mV Change Research Articles

A SrF2-based oxygen sensor operative at low temperature1Paper presented at the 2nd Asia Conference on Chemical Sensors, Xi'an, P.R. China, 1995.1

Abstract Potentiometric disk type oxygen sensors monitoring in low temperatures (200–500°C) were fabricated using SrF 2 and their sensing characteristics were investigated. The disk type oxygen sensors consist of a reference electrode: Air (O 2 )|Pt, a solid electrolyte SrF 2 and a sensing Pt electrode. The correlation between the electromotive force(EMF) and oxygen partial pressure at various temperatures between 200 and 500°C was obtained. The experimental results were found to follow the Nernst equation in the test range of 0.1–100% oxygen partial pressure. The sensor element of the disk type showed an EMF change of 60 mV for an oxygen concentration change of 1–10% at a device temperature of 300°C. The sensor with Ni/NiO reference electrode showed an EMF change of 55 mV change for an oxygen concentration change of 0.1–10% at the same temperature.

A tungsten oxide coated wire electrode used as a pH sensor in flow injection potentiometry

In this study, a tungsten oxide wire electrode is described for use as a pH electrode in a portable battery-powered flow injection analyser described previously.1 The tungsten oxide electrode exhibits a linear response of 44.8 ± 0.5 mV change per pH unit over a wide range, pH 2–11 in the steady-state mode. In the flow injection mode, the tungsten oxide wire electrode exhibited a slope of 42.4 ± 0.9 mV per pH unit. The tungsten oxide electrode was employed to determine the pH of various alcoholic beverages and environmental water samples.

A photo-cured coated-wire calcium ion selective electrode for use in flow injection potentiometry

A multi-sensor cell containing a new photo-cured calcium ion selective electrode sensor is reported. Four membranes containing different components are prepared to determine the one with optimum selectivity and sensitivity. This is shown to consist of the N,N,N′,N′-tetracyclohexyl-3-oxapentanediamide ligand (ETH 129) as the ionophore, 2-nitrophenyl octyl ether as the plasticiser and tetradodecyl ammonium tetrakis(4-chlorophenyl) borate as the lipophilic additive. The photo-curing process is applied after coating a thin membrane on a silver wire as substrate transducer to produce the calcium sensor. The curing process is demonstrated to be faster (1 min) than previous methods and does not require a nitrogen atmosphere for reproducible production of membrane response characteristics. Four sensors constructed with the identical optimum membrane are shown to function reproducibly in a multi-sensor flow-through cell using the steady-state mode of flow measurement, and an average calibration slope of 28.5±0.4 mV change per activity decade is observed over a log-linear concentration range between 0.01 and 10 mM. The sensor is also shown to respond to changes in pH. Hence, in the flow injection potentiometric mode, a constant carrier buffer composition of pH 8.3 is required for accurate potentiometric calcium determinations. The sensor is used to determine calcium in water samples by flow injection potentiometry. The accuracy of the electrode determination relative to atomic absorption spectroscopy was in the range 5–9% for three different water samples.

Effect of Pco 2 Changes Induced by Head-Upright Tilt on Transcranial Doppler Recordings

Transcranial Doppler (TCD) monitoring of mean blood flow velocity (mV) during head-upright tilt can allow testing of cerebral autoregulation. Nonetheless, head-upright tilt can induce changes in the ventilation-perfusion relationship and/or respiratory activity that might influence TCD data. Forty-eight healthy volunteers underwent monitoring of mV and end-tidal CO2 in the horizontal position and during head-upright tilt. Both mV and end-tidal CO2 significantly decreased in orthostasis (P < .01). Linear regression analysis showed a significant linkage between end-tidal CO2 and mV changes (r = .83, P < .01). Changes in ventilation-perfusion ratio and in the respiratory pattern induced by head-upright tilt can significantly influence TCD data by determining a PCO2 decrease.

