[Ca2+]i-induced augmentation of the inward rectifier potassium current (IK1) in canine and human ventricular myocardium

The inward rectifier K⁺ current (I_K1) plays an important role in terminal repolarization and stabilization of the resting potential in cardiac cells. Although I_K1 was shown to be sensitive to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), the nature of this Ca²⁺ sensitivity—in spite of its deep influence on action potential morphology—is controversial. Therefore, we aimed to investigate the effects of a nonadrenergic rise in [Ca²⁺]_i on the amplitude of I_K1 in canine and human ventricular myocardium and its consequences on cardiac repolarization. I_K1, defined as the current inhibited by 10 μM Ba²⁺, was significantly increased in isolated canine myocytes following a steady rise in [Ca²⁺]_i. Enhanced I_K1 was also observed when [Ca²⁺]_i was not buffered by ethylene glycol tetraacetic acid, and [Ca²⁺]_I transients were generated. This [Ca²⁺]_i-dependent augmentation of I_K1 was largely attenuated after inhibition of CaMKII by 1 μM KN-93. Elevation of [Ca²⁺]_o in multicellular canine and human ventricular preparations resulted in shortening of action potentials and acceleration of terminal repolarization. High [Ca²⁺]_o enhanced the action potential lengthening effect of the Ba²⁺-induced I_K1 blockade and attenuated the prolongation of action potentials following a 0.3-μM dofetilide-induced I_Kr blockade. Blockade of I_Ks by 0.5 μM HMR-1556 had no significant effect on APD₉₀ in either 2 mM or 4 mM [Ca²⁺]_o. It is concluded that high [Ca²⁺]_i leads to augmentation of the Ba²⁺-sensitive current in dogs and humans, regardless of the mechanism of the increase. This effect seems to be at least partially mediated by a CaMKII-dependent pathway and may provide an effective endogenous defense against cardiac arrhythmias induced by Ca²⁺ overload.     

Hydrogen peroxide Modulates Ca2+-Activation of Single permeabilized Fibres from Fast- and Slow-twitch Skeletal Muscles of Rats

We examined the effects of redox modulation on single membrane-permeabilized fibre segments from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of adult rats to determine whether the contractile apparatus was the redox target responsible for the increased contractility of muscles exposed to low concentrations of H₂O₂. The effects of H₂O₂ on maximum Ca²⁺-activated force were dose-dependent with 30 min exposure to 5 mM H₂O₂ causing a progressive decrease by 22 ± 3 and 13 ± 2% in soleus and EDL permeabilized muscle fibres, respectively. Lower concentrations of exogenous H₂O₂ (100 M and 1 mM) had no effect on maximum Ca²⁺-activated force. Subsequent exposure to the reductant dithiothreitol (DTT, 10 mM, 10 min) fully reversed the H₂O₂-induced depression of force in EDL, but not in soleus muscle fibres. Incubation with DTT alone for 10 min did not alter Ca²⁺-activated force in either soleus or EDL muscle fibres. The sensitivity of the contractile filaments to Ca²⁺ (pCa₅₀) was not altered by exposure to any concentration of exogenous H₂O₂. However, all concentrations of H₂O₂ diminished the Hill coefficient in permeabilized fibres from the EDL muscle, indicating that the cooperativity of Ca²⁺ binding to troponin is altered. H₂O₂ (5 mM) did not affect rigor force, which indicates that the number of crossbridges participating in contraction was not reduced. In conclusion, H₂O₂ may reduce the maximum Ca²⁺ activated force production in skinned muscle fibres by decreasing the force per crossbridge.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

