Characterization of acid-sensing ion channels in dorsal horn neurons of rat spinal cord

Acid-sensing ion channels (ASICs) are ligand-gated cation channels activated by extracellular protons. In periphery, they contribute to sensory transmission, including that of nociception and pain. Here we characterized ASIC-like currents in dorsal horn neurons of the rat spinal cord and their functional modulation in pathological conditions. Reverse transcriptase-nested PCR and Western blotting showed that three ASIC isoforms, ASIC1a, ASIC2a, and ASIC2b, are expressed at a high level in dorsal horn neurons. Electrophysiological and pharmacological properties of the proton-gated currents suggest that homomeric ASIC1a and/or heteromeric ASIC1a + 2b channels are responsible for the proton-induced currents in the majority of dorsal horn neurons. Acidification-induced action potentials in these neurons were compatible in a pH-dependent manner with the pH dependence of ASIC-like current. Furthermore, peripheral complete Freund's adjuvant-induced inflammation resulted in increased expression of both ASIC1a and ASIC2a in dorsal horn. These results support the idea that the ASICs of dorsal horn neurons participate in central sensory transmission/modulation under physiological conditions and may play important roles in inflammation-related persistent pain.

Calcium-permeable acid-sensing ion channel is a molecular target of the neurotoxic metal ion lead

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhib

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity

Development of chronic pain involves alterations in peripheral nociceptors as well as elevated neuronal activity in multiple regions of the CNS. Previous pharmacological and behavioral studies suggest that peripheral acid-sensing ion channels (ASICs) cont

Study of the ion channel behavior of didodecyldimethylammonium bromide formed bilayer lipid membrane stimulated by PF6-

Bilayer lipid membranes ( BLM) formed from didode-cyldimethylammonium bromide were made on the freshly exposed surface of a glassy carbon (GC) and were demonstrated by the ac impedance spectroscopy. The ion channels of membrane properties induced by PF6- were studied by the cyclic voltammetric methods. Experimental results indicated that the ion channel of BLM was open in the presence of the PF6- due to the interaction of PF6- with the BLM, while it was switched off in the absence of PF6-. Because the ion channel behavior was affected by the concentration of PF6-, a sensor for PF6- can be developed.

Ion channel behavior of amphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes investigated by electrochemistry and spectroscopy

Amphotericin B (AmB) is a popular drug frequently applied in the treatment of systemic fungal infections. In the presence of ruthenium (II) as the maker ion, the behavior of AmB to form ion channels in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes were studied by cyclic votammetry, AC impedance spectroscopy, and UV/visible absorbance spectroscopy. Different concentrations of AmB ranging from a molecularly dispersed to a highly aggregated state of the drug were investigated. In a fixed cholesterol or ergosterol content (5 mol %) in glassy carbon electrode-supported model membranes, our results showed that no matter what form of AmB, monomeric or aggregated, AmB could form ion channels in supported ergosterol-containing phosphatidylcholine bilayer model membranes. However, AmB could not form ion channels in its monomeric form in sterol-free and cholesterol-containing supported model membranes. On the one hand, when AmB is present as an aggregated state, it can form ion channels in cholesterol-containing supported model membranes; on the other hand, only when AmB is present as a relatively highly aggregated state can it form ion channels in sterol-free supported phosphatidylcholine bilayer model membranes. The results showed that the state of AmB played an important role in forming ion channels in sterol-free and cholesterol-containing supported phosphatidylcholine bilayer model membranes.

Mechanisms of H+ modulation of glycinergic response in rat sacral dorsal commissural neurons