The evaluation of the critical electrochemical potentials influencing environmentally assisted cracking of Al-Li-Cu alloys in selected environments

The electrochemical behavior of aluminium has been determined as a function of copper content using potentiodynamic polarization and scratching electrode techniques. This information has been correlated to trends in environmental cracking of Al-Li-Cu alloys in inhibited sodium chloride environments. Scratching electrode-constant extension tests of stressed tensile samples have revealed a change from no failures to rapid failures due to a 10 mV change in applied potential. The potentials ( E CRIT-EAC) where these transitions occur appear to be linked to the electrochemistry of a copper depleted region along microstructural boundaries. Similar values for E CRIT-EAC were observed for both Li-containing and Li-devoid Al-Cu-X alloys, and for a given alloy the values were a function of temper. These observations are used to support the theory of Cu-depletion controlling crack-tip electrochemistry as opposed to an active precipitate phase as the dominant feature.

Evaluation of a new wall‐jet flow‐through cell for commercial ion‐selective electrodes in flow injection potentiometry

AbstractAn improved wall‐jet configuration type flow‐through cell has been developed for use with commercial ion‐selective electrodes (ISEs) and has been evaluated in the flow injection potentiometric (FIP) mode. The flow‐through cell was designed to accommodate commercial ISEs that employ a range of sensing surfaces, including gas‐sensing, liquid polymer membrane and solid‐state based ISEs. Commercial ISEs employed for the evaluation of the flow‐through cell were ammonia, calcium and iodide ion‐selective electrodes. The observed electrode slopes were −58.8 ± 0.9, 25.2 ± 0.5 and −58.3 ± 0.9, mV change / activity decade, respectively, over a wide log‐linear range using a 100 μL sample volume in the FIP mode. Sample throughput rates as fast as 240 injections/h and analytically useful peak height responses can be achieved using injected sample volumes as low as 25 μL with all three types of ISEs.

Characterization of apamin-sensitive Ca(2+)-activated potassium channels in human leukaemic T lymphocytes.

1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellularly applied ions, toxins and drugs on voltage-independent, apamin-sensitive Ca(2+)-activated K+ channels, K(Ca), expressed in the Jurkat human leukaemic T cell line. 2. Extracellular Ba2+ and Sr+ produced a voltage-dependent block. The equilibrium dissociation constant of the Ba2+/K(Ca) channel complex increased e-fold for a 20 mV change of potential. Ba2+ block of Jurkat K(Ca) channels is therefore as steep as expected from the movement of a single divalent cation about half-way into the electric field of the membrane from the outside. 3. We determined the ion selectivity as well as the conductance of these channels. Calculated permeability ratios, PX/PK, for these K(Ca) channels were 1.0, 0.96, 0.26 and 0.53 for K+, Rb+, Cs+ and NH4+, respectively. Conductance ratios, gX/gK, for the same ions were 1.0, 1.0, 0.67 and 0.11, respectively. Most strikingly this channel can also carry significant current with Cs+ as current carrier. 4. Scyllatoxin (ScTX), a thirty-one amino acid peptide toxin, reduced current through these K(Ca) channels with a half-blocking concentration of approximately 0.3 nM independent of the pH. Other drugs that were able to reduce current through these channels include the classical calcium antagonists diltiazem and verapamil. In contrast, nifedipine, clotrimazole and kaliotoxin (100 nM) were unable to block current through these channels in Jurkat T cells.

Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells.