 We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (K_ATP channel) of guinea-pig ventricular myocytes. Single K_ATP channel currents were recorded from inside-out patches in the presence of symmetrical K⁺ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (·O₂ ^-) and hydrogen peroxide (H₂O₂). Exposure of the patch membrane to ·O₂ ^- and H₂O₂ increased the opening of K_ATP channels, but this activation was prevented by adding 1 μM glibenclamide to the bath solution. In the presence of ferric iron (Fe³⁺: 0.1 mM), the same procedure produced hydroxyl radicals (·OH) via the iron-catalysed Haber-Weiss reaction. ·OH also activated K_ATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 μM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on K_ATP channels is different and that ·OH is more potent than ·O₂ ^-/H₂O₂ in activating K_ATP channels in the heart. Received: 14 July 1998 / Received after revision: 5 September 1998 / Accepted: 14 September 1998     

Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells

Recent studies have demonstrated the importance of large-conductance Ca²⁺-activated K⁺ (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K⁺ symmetrical solution (containing 140 mM KCl and ~300 nM free Ca²⁺) showed single-channel conductance of 215–220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P _o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca²⁺] from ~300 nM to Ca²⁺-free decreased the P _o by ~85 %, whereas elevation to ~800 nM increased P _o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P _o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca²⁺] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.     

Detection of dihydropyridine- and voltage-sensitive intracellular Ca2+ signals in normal human parathyroid cells

We recently showed dihydropyridine- and voltage-sensitive Ca²⁺ entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. To determine whether normal parathyroid cells have a similar extracellular Ca²⁺ entry system, cells were isolated from normal (non-hyperplastic) human parathyroid glands. Fluorescence signals related to the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_I) were examined in these cells. Cells loaded with fluo-3/AM showed a transient increase in fluorescence (Ca²⁺ transient) following a 10-s exposure to a 150 mM K⁺ solution in the presence of millimolar concentrations of external Ca²⁺. The Ca²⁺ transient was reduced by dihydropyridine antagonists or 0.5 mM Cd²⁺, but enhanced by FPL-64176, an L-type Ca²⁺-channel agonist. Ca²⁺ transients induced by the 10-s exposure to 3.0 mM extracellular Ca²⁺ ([Ca²⁺]_o) were also inhibited by dihydropyridine antagonists or 0.5 mM Cd²⁺. These results provide the first evidence that normal human parathyroid cells express a dihydropyridine-sensitive Ca²⁺ entry system that may be involved in the [Ca²⁺]_o-induced change in [Ca²⁺]_I. This system might provide a compensatory pathway for negative feedback regulation of parathyroid hormone secretion under physiological conditions.     

Calcium and calcimimetics regulate paracellular Na+ transport in the thin ascending limb of Henle’s loop in mouse kidney

The effect of Ca²⁺ and calcimimetics on NaCl transport was investigated in the in vitro isolated microperfused mouse thin ascending limb of Henle’s loop. In the presence of a transmural NaCl gradient, the transepithelial diffusional potential was 13.7?±?0.4 mV (n?=?17). When the Ca²⁺ in the bath was increased from 1.5 to 4.5 mM at 37°C, the relative permeability of Na⁺ to Cl^? (P _Na /P _Cl) estimated from the diffusional voltage deflection due to the transepithelial NaCl gradient (V _d) changed from 0.371?±?0.017 to 0.341?±?0.015 (n?=?10, P?<?0.0001). When the Ca²⁺ in the lumen was increased from 1.5 to 4.5 mM, the P _Na /P _Cl decreased from 0.349?±?0.013 to 0.330?±?0.013 (n?=?5, P?<?0.002). The addition of 0.1 mM neomycin and 0.2 mM gentamicin to the bath or lumen also decreased the P _Na /P _Cl. The same effect on P _Na /P _Cl of Ca²⁺ and calcimimetics occurred in ClC-K1 (kidney-specific chloride channel) knockout mice. The addition of 300 μg/ml protamine to the bath strongly inhibited changes to P _Na /P _Cl induced by basolateral Ca²⁺. These data indicate that ambient Ca²⁺ and calcimimetics inhibit Na⁺ transport in the thin ascending limb, which is known to occur via the paracellular shunt pathway. Our observations strongly suggest that Ca²⁺ is involved in the regulation of paracellular Na⁺ permeability in the thin ascending limbs.     

