A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

Mechanosensitive Properties of BK Channels from Embryonic Rat Neuroepithelium

The mechanosensitive properties of large-conductance Ca²⁺-activated K⁺ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed in on-cell patches. This mechanosensitivity was not mediated by Ca²⁺ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment. Received: 1 February 1996/Revised: 25 March 1996     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

Chemical soil conditions in pristineNothofagus forests of New Zealand as compared to German forests

In many German forest soils low base saturation of CEC in deeper soil layers was reported and acidic deposition is seen as the major cause of these findings. To test this hypothesis we sampled 5 New Zealand forest soils from pristine beech (Nothofagus fusca, N. menziesii, N. solandri) sites under climatic and geological conditions comparable to higher elevations in Germany. The soils developed from granite and greywacke. Soil samples were analyzed for pH and the exchangeable cations were extracted with 1M NH₄Cl. The base saturation of all soil profiles was very low, even in deeper layers and was thus similar to the patterns found in many German forest soils. The pH was generally higher in the New Zealand soils as compared to Germany. The reason for the depletion of base cations in deeper soil layers of New Zealand forest soils is most likely the leaching of base cations with HCO₃ ^- resulting from the dissociation of carbonic acid in connection with high amounts of seepage. Thus, under high rainfall conditions, the low base saturation found in deeper layers of forest soils cannot exclusively be attributed to the effects of acidic depositions and land use. ei]Section editor: R F Huettl     

Caesium accumulation by microorganisms: uptake mechanisms,cation competition,compartmentalization and toxicity

Summary The continued release of caesium radioisotopes into the environment has led to a resurgence of interest in microbe-Cs interactions. Caesium exists almost exclusively as the monovalent cation Cs⁺ in the natural environment. Although Cs⁺ is a weak Lewis acid that exhibits a low tendency to form complexes with ligands, its chemical similarity to the biologically essential alkali cation K⁺ facilitates high levels of metabolism-dependent intracellular accumulation. Microbial Cs⁺ (K⁺) uptake is generally mediated by monovalent cation transport systems located on the plasma membrane. These differe widely in specificity for alkali cations and consequently microorganisms display large differences in their ability to accumulate Cs⁺; Cs⁺ appears to have an equal or greater affinity than K⁺ for transport in certain microorganisms. Microbial Cs⁺ accumulation is markedly influenced by the presence of external cations, e.g. K⁺, Na⁺, NH₄ ⁺ and H⁺, and is generally accompanied by an approximate stoichiometric exchange for intracellular K⁺. However, stimulation of growth of K⁺-starved microbial cultures by Cs⁺ is limited and its has been proposed that it is not the presence of Cs⁺ in cells that is growth inhibitory but rather the resulting loss of K⁺. Increased microbial tolerance to Cs⁺ may result from sequestration of Cs⁺ in vacuoles or changes in the activity and/or specificity of transport systems mediating Cs⁺ uptake. The precise intracellular target(s) for Cs⁺-induced toxicity has yet to be clearly defined, although certain internal structures, e.g. ribosomes, become unstable in the presence of Cs⁺ and Cs⁺ is known to substitute poorly for K⁺ in the activation of many K⁺-requiring enzymes.     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Temperature dependence of multiple high voltage activated Ca2+ channels in chick sensory neurones

The temperature dependence of high voltage activated Ca²⁺ channels has been investigated in cultured dorsal root ganglion neurones from chick embryos, using the cell-attached patch-clamp technique. The dihydropyridine sensitive L-type Ca²⁺ channel had a conductance of 23 pS, with 110 mM Ba²⁺ as charge carrier and in the presence of 3 M Bay K 8644. When the temperature was raised from 15 to 30 °C, the unitary channel current amplitude increased, with Q₁₀ value equal to 1.4. The rising phase of the averaged single-channel current became faster, with Q₁₀ value 2.7, whereas the decay phase showed a lower temperature sensitivity. Channel open probability decreased according to an exponential distribution of open and closed times. A second type of Ca²⁺ channel was identified, which was DHP-insensitive and had a lower conductance with a mean value equal to 13 pS. For the current amplitude, the Q₁₀ value was 1.3. Both activation and inactivation kinetics were strongly accelerated by an increase in temperature. The corresponding time constants gave Q₁₀ values equal to 5.9 for activation, and 2.0 for inactivation. Peak channel open probability was highly sensitive to a change in temperature, with a Q₁₀ value of 1.6. Finally, in -conotoxin GVIA pre-treated neurones, a non-inactivating DHP-insensitive Ca²⁺ channel with the lowest unitary conductance (10 pS) and a much lower temperature dependence was recorded. Single-channel current was increased by heating, with Q₁₀ value 1.3, whereas the channel kinetics were almost unaffected by temperature. Our data are consistent with the assumption that the different temperature dependence of the Ca²⁺ channel behaviours may be explained by separate gating processes of three types of Ca²⁺ channels.     

Free amino compounds and cell volume regulation in erythrocytes from different marine fish species under hypoosmotic conditions: the role of a taurine channel

Erythrocytes from the marine fish species ballan wrasse (Labrus berggylta Ascanius), bullhead (Myoxocephalus scorpius L.), cod (Gadus morhua L.), dab (Limanda limanda L.), eelpout (Zoarces viviparus L.), flounder (Platichthys flesus L.), lumpfish (Cyclopterus lumpus L.), plaice (Pleuronectes platessa L.), sole (Solea solea L.) and turbot (Scophthalmus maxima L.) possess the capacity for regulatory volume decrease. This property was demonstrated in vitro by reduction of the osmolality of the incubation medium from 330 to 255 mosmol·kg^-1. During the 4-h incubation period only the lumpfish cells completely regained the original volume. Twenty-seven free amino compounds were present in detectable amounts in the erythrocytes. At normal osmolality the taurine content was between 14.0 mol·g dry weight^-1 (lumpfish) and 147.4 mol·g dry weight^-1 (sole). Except in the bullhead, taurine was the quantitatively dominating amino compound in the erythrocytes from all species, and accounted for betwee 23% (lumpfish) and 88% (sole) of the total content of free amino compounds. In each species the regulatory volume decrease was associated with a reduction in the cellular content of taurine. Taurine contributed to between 6% (lumpfish) and 36% (flounder) of the cell shrinkage. There was a significant negative correlation, however, between the cellular concentration of taurine at normal osmolality and the capacity of the cells for regulatory volume decrease. Gamma-aminobutyric acid and/or glycine also contributed to the process of volume regulation, but to a lesser extent than taurine. The volume regulatory efflux of taurine and -aminobutyric acid were mediated by taurine channels. It is suggested that these channels also mediated the reduction in the cellular contents of glycine.Abbreviations cmp counts per minute - dw dry weight - GABA -amino-n-butyric acid - MW molecular weight - SD standard deviation - ww wet weight