1. The actions of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, memantine (1-amino-3,5-dimethyladamantane) and (+)-MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole-cell patch clamp technique. 2. Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells. 3. Memantine (0.3-30 microM) blocked L-glutamate (100 microM)-mediated currents in a concentration-dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at -70 mV) of 0.93 +/- 0.15 microM, 0.82 +/- 0.12 microM and 0.47 +/- 0.06 microM (mean +/- s.c. mean), respectively. 4. The memantine-induced block was strongly voltage-dependent. Alteration of the holding potential from -70 mV to +60 mV resulted in an e-fold increase in the IC50 values per 30-33 mV change in membrane potential, for all 3 subunit combinations investigated. 5. The kinetics of the actions of memantine (30 microM) were investigated for the NR1a/2A combination, in 6 cells (13-15 determinations). At -70 mV, the block and recovery from block were both best described by two exponentials with time-constants of 201 +/- 23 ms (81 +/- 2%) and 3.9 +/- 0.6 s and 597 +/- 94 ms (18 +/- 1%) and 18.6 +/- 2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time-constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models. 6. (+)-MK-801 was studied briefly for comparative purposes. (+)-MK-801 (200 nM) preferentially blocked NMDA receptor currents (at -70 mV) in NR1a/NR2A and NR1a/NR2B (82 +/- 10% and 93 +/- 2% depressions) compared to NR1a/NR2D (38 +/- 7%) transfected cells. (+)-MK-801 appeared to be less voltage-dependent than memantine on all three receptor combinations. 7. In conclusion, memantine was a voltage-dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.

A photo‐cured calcium ion‐selective electrode for use in flow injection potentiometry that tolerates high perchlorate levels

AbstractAn improved photo‐cured membrane selective to calcium was developed based on the calcium bis[4‐(l, 1,3,3‐tetramethylbutyl)phenyl] phosphate ionophore and incorporates the lipophilic additive, potassium tetrakis(p‐chloropheny1)borate. The calcium electrode exhibited a hyper‐Nernstian response of 31.0 mV change per activity decade and a log‐linear range between 1 × 10−5 to 0.1 M in pure calcium chloride solution, in steady‐state mode. The calcium electrode was fast responding, reaching 90% of steady‐state value in 5s making it ideal for flow injection measurements. The photo‐cured electrode can measure calcium in a high perchlorate background, and was used to determine the total calcium level in full cream milk that was digested by a nitric/perchloric acid mixture. The results obtained were in good agreement with atomic absorption spectroscopy measurements.

The [4Fe-4S] cluster domain of the nitrogenase iron protein facilitates conformational changes required for the cooperative binding of two nucleotides.

MgATP binding and hydrolysis are central to all reduction reactions catalyzed by nitrogenase. The iron (Fe) protein component of nitrogenase is a homodimeric protein with a bridging [4Fe-4S] cluster and two nucleotide binding sites, one on each subunit. This work presents evidence that the [4Fe-4S] cluster domain of the nitrogenase Fe protein functions as a hinge region between the two nucleotide binding domains, participating in the cooperative binding of two nucleotides. Alanine residues at position 98 (located near the [4Fe-4S] cluster) of the Azotobacter vinelandii Fe protein were changed by means of site-directed mutagenesis to Val (V) and Gly (G), and the resulting altered proteins were purified and characterized. While the wild-type and A98G Fe proteins were found to bind two nucleotides (MgATP or MgADP) with strong cooperativity (Hill coefficient of 2), the A98V Fe protein was found to bind one nucleotide with no apparent cooperativity. The binding of two nucleotides to the wild-type Fe protein is known to induce protein conformational changes which are reflected as changes in the properties of the [4Fe-4S] cluster, including a change in the redox potential of the [4Fe-4S] cluster of -120 mV for MgATP binding (-300 to -420 mV) and of -160 mV for MgADP binding (-300 to -460 mV). The binding of one nucleotide to the A98V Fe protein was found to result in only half the lowering of the redox potential, with MgATP binding resulting in a -80 mV change (-280 to -360 mV) and MgADP binding resulting in a -50 mV change (-280 to -330 mV). Results from 1H NMR, EPR, and CD spectra, along with Fe chelation rates, were all consistent with the binding of a single nucleotide to the A98V Fe protein inducing a partial conformational change. Finally, the A98V Fe protein with one nucleotide bound, still bound to the molybdenum-iron protein but did not support MgATP hydrolysis, electron transfer, or substrate reduction. A model is discussed in which the [4Fe-4S] cluster domain can be viewed as a hinge region between the two nucleotide binding domains which facilitates conformational rearrangements required for the cooperative binding of a second nucleotide.

