidative and cytotoxic activities and results pointed to selective antiradical activity of palladium(II) complexes towards •OH and -•OOH radicals and anti-ABTS (2,2 ′ -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical) activity comparable to that of ascorbate. Results indicated the effect of C1 and C2 on the enzymatic activity of the antioxidative defense system. In vitro cytotoxicity assay performed on different carcinoma cell lines (HCT166, A375, and MIA PaCa-2), and one healthy fibroblast cell line (MRC-5) showed a cytotoxic effect of both C1 and C2, expressed as a decrease in carcinoma cells’ viability, mostly by induction of apoptosis. In vivo toxicity tests performed on zebrafish embryos indicated different effects of C1 and C2, ranging from adverse developmental effect to no toxicity, depending on tested concentration. According to docking studies, both complexes (C1 and C2) showed better inhibitory activity in comparison to other palladium(II) complexes.

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The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na(+)/K(+) pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15 min. Biochemical data showed that Na(+)/K(+)-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, (31)P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na(+)/H(+) exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na(+)/K(+) pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na(+)/K(+) pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field.

In the present report, by using a patch clamp technique, we provide, to our knowledge, the first detailed description of an anionic channel from filamentous fungi. The characterized channel, an outwardly rectifying anionic channel ( The molecular identities and electrophysiological and structural properties of anion-selective channels are well characterized in animal cells and, to a lesser extent, in plant cells (7,22,29,31). In fungi, on the other hand, data on anion channels are very scarce. Up to now, only two electrophysiological studies have demonstrated the existence of anion channels in fungal membranes. Both of these studies, however, were mainly methodological, dealing with the development of techniques for successful patch clamping (i.e., gigaseal formation) of fungal plasma membranes. The channel activity recordings primarily illustrated the validity of the techniques. The available data show that anion channels from cell membranes of Neurospora crassa (37) and Aspergillus niger (33) are selective for Cl Ϫ over K ϩ , with single-channel conductances of 17 pS and 43 pS, respectively, and that the one from N. crassa is a weak outward rectifier while the one from A. niger is a strong rectifier. No data on selectivity among anions, kinetic parameters, or blockers of these channels are available.The lack of knowledge about fungal anion channels is mainly the result of technical difficulties in the formation of a highresistance seal, a"gigaohm seal" (Ͼ1 G⍀) between hyphal plasma membranes and patch clamp pipettes. The "gigaohm seal" is necessary for detailed analysis of ion channel properties (i.e., selectivity and gating). These difficulties are a consequence of the specific structure of the fungal cell wall. The only readily patchable fungal cell membrane is that of a yeast (Saccharomyces cerevisiae), the experimental model used in virtually all electrophysiological characterizations of fungal cationic channels (3). None of these experiments showed anion channel activity, indicating the absence of anionic channels. However, unicellular organization and cell walls composed of mannans and glucans clearly separate yeasts from the majority of other fungi denoted filamentous fungi. Filamentous fungi are characterized by multicelullar mycelia, polarized hyphal growth, and chitin cell walls. Electrophysiological study of the only cationic channel from a filamentous fungus (N. crassa) that has been well characterized was conducted by cloning and expression of the channel in yeast plasma membranes (32).Removal of the cell wall with cell wall-degrading enzymes, which gave good results in plants, failed to solve the problem in fungi, since patch clamping of the obtained protoplasts resulted only in subgigaohm seals that were not suitable for detailed examination of ionic channels (14,25). The only exception is the report of a gigaohm seal on enzyme-derived germling protoplast from Uromyces (40). In another report, the problem of cell wall removal was resolved by using a wall-less slime mutant of N. crassa (24). Desp...

The effect of extremely low frequency magnetic fields (50 Hz, 0.5 mT) -ELF-MF, on phosphate metabolism has been studied in the isolated ganglions of the garden snail Helix pomatia, after 7 and 16 days of snail exposure to ELF-MF. The influence of ELF-MF on the level of phosphate compounds and intracellular pH was monitored by 31 P NMR spectroscopy. Furthermore, the activity of enzymes involved in phosphate turnover, total ATPases, Na + /K + -ATPase and acid phosphatase has been measured. The exposure of snails to the ELF-MF for the period of 7 days shifted intracellular pH toward more alkaline conditions, and increased the activity of investigated enzymes. Prolonged exposure to the ELF-MF for the period of 16 days caused a decrease of PCr and ATP levels and decreased enzyme activity, compared to the 7-day treatment group. Our results can be explained in terms of: 1. increase in phosphate turnover by exposure to the ELF-MF for the period of 7 days, and 2. adaptation of phosphate metabolism in the nervous system of snails to prolonged ELF-MF exposure.