H2O2-stimulated Ca2+ influx via TRPM2 is not the sole determinant of subsequent cell death

Activation of transient receptor potential melastatin 2 (TRPM2), a non-selective, Ca²⁺-permeable cation channel, is implicated in cell death. Channel opening is stimulated by oxidative stress, a feature of numerous disease states. The wide expression profile of TRPM2 renders it a potentially significant therapeutic target in a variety of pathological settings including cardiovascular and neurodegenerative diseases. HEK293 cells transfected with human TRPM2 (HEK293/hTRPM2) were more vulnerable to H₂O₂-mediated cell death than untransfected controls in which H₂O₂-stimulated Ca²⁺ influx was absent. Flufenamic acid partially reduced Ca²⁺ influx in response to H₂O₂ but had no effect on viability. N-(p-Amylcinnamoyl) anthranilic acid substantially attenuated Ca²⁺ influx but did not alter viability. Poly(adenosine diphosphate ribose) polymerase inhibitors (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone and nicotinamide) reduced Ca²⁺ influx and provided a degree of protection but also had some protective effects in untransfected controls. These data suggest H₂O₂ triggers cell death in HEK293/hTRPM2 cells by a mechanism that is in part Ca²⁺ independent, as blockade of channel opening (evidenced by suppression of Ca²⁺ influx) did not correlate well with protection from cell death. Determining the underlying mechanisms of TRPM2 activation is pertinent in elucidating the relevance of this channel as a therapeutic target in neurodegenerative diseases and other pathologies associated with Ca²⁺ dysregulation and oxidative stress.     

A study of the mechanisms of excitation–contraction coupling in frog skeletal muscle based on measurements of [Ca<Superscript>2+</Superscript>] transients inside the sarcoplasmic reticulum

[Ca²⁺] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca²⁺ release of extrinsic Ca²⁺ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca²⁺ buffer is added to the myoplasm, part of the released Ca²⁺ binds to it, reducing the Ca²⁺ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca²⁺ released. Monitoring release by measuring [Ca²⁺] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca²⁺] in the SR ([Ca²⁺]_SR) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca²⁺]_SR on the Ca²⁺ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca²⁺] ([Ca²⁺]_myo) transients were simultaneously recorded with Rhod2. The Ca²⁺ release flux was calculated from [Ca²⁺]_myo and divided by [Ca²⁺]_SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026?±?0.005 ms^?1 at resting [Ca²⁺]_SR?=?372?±?5 μM to 0.021?±?0.004 ms^?1 at resting [Ca²⁺]_SR?=?120?±?16 μM (n?=?4, p?=?0.03). The time averaged permeability was not significantly changed (0.009?±?0.003 and 0.010?±?0.003 ms^?1, at the higher and lower [Ca²⁺]_SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca²⁺]_SR (Δ[Ca²⁺]_SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca²⁺]_SR?=?30.2?±?3.5 μM from resting [Ca²⁺]_SR?=?88.8?±?13.6 μM, n?=?5) than in 40 mM EGTA (Δ[Ca²⁺]_SR?=?72.2?±?10.4 μM from resting [Ca²⁺]_SR?=?98.2?±?15.6 μM, n?=?4) suggesting that a Ca²⁺ activated component of release was suppressed by BAPTA.     