A portable flow injection analyzer for use with ion‐selective electrodes

AbstractA battery powered portable monitor based on flow injection potentiometry has been developed weighing 1.8 kg, constructed in a carry‐case and applicable for remote site monitoring. Portability has been achieved using a light weight rechargeable Ni‐Cd battery‐pack (7.2 V). The flow injection manifold incorporates a low powered peristaltic pump, a wall‐jet type flow cell containing a commercial iodide ion‐selective electrode and a Ag/AgCl reference electrode connected to an analog‐to‐digital converter, also powered by the battery pack, and a RS232‐serial output to a notebook computer for real‐time data display and storage. The system performance has been evaluated using the iodide ion‐selective electrode, including the effects of various carrier solutions on sample peak heights, peak widths and the calibration slopes and working ranges. Fast response with peak widths as low as approximately 10 s. was observed with near Nernstian response of −53.8 mV change per activity decade and a log‐linear range between 5 × 10−6 and 1 × 10−2M in a 0.1 M sodium acetate carrier stream.

Electrophysiologic characteristics of M cells in the canine left ventricular free wall.

Recent studies have described the existence of M cells in the deep structures of the canine and human ventricle. The present study was designed to further characterize the M cell with respect to its distribution across the canine left ventricular free wall and the dependence of its action potential on [K+]o. We used standard microelectrode techniques to record transmembrane activity from deep subepicardial or transmural strips isolated from the canine left ventricular free wall near the base as well as subendocardial Purkinje fibers. M cells behavior (steep APD-rate relation) was observed at depths of 1 to 7 mm from the epicardial surface (deep subepicardium to mid-myocardium). M cells were found to be distributed uniformly in the deep subepicardium and did not appear in discrete bundles. We observed transitional behavior throughout the wall. The maximum rate of rise of the action potential upstroke, Vmax, increased sharply between epicardium and deep subepicardium (176 +/- 13 to 332 +/- 61 V/sec), remained high throughout the mid-myocardium and deep subendocardium, and returned to lower values only in the superficial layers of the endocardium (205 +/- 21 V/sec). The relationship between Vmax and takeoff potential in the M cell was fit by a Boltzmann equation with a V0.5 of -68.6 +/- 1.5 mV and k of 3.4 +/- 0.5. The relationship between resting membrane potential (RMP) and [K+]o in the M cell was exponential from 8 to 20 mmol/L (58 mV change in RMP per 10-fold change in [K+]o), deviating from K+ electrode behavior at [K+]o < 8 mmol/L. RMP in M cells continued to hyperpolarize at [K+]o < 2.5 mmol/L, reaching potentials of approximately -110 mV at [K+]o of 1 mmol/L. In contrast, subendocardial Purkinje fibers depolarized at these low levels of [K+]o. Unlike endocardium and epicardium, M cells developed early afterdepolarizations at low [K+]o and slow rates. Our data indicate that the M cells are widely distributed in the intramural layers of the canine left ventricular free wall. M cells and transitional cells occupy 30% to 40% of the left ventricular wall and an estimated 20% to 40% of the mass of the ventricles of the normal canine heart. They display characteristics common to both myocardial and specialized conducting cells. Like Purkinje fibers, M cells exhibit a relatively large Vmax and steep APD-rate relations that are modulated by [K+]o. Unlike Purkinje fibers, M cells do not appear in bundles, they do not depolarize at [K+]o < 2.5 mmol/L, nor do they exhibit phase 4 depolarization.

Kinetic properties of the sodium‐calcium exchanger in rat brain synaptosomes.