Many ionotropic receptors are modulated by extracellular H+. So far, few studies have directly addressed the role of such modulation at synapses. In the present study, we investigated the effects of changes in extracellular pH on glycinergic miniature inhibitory postsynaptic currents (mIPSCs) as well as glycine-evoked currents (I-Gly) in mechanically dissociated spinal neurons with native synaptic boutons preserved. H+ modulated both the mIPSCs and I-Gly, biphasically, although it activated an amiloride-sensitive inward current by itself. Decreasing extracellular pH reversibly inhibited the amplitude of the mIPSCs and I-Gly, while increasing external pH reversibly potentiated these parameters. Blockade of acid-sensing ion channels (ASICs) with amiloride, the selective antagonist of ASICs, or decreasing intracellular pH did not alter the modulatory effect of H+ on either mIPSCs or I-Gly, H+ shifted the EC50 of the glycine concentration-response curve from 49.3 +/- 5.7 muM at external pH 7.4 to 131.5 +/- 8.1 muM at pH 5.5, without altering the Cl- selectivity of the glycine receptor (GlyR), the Hill coefficient and the maximal I-Gly, suggesting a competitive inhibition of I-Gly by H+. Both Zn2+ and H+ inhibited I-Gly. However, H+ induced no further inhibition of I-Gly in the presence of a saturating concentration of Zn2+. In addition, H+ significantly affected the kinetics of glycinergic mIPSCs and I-Gly. It is proposed that H+ and/or Zn2+ compete with glycine binding and inhibit the amplitude of glycinergic mIPSCs and I-Gly. Moreover, binding of H+ induces a global conformational change in GlyR, which closes the GlyR Cl- channel and results in the acceleration of the seeming desensitization of IGly as well as speeding up the decay time constant of glycinergic mIPSCs. However, the deprotonation rate is faster than the unbinding rate of glycine from the GlyR, leading to reactivation of the undesensitized GlyR after washout of agonist and the appearance of a rebound I-Gly. H+ also modulated the glycine cotransmitter, GABA-activated current (I-GABA). Taken together, the results support a 'conformational coupling' model for H+ modulation of the GlyR and suggest that W may act as a novel modulator for inhibitory neurotransmission in the mammalian spinal cord.

Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons

The characterization of acid-sensing ion channel (ASIC)-like currents has been reported in hippocampal neurons in primary culture. However, it is suggested that the profile of expression of ASICs changes in culture. In this study, we investigated the properties of proton-activated current and its modulation by extracellular Ca2+ and Zn2+ in neurons acutely dissociated from the rat hippocampal CA1 using conventional whole-cell patch-clamp recording. A rapidly decaying inward current and membrane depolarization was induced by exogenous application of acidic solution. The current was sensitive to the extracellular proton with a response threshold of pH 7.0-6.8 and the pH(50) Of 6.1, the reversal potential close to the Na+ equilibrium potential. It had a characteristic of acid-sensing ion channels (ASICs) as demonstrated by its sensitivity to amiloride (IC50 = 19.6 +/- 2.1 muM). Either low [Ca2+](0) or high [Zn2+](0) increased the amplitude of the current. All these characteristics are consistent with a current mediated through a mixture of homomeric ASIC1a and heteromeric ASIC1a + 2a channels and closely replicate many of the characteristics that have been previously reported for hippocampal neurons cultured for a week or more, indicating that culture artifacts do not necessarily flaw the properties of ASICs. Interestingly, we found that high [Zn2+] (>10(-4) M) slowed the decay time constant of the ASIC-like current significantly in both acutely dissociated and cultured hippocampal neurons. In addition, the facilitating effects of low [Ca2+](0) and high [Zn2+](0) on the ASIC-like current were not additive. Since tissue acidosis, extracellular Zn elevation and/or Ca2+ reduction occur concurrently under some physiological and/or pathological conditions, the present observations suggest that hippocampal ASICs may offer a novel pharmacological target for therapeutic invention. (C) 2004 Elsevier B.V. All rights reserved.

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li+, Na+, NH4+, K+, Mg2+, and Ca2+) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na+, NH4+, K+, Mg2+, and Ca2+ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K+, Mg2+, and Ca2+ significantly, decreased after exposure to Az, except that Na+ and NH4+ remained constant. Li+ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.

Electrochemical study of gramicidin D forming ion-permeable channels in the bilayer lipid membranes

Gramicidin within the lipid bilayer matrix is a well-known channel-forming polypeptide, but the mechanism of the ions across the membrane induced by gramicidin is not well understood. We found that at very low concentration of gramicidin in a bilayer lipid membrane, the channel behavior was controlled by the voltage applied across the membrane. When the voltage is higher than 75 mV, the channel is closing, while lower than 75 mV, the channel is opening. But when the concentration of the gramicidin in the BLMs is high, the channel behavior is changed into voltage-independent. (C) 1998 Elsevier Science Ltd. All rights reserved.