H2O2-mediated modulation of cytosolic signaling and organelle function in rat hippocampus

Reactive oxygen species (ROS) released from (dys-)functioning mitochondria contribute to normal and pathophysiological cellular signaling by modulating cytosolic redox state and redox-sensitive proteins. To identify putative redox targets involved in such signaling, we exposed hippocampal neurons to hydrogen peroxide (H₂O₂). Redox-sensitive dyes indicated that externally applied H₂O₂ may oxidize intracellular targets in cell cultures and acute tissue slices. In cultured neurons, H₂O₂ (EC₅₀ 118 μM) induced an intracellular Ca²⁺ rise which could still be evoked upon Ca²⁺ withdrawal and mitochondrial uncoupling. It was, however, antagonized by thapsigargin, dantrolene, 2-aminoethoxydiphenyl borate, and high levels of ryanodine, which identifies the endoplasmic reticulum (ER) as the intracellular Ca²⁺ store involved. Intracellular accumulation of endogenously generated H₂O₂—provoked by inhibiting glutathione peroxidase—also released Ca²⁺ from the ER, as did extracellular generation of superoxide. Phospholipase C (PLC)-mediated metabotropic signaling was depressed in the presence of H₂O₂, but cytosolic cyclic adenosine-5′-monophosphate (cAMP) levels were not affected. H₂O₂ (0.2–5 mM) moderately depolarized mitochondria, halted their intracellular trafficking in a Ca²⁺- and cAMP-independent manner, and directly oxidized cellular nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂). In part, the mitochondrial depolarization reflects uptake of Ca²⁺ previously released from the ER. We conclude that H₂O₂ releases Ca²⁺ from the ER via both ryanodine and inositol trisphosphate receptors. Mitochondrial function is not markedly impaired even by millimolar concentrations of H₂O₂. Such modulation of Ca²⁺ signaling and organelle interaction by ROS affects the efficacy of PLC-mediated metabotropic signaling and may contribute to the adjustment of neuronal function to redox conditions and metabolic supply. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Florian J. Gerich and Frank Funke contributed evenly to this study.     

Electrophysiological characterization of store-operated and agonist-induced Ca2+ entry pathways in endothelial cells

In endothelial cells, agonist-induced Ca²⁺ entry takes place via the store-operated Ca²⁺ entry pathway and/or via channel(s) gated by second messengers. As cell stimulation leads to both a partial Ca²⁺ store depletion as well as the production of second messengers, these two pathways are problematic to distinguish. We showed that passive endoplasmic reticulum (ER) depletion by thapsigargin or cell stimulation by histamine activated a similar Ca²⁺-release-activated Ca²⁺ current (CRAC)-like current when 10 mM Ba²⁺/2 mM Ca²⁺ was present in the extracellular solution. Importantly, during voltage clamp recordings, histamine stimulation largely depleted the ER Ca²⁺ store, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba²⁺ medium. On the contrary, in the presence of 10 mM Ca²⁺, the ER Ca²⁺ content remained elevated, and histamine induced an outward rectifying current that was inhibited by Ni²⁺ and KB-R7943, two blockers of the Na⁺/Ca²⁺ exchanger (NCX). Both blockers also reduced histamine-induced cytosolic Ca²⁺ elevation. In addition, removing extracellular Na⁺ increased the current amplitude which is in line with a current supported by the NCX. These data are consistent with the involvement of the NCX working in reverse mode (Na⁺ out/Ca²⁺ in) during agonist-induced Ca²⁺ entry in endothelial cells.     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

External divalent cations increase anion–cation permeability ratio in glycine receptor channels

The functional role of ligand-gated ion channels in the central nervous system depends on their relative anion–cation permeability. Using standard whole-cell patch clamp measurements and NaCl dilution potential measurements, we explored the effect of external divalent ions on anion–cation selectivity in α1-homomeric wild-type glycine receptor channels. We show that increasing external Ca²⁺ from 0 to 4 mM resulted in a sigmoidal increase in anion–cation permeability by 37%, reaching a maximum above about 2 mM. Our accurate quantification of this effect required rigorous correction for liquid junction potentials (LJPs) using ion activities, and allowing for an initial offset potential. Failure to do this results in a considerable overestimation of the Ca²⁺-induced increase in anion–cation permeability by almost three-fold at 4 mM external Ca²⁺. Calculations of LJPs (using activities)_ were validated by precise agreement with direct experimental measurements. External SO ₄ ^2? was found to decrease anion–cation permeability. Single-channel conductance measurements indicated that external Ca²⁺ both decreased Na⁺ permeability and increased Cl^? permeability. There was no evidence of Ca²⁺ changing channel pore diameter. Theoretical modeling indicates that the effect is not surface charge related. Rather, we propose that, under dilution conditions, the presence of an impermeant Ca²⁺ ion in the channel pore region just external to the selectivity filter tends to electrostatically retard outward movement of Na⁺ ions and to enhance movement of Cl^? ions down their energy gradients.     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