1. The kinetic properties of the internal Na+ (Na+i)- dependent 45Ca2+ influx and external Na+ (Na+o)-dependent 45Ca2+ efflux were determined in isolated rat brain nerve terminals (synaptosomes) under conditions which the concentrations of internal Na+ ([Na+]i), external Na+ ([Na+]o), external Ca2+ (Ca2+]o), and external K+ ([K+]o) were varied. Both fluxes are manifestations of Na(+)-Ca2+ exchange. 2. Ca2+ uptake was augmented by raising [Na+]i and / or lowering [Na+]o. The increase in Ca2+ uptake induced by removing external Na+ was, in most instances, quantitatively equal to the Na+i-dependent Ca2+ uptake. 3. The Na+i-dependent Ca2+ uptake (measured at 1 s) was activated with an apparent half-maximal [Ca2+]o (KCa(o)) of about 0.23 mM. External Na+ inhibited the uptake in a non- competitive manner: increasing [Na+]o from 4.7 to 96 mM reduced the maximal Na+(i)-dependent Ca2+ uptake but did not affect KCa(o). 4. The inhibition of Ca2+ uptake by Na+o was proportional to ([Na+]o)2, and had a Hill coefficient (nH) of approximately 2.0. The mean apparent half-maximal [Na+]o for inhibition (KI(Na)) was about 60mM, and was independent of [Ca2+]o between 0.1 and 1.2mM; this, too, is indicative of non-competitive inhibition. 5. Low concentrations of alkali metal ions (M+) in the medium, including Na+, stimulated the Na+i-dependent uptake. The external Na+ and K+ concentrations required for apparent half-maximal activation (KM(Na) and KM(K), respectively) were 0.12 and 0.10mM. Thus, the relationship between Ca2+ uptake and [Na+]o was biphasic: uptake was stimulated by [Na+]o < or = 10 mM, and inhibited by higher [Na+]o. 6. The calculated maximal Na+i-dependent Ca2+ uptake (Jmax) was about 1530 pmol (mg protein) -1s-1 at 30 degrees C saturating [Ca2+]o and external M+ concentration ([M+]o), and with negligible inhibition by external Na+. 7. Internal Na+ activated the Ca2+ uptake with an apparent half-maximal concentration (KNa(i)) of about 20 mM and a Hill coefficient, nH, of approximately 3.0. 8. The Jmax for the Na+o-dependent efflux of Ca2+ from 45Ca(2+)-loaded synaptosomes treated with carbonyl cyanide p-trifluormethoxy-phenylhydrazone (FCCP) and caffeine (to release stored Ca2+ and raise the internal Ca2+ concentration ([Ca2+]i) was about 1800-2000 pmol (mg protein -1s-1 at 37 degrees C. 9. When the membrane potential (Vm) was reduced (depolarized) by increasing [K+]o, the Na+i-dependent Ca2+ influx increased, and the Na+o-dependent Ca2+ efflux declined. Both fluxes changed about 2-fold per 60 mV change in Vm. This voltage sensitivity corresponds to the movement of one elementary charge through about 60% of the membrane electric field. The symmetry suggests that the voltage-sensitive step is reversible. 10. The Jmax values for both Ca2P influx and efflux correspond to a Na+-Ca2+ exchange-mediated flux of about 425-575 jumol Ca2P (1 cell water)-' s-' or a turnover of about one quarter of the total synaptosome Ca2P in 1 s. We conclude that the Na+-Ca2P exchanger may contribute to Ca2P entry during nerve terminal depolarization; it is likely to be a major mechanism mediating Ca2P extrusion during subsequent repolarization and recovery.

Resistive properties of the epithelial membranes of the urinary bladder of the toad, Bufo marinus, determined using the fluorescent dye, RH160.

A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range +/- 200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 mumol.l-1), reduced by bretylium (5 mmol.l-1) and abolished in the presence of nystatin (730 U.ml-1). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 k omega.microF, and the basolateral membrane resistance was 1.5 k omega.microF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.

