G protein {beta}{gamma} gating confers volatile anesthetic inhibition to Kir3 channels

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the βγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gβγ available to the channels, we asked whether reducing available Gβγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gβγ using the C-terminal domain of β-adrenergic receptor kinase (cβARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gβγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gβγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gβγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.     

A New Kv1.2 Channelopathy Underlying Cerebellar Ataxia

A forward genetic screen of mice treated with the mutagen ENU identified a mutant mouse with chronic motor incoordination. This mutant, named Pingu (Pgu), carries a missense mutation, an I402T substitution in the S6 segment of the voltage-gated potassium channel Kcna2. The gene Kcna2 encodes the voltage-gated potassium channel α-subunit Kv1.2, which is abundantly expressed in the large axon terminals of basket cells that make powerful axo-somatic synapses onto Purkinje cells. Patch clamp recordings from cerebellar slices revealed an increased frequency and amplitude of spontaneous GABAergic inhibitory postsynaptic currents and reduced action potential firing frequency in Purkinje cells, suggesting that an increase in GABA release from basket cells is involved in the motor incoordination in Pgu mice. In line with immunochemical analyses showing a significant reduction in the expression of Kv1 channels in the basket cell terminals of Pgu mice, expression of homomeric and heteromeric channels containing the Kv1.2(I402T) α-subunit in cultured CHO cells revealed subtle changes in biophysical properties but a dramatic decrease in the amount of functional Kv1 channels. Pharmacological treatment with acetazolamide or transgenic complementation with wild-type Kcna2 cDNA partially rescued the motor incoordination in Pgu mice. These results suggest that independent of known mutations in Kcna1 encoding Kv1.1, Kcna2 mutations may be important molecular correlates underlying human cerebellar ataxic disease.     

Depolarization Increases Phosphatidylinositol (PI) 4,5-Bisphosphate Level and KCNQ Currents through PI 4-Kinase Mechanisms

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P², PIP²) play an important role in cell signaling. The presence of PIP² is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP² levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP²-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP² modulation. Membrane depolarization induces an elevation in membrane PIP², and subsequently increases functions of PIP²-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP² occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP² in cell function, the effect of membrane depolarization in PIP² metabolism is destined to have important physiological implications.     

��5��1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

Large conductance, calcium-activated K⁺ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous α5β1 integrin ligand, enhances BK channel current through both Ca²⁺- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel α-subunits (mSlo) are acutely potentiated following α5β1 integrin activation. The effect occurs in a Ca²⁺-dependent manner, 1–3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca²⁺ concentrations ≥1 μm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca²⁺ sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by α5β1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel α-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca²⁺ sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.     

A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode disks

Internode disks of tomato (Lycopersicon esculentum cv. Moneymaker) were shaken in glutamine and sucrose solutions. At low external pH (<±5.5), the uptake of these substances was accompanied with K⁺ efflux, at high pH (>±5.5) with K⁺ influx. Low concentrations of external K⁺ (2 mmol l^-1) stimulated the uptake of glutamine, which was strongly inhibited by the supply of high K⁺ concentrations (20 mmol l^-1). The effect of K⁺ was particularly pronounced at high pH-values. Addition of CCCP in light reduced the uptake of glutamine to the same level as in the dark, and stopped the K⁺ fluxes which coincided with the uptake. A model is presented wherein the movements of K⁺ across the membrane are related to co-transport, depending on the membrane potential and the Nernst potential of K⁺.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone     

Mechanically enforced bond dissociation reports synergistic influence of Mn2+ and Mg2+ on the interaction between integrin α7β1 and invasin

Integrins require the divalent ions magnesium and manganese for ligand recognition. Here we mechanically enforced bond dissociation to explore the influence of these ions on the mechanical strength of the specific bond between α(7) β(1) integrin and its pathologically relevant ligand invasin. Upon addition of these cations to the measurement buffer, we observe a pronounced increase in the force necessary to separate integrin and invasin coated beads. Both ions were found to work synergistically. With free invasin in the measurement buffer we furthermore observe that competitive blocking of binding sites overrides the increase in binding strength of individual beads. We show that this is due to a very strong dependence of bond affinity on divalent ions. Our study illustrates the importance of divalent ions for the regulation of force transmission by integrin ligand bonds on the molecular level.     

Influence of EDTA complexing agent on biopreparation of linen fabric

In this work the ‘EDTA–enzyme–substrate’ interaction was investigated in linen biopreparation. The effect of EDTA on the degradation of non-cellulosic constituents during bioscouring with pectinase enzyme was investigated in detail. Greige linen fabric was treated with pectinase, pectinase supplemented with ethylenediamine-tetra-acetic acid (EDTA), and EDTA pre-treatment followed by pectinase, in a tumble agitated bath. Adding EDTA to the enzyme solution accelerated the degree of hydrolysis and resulted in higher reducing sugar liberation and more efficient calcium ion extraction, indicating a synergistic effect of enzyme and EDTA. However, when EDTA was applied as a pre-treatment, a decrease in the efficiency of the subsequent enzymatic hydrolysis was observed.     

Copper- and Arene-Catalyzed Cleavage of DNA

DNA was found to be cleaved by arenes and copper(II) salts in neutral solutions. The efficiency of this reaction is comparable with the DNA cleavage by such systems as Cu(II)–phenanthroline and Cu(II)–ascorbic acid in efficiency, but, unlike them, it does not require the presence of an exogenous reducing agent or hydrogen peroxide. The Cu²⁺–arene system does not cleave DNA under anaerobic conditions. Catalase, sodium azide as well as bathocuproine, a specific chelator of Cu(I), completely inhibit the reaction. Our results suggest that Cu(I) ions, superoxide radical and singlet oxygen participate in this reaction. It was shown by EPR and spin traps that the reaction proceeds with the formation of alkoxyl radicals capable of inducing breaks in DNA molecules. An efficient cleavage of DNA in the Cu(II)–o-bromobenzoic acid system requires the generation of radicals under the conditions of formation of a specific copper–DNA–o-bromobenzoic acid complex, in which copper ions are likely to be coordinated with oxygen atoms of the DNA phosphate groups.     

Thallium transport and the evaluation of olfactory nerve connectivity between the nasal cavity and olfactory bulb

Little is known regarding how alkali metal ions are transported in the olfactory nerve following their intranasal administration. In this study, we show that an alkali metal ion, thallium is transported in the olfactory nerve fibers to the olfactory bulb in mice. The olfactory nerve fibers of mice were transected on both sides of the body under anesthesia. A double tracer solution (thallium-201, (201)Tl; manganese-54, (54)Mn) was administered into the nasal cavity the following day. Radioactivity in the olfactory bulb and nasal turbinate was analyzed with gamma spectrometry. Auto radiographic images were obtained from coronal slices of frozen heads of mice administered with (201)Tl or (54)Mn. The transection of the olfactory nerve fibers was confirmed with a neuronal tracer. The transport of intranasal administered (201)Tl/(54)Mn to the olfactory bulb was significantly reduced by the transection of olfactory nerve fibers. The olfactory nerve transection also significantly inhibited the accumulation of fluoro-ruby in the olfactory bulb. Findings indicate that thallium is transported by the olfactory nerve fibers to the olfactory bulb in mice. The assessment of thallium transport following head injury may provide a new diagnostic method for the evaluation of olfactory nerve injury.