Ca2+ activation of hSlo K+ channel is suppressed by N-terminal GFP tag

The human slow poke (hSlo) K+ channel was tagged with GFP (green fluorescent protein) at the N-terminus of its alpha-subunit. The fusion protein was expressed transiently in HEK293 cells; it formed functional voltage-gated channels as shown by whole cell patch-clamp measurements. However, the tag lowered the voltage dependence of gating and it suppressed the typical left-shift of gating by intracellular binding of Ca2+. The location of the GFP-tagged N-terminus was confirmed to be on the extracellular side by application of a monoclonal antibody to nonpermeabilized cells. Structural interpretations of the effects are discussed.     

Intracellular ATP depletion inhibits swelling-induced -[H]aspartate release from primary astrocyte cultures

Volume expansion-sensing outward rectifier (VSOR) anion channel, also referred to as volume-sensitive organic osmolyte-anion channel (VSOAC), appears to be responsible for cell swelling-induced amino acid release in a variety of cells. One prominent feature of the VSOR/VSOAC is that non-hydrolyzed intracellular ATP binding to the channel or an accessory protein is required for its activation. In this study, the effect of intracellular ATP depletion on the swelling-induced release of -[³H]aspartate from rat primary astrocyte cultures due to exposure to either high K⁺ or hypotonic media was studied. When the cells were pretreated for 10 min with a combination of the metabolic inhibitors 2-deoxyglucose and rotenone, 100 mM K⁺ media- or hypotonic media-induced -[³H]aspartate release was completely suppressed. Added separately, each inhibitor showed only partial or no inhibition of -[³H]aspartate release, which correlated with its relative effectiveness in decreasing intracellular ATP levels. These data are consistent with the view that during high [K⁺]_o or hypotonic media-induced swelling of primary astrocyte cultures an ATP-dependent swelling-activated VSOAC channel is responsible for -[³H]aspartate release and close to normal ATP is required for full channel activation.     

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.     

A molecular scheme for the reaction between γ-aminobutyric acid and the most abundant chloride channel on crayfish deep extensor abdominal muscle

Single-channel measurements were performed with the aim of constructing a detailed molecular scheme for the reaction between -aminobutyric acid (GABA) and a chloride channel of crayfish deep extensor abdominal muscle (DEAM). GABA was applied in pulses to outside-out patches of muscle membrane, and, based on the dose-response of the peak currents and of their rise times, a linear model with five binding steps has been proposed. Evaluation of the single-channel kinetics indicated at least three open states. Two of them originate most probably from the fully liganded receptor state and are grouped in mixed bursts due to their different life times. The third one appears independently, outside the bursts, and originates from a lower liganded receptor state. Simulations of the dose-responses and the open time distributions with this model led to a set of rate constants which generated relatively optimal fits.     

SYMPOSIUM: Experimental Biology 1995 Role of Mesangial Cell Ion Transport in Glomerular Physiology and Disease: ANGIOTENSIN II-INDUCED TYROSINE PHOSPHORYLATION IN MESANGIAL AND VASCULAR SMOOTH MUSCLE CELLS

• 1 Angiotensin II (AngII)-induced, activation of phospholipase C (PLC) and Ca²⁺-dependent Cl^? channels is an important signal transduction pathway for the regulation of vascular smooth muscle cell (VSMC) and glomerular mesangial cell contraction and growth. While AT receptors are traditionally thought to be G-protein coupled to the β isoform of PLC, recent evidence suggests that in some tissues AT receptors may also activate the PLC-γ isoform via tyrosine phosphorylation.
• 2 By western analysis, we identified PLC-γ1 in the above cell types. We found that within 3 min of exposure to 10^?7 mol/L AngII, tyrosine phosphorylation of PLC-γ1 was observed; however, peak response (> 3-fold increase) occurred within 0.5 min. In addition, pre-incubation of these cells with the tyrosine kinase inhibitor genistein blocked the tyrosine phosphorylation of PLC-γ1 by AngII. In contrast, preincubation with the tyrosine phosphatase inhibitor sodium vanadate increased the levels of tyrosine phosphorylation of PLC-γ1. Similar results were found when intracellular levels of 1,4,5-IP₃ were measured after AngII exposure.
• 3 By using patch clamp techniques on cultured rat mesangial cells exposed to AngII, we found that the release of 1,4,5-IP₃-sensitive intracellular Ca²⁺ stores stimulated low conductance Cl^? channels. Preincubation with genistein, abolished the usual 10-fold increase in Cl^? channel activity observed with AngII.
• 4 Therefore, we conclude that in VSMC and glomerular mesangial cells (i) AngII transiently stimulates PLC activity via tyrosine phosphorylation of the γ1 isoenzyme, (ii) tyrosine phosphorylation of PLC-γ1 and production of 1,4,5-IP₃ in response to AngII is dramatically inhibited by tyrosine kinase inhibition and stimulated by tyrosine phosphatase inhibition, (iii) activation of Ca²⁺-dependent Cl^? channels by AngII-induced release of 1,4,5-IP₃-dependent intracellular Ca²⁺ stores is also abolished by tyrosine kinase inhibition. In summary, this AngII-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of AngII on VSMC and glomerular mesangial cells.