Local anaesthetic blockade of neuronal nicotinic ACh receptor-channels in rat parasympathetic ganglion cells.

1 The effects of the local anaesthetics QX-222 and procaine on nicotinic acetylcholine (ACh)-evoked currents in cultured parasympathetic cardiac neurones of the rat were investigated by use of the whole-cell, perforated-patch, and outside-out recording configurations of the patch clamp method. 2 QX-222 and procaine, applied to the extracellular surface, reversibly inhibited the peak amplitude of the whole-cell nicotinic ACh-evoked current in a concentration-dependent manner, with half-maximal inhibitory concentrations (IC50) of 28 microM and 2.8 microM, respectively, at -80 mV. In these neurones, the sustained inward current mediated by M1 muscarinic receptor activation was unaltered by QX-222, and neither local anaesthetic affected the adenosine 5'-triphosphate (ATP)-evoked current. 3 QX-222 and procaine block of nicotinic ACh-evoked inward current was voltage-dependent and enhanced by hyperpolarization. An e-fold change in their dissociation equilibrium constants (Kd) resulted from a 62 mV and a 122 mV change in membrane potential, respectively. 4 Both local anaesthetics produce a concentration-dependent increase in the half-time of decay of the nicotinic ACh-evoked inward current. 5 Measurements of unitary currents in outside-out patches showed that QX-222 reversibly increased the mean burst duration and closed time and reduced the mean channel open time and open-state probability of the nicotinic ACh receptor-channel (AChR) in a concentration-dependent manner. 6 The Kd and voltage sensitivity of local anaesthetic block of the nicotinic AChR in rat intracardiac neurones suggests that the pore-forming region of this channel differs from that of the AChR in frog and rat skeletal muscle and from the neuronal alpha 4 beta 2 ACh receptor-channel.

A calcium-activated potassium channel causes frequency-dependent action-potential failures in a mammalian nerve terminal

1. The contribution of a calcium-activated potassium channel to action-potential failure was studied in nerve terminals of the rat posterior pituitary. 2. Depolarizing current injections under current clamp were faithfully followed by action potentials for stimulation frequencies of < or = 12 Hz. Further increases in frequency resulted in action-potential failure within a few hundred milliseconds. The fraction of failures increased with stimulation frequency. This decrease in excitability was concomitant with a hyperpolarization from -57.3 +/- 1.4 to -61.3 +/- 1.4 (SE) mV. 3. The decrease in excitability was dependent on calcium influx through voltage-dependent calcium channels, because action-potential failures did not occur at frequencies < or = 30 Hz in the presence of cadmium. The dihydropyridine agonist BayK 8644 increased the fraction of failed action potentials. 4. Depolarizations from -80 to 10 mV for 3 s evoked macroscopic potassium currents with a rapidly activated, transient component and a slowly developing, noninactivating component. The late outward current was dependent on calcium influx, because it was reduced by cadmium and enhanced by BayK 8644. 5. Tetraethylammonium and 4-aminopyridine effectively blocked potassium outward currents but failed to distinguish this calcium-dependent potassium channel from the other two potassium channels in this preparation. Charybdotoxin and apamin did not affect potassium currents in this preparation. 6. In excised inside-out patches, the calcium-dependent potassium channel had a slope conductance of 193 pS. The open probability changed e-fold per 14.8 mV change in membrane potential with a calcium concentration at the cytoplasmic membrane face ([Ca]i) of 100 nM. 7. The channel was highly sensitive to [Ca]i. Depolarizations to 100 mV at 10 nM [Ca]i activated the channel half-maximally. When [Ca]i was raised to 250 nM, the voltage for half-maximal activation shifted to -16 mV. Calcium also decreased the steepness of the voltage activation curve. 8. At a constant membrane potential, pressure ejection of calcium to the cytosolic face of an excised patch activated the channel with a delay of 82 ms. This slow activation in excised patches was consistent with the slow activation of the delayed component of the macroscopic current. 9. At constant calcium concentration, the time course of activation exhibited a strong voltage dependence. Most of the channels did not inactivate during depolarizations lasting < or = 300 ms. 10. The channel exhibited complex gating, with at least two distinct open and closed states.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of a glutamate-activated chloride current in Xenopus oocytes injected with Caenorhabditis elegans RNA: evidence for modulation by avermectin