Selective and lithography-independent fabrication of 20 nm nano-gap electrodes and nano-channels for nanoelectrofluidics applications

A new method has been developed to selectively fabricate nano-gap electrodes and nano-channels by conventional lithography. Based on a sacrificial spacer process, we have successfully obtained sub-100-nm nano-gap electrodes and nano-channels and further reduced the dimensions to 20 nm by shrinking the sacrificial spacer size. Our method shows good selectivity between nano-gap electrodes and nano-channels due to different sacrificial spacer etch conditions. There is no length limit for the nano-gap electrode and the nano-channel. The method reported in this paper also allows for wafer scale fabrication, high throughput, low cost, and good compatibility with modern semiconductor technology.

Photo-induced reactions in the ion-molecule complex Mg+-OCNC2H5

Ion - molecule complexes of magnesium cation with ethyl isocyanate were produced in a laser- ablation supersonic expansion nozzle source. Photo- induced reactions in the 1: 1 complexes have been studied in the spectral range of 230 - 410 nm. Photodissociation mass spectrometry revealed the persistent product Mg+ from nonreactive quenching throughout the entire wavelength range. As for the reactive channels, the photoproducts, Mg+OCN and C2H5+, were produced only in the blue absorption band of the complex with low yields. The action spectrum of Mg+(OCNC2H5) consists of two pronounced peaks on the red and blue sides of the Mg+ 3(2)P <-- 3(2)S atomic transition. The ground state geometry of Mg+ - OCNC2H5 was fully optimized at B3LYP/6- 31 - G** level by using GAUSSIAN 98 package. The calculated absorption spectrum of the complex using the optimized structure of its ground state agrees well with the observed action spectrum. Photofragment branching fractions of the products are almost independent of the photolysis photon energy for the 3P(x,y,z) excitations. The very low branching ratio of reactive products to nonreactive fragment suggests that evaporation is the main relaxation pathway in the photo- induced reactions of Mg+ (OCNC2H5). (C) 2003 American Institute of Physics.

Proton transfer reactions within the pyridazine-methanol cluster ion

The multiphoton ionization of the hydrogen-bonding cluster pyridazine-methanol (C4H4N2-CH3OH) was studied using a time-of-flight mass spectrometer at the wavelengths of 355 and 532 nm. At both wavelengths, a series of protonated C4H4N2-(CH3OH)(n)-H+ cluster ions were obtained. Relevant ab initio calculations were performed with HF and B3LYP methods. Equilibrium geometries of both neutral and ionic C4H4N2-CH3OH clusters, and dissociation channels and dissociation energies of ionic clusters, are presented. The results show that when C4H4N2-CH3OH is vertically ionized, C4H4N2H+ and CH3O are the dominant products via proton transfer reaction. A high energy barrier makes another channel corresponding to the production of C4H4N2H+ and CH2OH disfavored. (C) 2002 Elsevier Science B.V. All rights reserved.

PH-Dependent syntheses and structures of two Copper(II)/Phenanthroline/p-Sulfonatocalix[4]arene supramolecular compounds with 1D water-filled channels

Two copper-organic framework supramolecular assemblies of p-sulfonatocalix[4]arene and 1,10-phenanthroline Cu-2[C12H8N2][C28H20S4O16][H2O](23.5) (1) and Cu-3[C12H8N2](3)[C28H19S4O16]Cl[H2O](17.6) (2) were obtained by pH-dependent synthesis at room temperature. Both structures show ID water-filled channels (rectangular shape in I and triangular in 2) with the solvent-accessible volume occupying 30.8% (1) and 24.2% (2) of the unit-cell volume, respectively. The calixarene molecules in both structures assume analogous cone shapes of C-2 nu symmetry instead of the conventional C-4 nu symmetry. Their connecting to different amounts of copper/phenanthroline cations leads to the formation of different structures.