     

Unitary delayed rectifier channels of rat hippocampal neurons: properties of block by external tetraethylammonium ions

Patch-clamp recording was used to characterise a delayed rectifier potassium channel and the effects of external tetraethylammonium (TEA) in neurons isolated from the CA1 region of cultured neonatal rat hippocampus. A preliminary kinetic analysis is presented. Very low concentrations of TEA included in the patch pipette solution had two effects on unitary currents: first unitary currents were reduced in amplitude, with an associated increase in open channel noise, and second channel mean open time was reduced. The reduction in unitary amplitude was consistent with a single TEA molecule blocking the channel with a voltage-independent K _d of 53.4 M. The blocking and unblocking rate constants, estimated using two independent methods, were approximately 350 mM^–1 ms^–1 and 20 ms^–1, both rate constants being independent of voltage. Channels blocked in this way appeared able to close normally without first having to become unblocked. The reduction in mean channel open time was probably due to a second, kinetically slower blocking reaction with a much lower K _d, probably between 300 and 800 M. The voltage-independent blocking rate constant of the slower block was at least 25 times slower than that of the faster block.     

Sodium channel blockade enhances dispersion of the cardiac action potential duration

Summary The goal of this study was to elucidate the causes why the proarrhythmic activity of sodium channel blocking drugs is enhanced during the post-infarction period. Therefore, we studied the effects of a reduction in sodium conductance on the action potential duration and its dispersion in a simulated array of 1600 ventricular myocytes. Cardiac tissue is known to possess anisotropic properties with regard to the intercellular electrical resistance (R). Infarction as well as aging causes deposition of collagen in the cardiac tissue, thereby inducing zones of high electrical resistance leading to a non-uniform anisotropy (Spach et al., Circ Res 62811, 1988). For our study an array of 40*40 ventricular myocytes was simulated using Beeler-Reuter-algorithms. Physical tissue properties were assumed to be either a) uniform anisotropic (i.e., all longitudinal R=5000 cm, all transversal R=20000 cm; UA) or b) non-uniform anisotropic (i.e., transversal R for the inner 10*10 cells was set to 10¹⁰ cm; NUA). Mean action potential duration (APD) was increased under UA (287 ms, dispersion: 0,8 ms) when compared to NUA (285 ms, disp.: 3,2 ms). Assuming a 25% decrease in sodium conductance, we found the total activation time (TAT) to be increased (from 99 to 139 ms), indicating slowing of conduction, APD to be shortened (from 287 to 259 ms), and the APD-dispersion to be increased (from 0.8 to 29 ms) in UA. These changes were more pronounced in the case of NUA: increase in TAT from 103 to 150 ms, APD-shortening from 285 to 214 ms and a marked increase in APD-Dispersion from 3.2 to 53 ms). From these results it is concluded that a) the effects of a reduced sodium conductance are more pronounced in NUA tissue, and b) that the resulting increase in dispersion may provoke arrhythmia by local differences in APD.This may be one of the mechanisms underlying the increased proarrhythmic risk of class I antiarrhythmic drugs in the postinfarction period.     