Membrane currents were recorded from Xenopus laevis oocytes injected with C. elegans poly(A) + RNA. In such oocytes glutamate activated an inward membrane current that desensitized in the continued presence of glutamate. Glutamate-receptor agonists quisqualate, kainate, and N- methyl- d-aspartate were inactive. The reversal potential of the glutamate-sensitive current was −22 mV, and exhibited a strong dependence on external chloride with a 48 mV change for a 10-fold change in chloride. The chloride channel blockers flufenamate and picrotoxin inhibited the glutamate-sensitive current. Ibotenate, a structural analog of glutamate, also activated a picrotoxin-sensitive chloride current. Ibotenate was inactive when current was partially desensitized with glutamate, and the responses to low concentrations of glutamate and ibotenate were additive. The anthelmintic/insecticide compound avermectin directly activated the glutamate-sensitive current. In addition, avermectin increased the response to submaximal concentrations of glutamate, shifted the glutamate concentration-response curve to lower concentrations, and slowed the desensitization of glutamate-sensitive current. We propose that the glutamate-sensitive chloride current and the avermectin-sensitive chloride current are mediated via the same channel.

Properties of the inactivating outward current in single smooth muscle cells isolated from the rat anococcygeus.

The properties of the voltage- and time-dependent outward current in single smooth muscle cells isolated from the rat anococcygeus were studied. The outward current was activated by depolarizations to membrane potentials positive to -40 mV. Activation followed third order kinetics; at +20 mV, the time for the current to reach half its maximal amplitude was around 55 ms. The current inactivated with a time course that could best be described by a single exponential with a time constant around 1500 ms. The steady-state inactivation curve was voltage dependent over the range -110 to -30 mV, with a half-inactivation point of -67 mV. Recovery from inactivation followed an exponential time course with a time constant of around 770 ms at -90 mV. Deactivating tail current analysis revealed that a 10-fold change in the extracellular potassium ion concentration resulted in a 42 mV change in the reversal potential of the current. The current was blocked by 4-aminopyridine, tetraethylammonium, quinine and verapamil with IC50's--the concentrations producing 50% inhibition of the peak current--of 2 mM, 4 mM, 12 microM and 20 microM respectively. The current was not blocked by Toxin I (100 nM) or glibenclamide (10 microM). The current was still present in cells containing 5 mM EGTA; in these cells, replacing extracellular calcium with cadmium depressed the peak current by around 12%. This could be explained, at least in part, by a negative shift in the voltage dependence of inactivation.

Voltage activation of heart inner mitochondrial membrane channels.

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above +/- 60 mV [Kinnally et al., Biochem. Biophys. Res. Commun. 176, 1183-1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin A are likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.

A basolateral K+ conductance modulated by carbachol dominates the membrane potential of small intestinal crypts

The contribution of possible ionic conductances to the membrane potential (Em) of cells in guinea-pig small intestinal crypts has been studied using the nystatin "perforated-patch" approach in current-clamp experiments. Changes in extracellular K+ produced shifts in Em, with a 37 mV change in potential per ten-fold increase in extracellular K+ concentration. Reduction of extracellular Cl- by 130 mM led to a 7 mV hyperpolarisation while Na+ replacement was without effect on Em. The muscarinic agonist carbachol produced a hyperpolarisation which could be ascribed to an increase in basolateral K+ conductance. This effect was sustained in the presence of extracellular Ca2+ but was transient in its absence. We conclude that the conductance of the basolateral membrane of small intestinal crypts is mainly K+ selective and can be reversibly increased by muscarinic activation.