Characterization of secophalloidin-induced force loss in cardiac myofibrils

Secophalloidin (SPH) is known to cause in cardiac myofibrils force without Ca²⁺ (half-maximal effect ~2 mM) followed by irreversible loss of Ca²⁺-activated force. At maximal Ca²⁺ activation, SPH increases force (half-maximal effect < 0.1 mM). We found that SPH at low concentration (0.5 mM) did not cause either force activation or force loss at pCa 8.7, but both of these effects did occur when force was activated by Ca²⁺. The force loss was prevented when SPH was applied during rigor or in the presence of 2,3-butanedione monoxime (85 mM). Furthermore, studying muscle in which the force was previously reduced by SPH (up to 50%) did not reveal significant changes in Ca²⁺ sensitivity and cooperativity of Ca²⁺ activation or qualitative alterations in SPH-induced changes in Ca²⁺-activated contraction. Data suggest that the force loss is mediated by cycling cross-bridges, and might reflect a reduction in force generated by individual cross-bridges.     

Regulation of fast skeletal muscle activity by SERCA1 vicinal-cysteines

During prolonged skeletal muscle contractions free radicals are produced that may lead to fatigue. Vicinal cysteines, known as a Vicinal-thiol groups react preferentially among them depending on redox potential. Therefore, we examined the role of VT groups on the activity and conformational changes of sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA1) from rabbit skeletal muscle isolated SR, by selective oxidation–reduction of VT-groups. After Ca²⁺ is released from the SR to start contraction, SERCA1 pumps this cytosolic Ca²⁺ back to the SR leading to muscle relaxation. Phenylarsine oxide (PAO) reacts selectively with VT-proteins forming dithioarsines, which are stable but exchanges rapidly with 2,3-dimercaptopropanol (BAL). When 0.1 mM PAO is added to isolated SR, 60 and 67% inhibition of SERCA1 hydrolytic and Ca²⁺ uptake activities, respectively is observed. ATPase activity was fully reversible with 1 mM BAL. The SERCA1 thermal inactivation determined from isolated SR from muscle at rest showed a single transition for inactivation (T _i) at 49 ± 1.12°C. In the presence of 0.1 mM PAO, SERCA1 shows two transitions at T _i 34 ±0.9°C and at 27 ±1.2°C. The thermal denaturation profile of SERCA1 from muscle at rest, showed two transitions at T _m = 51.5 ±1.3°C and 63 ±1.02°C related to nucleotide and Ca²⁺ binding domains, respectively. Whereas isolated SR obtained after a protocol of tetanic stimulation to produce muscle fatigue, showed three transitions in the SERCA1 denaturation profile similar to the effect of PAO, addition of 1 mM BAL reverted the effect of fatigue on SERCA1 denaturation profile. These results indicate a mechanism relating VT group’s oxidation to muscle fatigue.     