Noradrenaline transport by rat heart sympathetic nerves: A re-examination of the role of sodium ions

Summary The effect of sodium ion on ³H-(–)-noradrenaline (0.0875 to 0.5 M) transport by rat heart atrial hemi-appendages incubated in vitro has been studied, and the following observations made: a) When sodium was omitted (choline and lithium substitution) there was no evidence for active noradrenaline transport, and only a component that did not show saturation kinetics up to 1 M noradrenaline, remained. b) Omission of sodium or addition of 4×10^–5 M desipramine inhibited noradrenaline transport to exactly the same extent, and their effects were not additive. Alprenolol did not reduce this sodium-independent transport, but tropolone lowered it somewhat. c) No evidence for corticosterone-sensitive noradrenaline transport (uptake-2) was found in this preparation at the low amine concentrations used. d) In control medium, the kinetic parameters of transport were: K _m: 0.59 ± 0.063 M and V _max: 2.44 ± 0.43 (pmoles/mg protein/min). With 26 mM sodium and the rest substituted by choline, K _m:2.26 ± 0.70 M (P0.001) and V _max: 2.74 ± 0.43 (pmoles/mg protein/min) (not significant). Also with 26 mM sodium, but with sucrose substitution, K _m: 0.76 ± 0.13 M (N.S.) and V _max: 1.06 ± 0.13 (pmol/mg/min) (P<0.05). Such results indicate that sodium only modifies the affinity of the transport system for noradrenaline, without changing V _max, and that changes in the latter are only a consequence of a reduction of the ionic strength. e) When noradrenaline transport was studied at different concentrations of external sodium, at constant ionic strength and with precautions to minimize the noradrenaline-releasing effect of low sodium, it was found that the data could be best represented by two hyperbolas placed in series. This suggests that the noradrenaline carrier has two sites for sodium, that do not interact with each other. When the same experiments were repeated in the absence of chloride, it was found that the noradrenaline transport system had lost virtually all its affinity for sodium. f) The effect of prolonged tissue incubation in the absence of sodium was found to produce a relatively small inactivation of noradrenaline transport. Such phenomenon was enhanced by raising the calcium concentration to 2 mM.     

Aprindine blocks the sodium current in guinea-pig ventricular myocytes

Summary Aprindine is a class Ib antiarrhythmic agent. We studied effects of aprindine (3 µmol/l) on the Na⁺ current using whole cell voltage clamp (tip resistance = 0.5 , [Na]_i and_o = 10 mmol/l at 18°C). Aprindine revealed tonic block (Kd_rest = 37.7 µmol/l, Kd_i = 0.74 µmol/l; n = 4). Aprindine, shifted inactivation curve to hyperpolarizing direction by 11.4 ± 3.5 mV (n = 4) without changes in slope factor. In the presence of 3 µmol/l aprindine, aprindine showed phasic block, i.e., duration-dependent block at 2 Hz (64% ±3070 at 1.5 ms, 82%±6% at 20 ms, 93%±7% at 200 ms; n = 4). Short single prepulse also produced aprindine-induced phasic block (12% at 1.5 ms, 22% at 100 ms; n = 2). After removal of fast inactivation of Na⁺ current by 3 mmol/l tosylchloramide sodium, aprindine revealed phasic block, independent of holding potential. The recovery time constant from aprindine-induced phasic block was 4.8 s at holding potential = –100 mV and 5.0 s at holding potential = –140 mV. This use-dependent block of aprindine had pH dependency. Under acidic condition (pH 6.0), 3 µmol/l aprindine showed smaller use-dependent block (14% ± 7% at 2 Hz; n = 4) comparing with either at pH 7,4 (68% ± 13%; n = 4) or at pH 8.0 (90% ±12%; n = 4).The results suggest that aprindine could bind to the receptor via activation process through channel pore, resulting in decrease of Na⁺ current, and egress from the receptor through the lipid bilayer. These effects might be attenuated under acidic condition due to changes in intracellular ratio of charged to neutralized form of drug molecule. Send offprint requests to: R. Sato at the above address     

The cardiotonic bipyridine AWD 122-60 inhibits adenosine triphosphate-sensitive potassium channels of mouse skeletal muscle

Summary The inhibition of ATP-sensitive potassium channels in mouse skeletal muscle by the cardiotonic bipyridine AWD 122-60 was investigated with the patch-clamp technique. In excised patches of the inside-out configuration, internally applied AWD 122-60 (10^–6–10^–3 mol/1) reversibly reduced the open-probability of single ATP-sensitive potassium channels. The agent shortened the periods of channel activity but did not affect the channel conductance. At positive membrane potentials channel inhibition by AWD 122-60 was more pronounced than at negative potentials, the drug concentrations producing 50% channel inhibition were 11 mol/l at + 40 mV and 29 mol/l at –40 mV. The Hill coefficients of the concentration-response curves were in the range between 0.5 and 0.6 for both potentials. milrinone (10^–4 mol/I), had no effects on ATP-sensitive potassium channels in skeletal muscle. potassium channels in heart muscle by AWD 122-60 are discussed. Send offprint requests to B. Neumcke at the above address