Characterization of distinct single-channel properties of Ca2+ inward currents in mitochondria

Previous studies have demonstrated several molecularly distinct players involved in mitochondrial Ca²⁺ uptake. In the present study, electrophysiological recordings on mitoplasts that were isolated from HeLa cells were performed in order to biophysically and pharmacologically characterize Ca²⁺ currents across the inner mitochondrial membrane. In mitoplast-attached configuration with 105 mM Ca²⁺ as a charge carrier, three distinct channel conductances of 11, 23, and 80 pS were observed. All types of mitochondrial currents were voltage-dependent and essentially depended on the presence of Ca²⁺ in the pipette. The 23 pS channel exhibited burst kinetics. Though all channels were sensitive to ruthenium red, their sensitivity was different. The 11 and 23 pS channels exhibited a lower sensitivity to ruthenium red than the 80 pS channel. The activities of all channels persisted in the presence of cylosporin A, CGP 37187, various K⁺-channel inhibitors, and Cl^? channel blockers disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate and niflumic acid. Collectively, our data identified multiple conductances of Ca²⁺ currents in mitoplasts isolated from HeLa cells, thus challenging the dogma of only one unique mitochondrial Ca²⁺ uniporter.     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Calcium dependent gating of thel-glutamate activated,excitatory synaptic channel on crayfish muscle

Excitatory, glutamate-activated single channel currents were measured in outside-out patches of crayfish muscle. The open time of single channel openings, and the durations and rates of bursts were evaluated. These kinetic parameters were not appreciably affected by replacement of extracellular Na⁺ by Li⁺ or choline. Changes in extracellular Ca²⁺ concentration Ca_o also did not influence the duration of single openings. However the mean burst duration decreased for Ca_o<13.5 mM and the rate of bursts declined with a power of almost 2 in low Ca_o. At Ca_o<1 mM practically no channel openings were observed in presence of glutamate. In order to exclude more rapid desensitization of the glutamate receptors in low Ca_o as the cause of disappearance of channel openings, glutamate was applied in short pulses with a liquid-filament switch. In 0 Ca_o also a glutamate pulse did not trigger channel openings. In presence of 13.5 mM Ca_o, the inorganic Ca-channel blockers La³⁺ and Cd²⁺ diminished the duration and rate of bursts of channel openings in a similar manner as low Ca_o. The effects of low Ca_o and of Cd²⁺ were tested also on quantal postsynaptic currents, EPSCs, which were recorded through a perfused macro-patch-clamp electrode. At 1.4 mM Ca_o in the perfused electrode tip, spontaneous EPSCs were reduced at least by a factor of 4, and elicited EPSCs by a factor of 16. Application of Cd²⁺ had similarly strong effects on the EPSCs. Also the decay of EPSCs was shortened substantially in 1.4 mM Ca_o or 5 mM Cd²⁺.The inhibitory Cl^–-channel of crayfish muscle, activated by glutamate or GABA, also was studied in outside-out patches. The openings of this channel persisted in 0 Ca_o solutions; the block of channel openings in low Ca_o thus is a specific property of the excitatory channel. The action of Ca_o on the excitatory channel may be described as that of a cofactor to glutamate. A possible reaction scheme is proposed.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 220     

Interleukin-1β suppresses activity of an inwardly rectifying K+ channel in human renal proximal tubule cells

We investigated the effect of interleukin-1β (IL-1β) on activity of an inwardly rectifying K⁺ channel in cultured human proximal tubule cells (RPTECs), using the patch-clamp technique and Fura-2 Ca²⁺ imaging. IL-1β (15 pg/ml) acutely reduced K⁺ channel activity in cell-attached patches. This effect was blocked by the IL-1 receptor antagonist (20 ng/ml), an inhibitor of phospholipase C, neomycin (300 μM), and an inhibitor of protein kinase C (PKC), GF109203X (500 nM). The Fura-2 Ca²⁺ imaging revealed that IL-1β increased intracellular Ca²⁺ concentration even after removal of extracellular Ca²⁺, which was blocked by an inhibitor of inositol 1,4,5-trisphosphate receptors, 2-aminoethoxydiphenyl borate (2-APB, 1 μM). Moreover, IL-1β suppressed channel activity in the presence of 2-APB without extracellular Ca²⁺. These results suggest that IL-1β suppresses K⁺ channel activity in RPTECs through binding to its specific receptor and activation of the PKC pathway even though intracellular Ca²⁺ does not increase.