Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

Distinct responses to ozone of abaxial and adaxial stomata in three Euramerican poplar genotypes

Ozone induces stomatal sluggishness, which impacts photosynthesis and transpiration. Stomatal responses to variation of environmental parameters are slowed and reduced by ozone and may be linked to difference of ozone sensitivity. Here we determine the ozone effects on stomatal conductance of each leaf surface. Potential causes of this sluggish movement, such as ultrastructural or ionic fluxes modification, were studied independently on both leaf surfaces of three Euramerican poplar genotypes differing in ozone sensitivity and in stomatal behaviour. The element contents in guard cells were linked to the gene expression of ion channels and transporters involved in stomatal movements, directly in microdissected stomata. In response to ozone, we found a decrease in the stomatal conductance of the leaf adaxial surface correlated with high calcium content in guard cells compared with a slight decrease on the abaxial surface. No ultrastructural modifications of stomata were shown except an increase in the number of mitochondria. The expression of vacuolar H⁺/Ca²⁺‐antiports (CAX1 and CAX3 homologs), β‐carbonic anhydrases (βCA1 and βCA4) and proton H⁺‐ATPase (AHA11) genes was strongly decreased under ozone treatment. The sensitive genotype characterized by constitutive slow stomatal response was also characterized by constitutive low expression of genes encoding vacuolar H⁺/Ca²⁺‐antiports.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.     

Existence,properties, and functional expression of “Maxi-K”-type,Ca2+-activated K+ channels in short-term cultured hepatocytes

A large-conductance, Ca²⁺-activated K⁺ channel was identified and characterized in embryonic chick hepatocytes using the patch-electrode voltage-clamp technique. The channel conductance was 213 pS in excised patches bathed in symmetrical 145 mM KCl and 1 mM Ca²⁺. Current-voltage relationships were linear with high K⁺ on both sides of the membrane but showed constant field rectification as the K⁺ gradient was increased. The reversal potential shifted 58 mV per 10-fold change in the ratio of external to internal K⁺. Channel openings occurred at potentials higher than +50 mV in cell-attached patches. The open probability × voltage relationship shifted to more negative potentials in excised, inside-out patches exposed to a solution containing high Ca²⁺. The voltage sensitivity of the channel was not significantly affected by changes in internal Ca²⁺ concentration. Conversely, channel gating, reflected in the half-activation potential, shifted 118 mV per 10-fold change in internal Ca²⁺ at concentrations less than ∼2 μM, although at higher Ca²⁺, this parameter was Ca²⁺ insensitive. Channel open probability in cell-attached patches increased significantly following exposure of the cells to either the Ca²⁺ ionophore A-23187 or L-alanine, a cell-volume modulator. Channel density increased with time spent in culture from no observations in 10-hr cells, through 13 and 80% of patches in 24-and 48-hr cultured cells, respectively. The implications of delayed functional expression for ion channel studies in acutely dissociated cells is discussed. J. Cell. Physiol. 171:87–94, 1997. © 1997 Wiley-Liss, Inc.     

Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca²⁺ uptake and H⁺ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake. Conversely, ruthenium red blocks Ca²⁺ uptake and H⁺ production but does not prevent dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake by mitochondria. These results suggest that mitochondrial Ca²⁺ uptake and release exist as two independent pathways. The efflux of Ca²⁺ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca²⁺ uptake carrier.     

Mitogen-activated protein kinase 6 controls root growth in Arabidopsis by modulating Ca-based Na flux in root cell under salt stress

Little is known about the role of mitogen-activated protein kinase 6 (MPK6) in Na⁺ toxicity and inhibition of root growth in Arabidopsis under NaCl stress. In this study, we found that root elongation in seedlings of the loss-of-function mutants mpk6-2 and mpk6-3 was less sensitive to NaCl or Na-glutamate, but not to KCl or mannitol, as compared with that of wild-type (WT) seedlings. The less sensitive characteristic was eliminated by adding the Ca²⁺ chelator EGTA or the Ca²⁺ channel inhibitor LaCl₃, but not the Ca²⁺ ionophore A23187. This suggested that the tolerance of mpk6 to Na⁺ toxicity was Ca²⁺-dependent. We measured plasma membrane (PM) Na⁺-conducted currents (NCCs) in root cells. Increased concentrations of NaCl increased the inward NCCs while decreased the outward NCCs in WT root cells, attended by a positive shift in membrane potential. In mpk6 root cells, NaCl significantly increased outward but not inward NCCs, accompanied by a negative shift in membrane potential. That is, mpk6 decreased NaCl-induced the Na⁺ accumulation by modifying PM Na⁺ flux in root cells. Observations of aequorin luminescence revealed a NaCl-induced increase of cytosolic Ca²⁺ in mpk6 root cells, resulting from PM Ca²⁺ influx. An increase of cytosolic Ca²⁺ was required to alleviate the NaCl-increased Na⁺ content and Na⁺/K⁺ ratio in mpk6 roots. Together, these results show that mpk6 accumulated less Na⁺ in response to NaCl because of the increased cytosolic Ca²⁺ level in root cells; thus, its root elongation was less inhibited than that of WT by NaCl.     

Tetrodotoxin-resistant sodium channels

Summary 1. Tetrodotoxin (TTX) has been widely used as a chemical tool for blocking Na⁺ channels. However, reports are accumulating that some Na⁺ channels are resistant to TTX in various tissues and in different animal species. Studying the sensitivity of Na⁺ channels to TTX may provide us with an insight into the evolution of Na⁺ channels.2. Na⁺ channels present in TTX-carrying animals such as pufferfish and some types of shellfish, frogs, salamanders, octopuses, etc., are resistant to TTX.3. Denervation converts TTX-sensitive Na⁺ channels to TTX-resistant ones in skeletal muscle cells, i.e., reverting-back phenomenon. Also, undifferentiated skeletal muscle cells contain TTX-resistant Na⁺ channels. Cardiac muscle cells and some types of smooth muscle cells are considerably insensitive to TTX.4. TTX-resistant Na⁺ channels have been found in cell bodies of many peripheral nervous system (PNS) neurons in both immature and mature animals. However, TTX-resistant Na⁺ channels have been reported in only a few types of central nervous system (CNS). Axons of PNS and CNS neurons are sensitive to TTX. However, some glial cells have TTX-resistant Na⁺ channels.5. Properties of TTX-sensitive and TTX-resistant Na⁺ channels are different. Like Ca²⁺ channels, TTX-resistant Na⁺ channels can be blocked by inorganic (Co²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Zn²⁺, La³⁺) and organic (D-600) Ca²⁺ channel blockers. Usually, TTX-resistant Na⁺ channels show smaller single-channel conductance, slower kinetics, and a more positive current-voltage relation than TTX-sensitive ones.6. Molecular aspects of the TTX-resistant Na⁺ channel have been described. The structure of the channel has been revealed, and changing its amino acid(s) alters the sensitivity of the Na⁺ channel to TTX.7. TTX-sensitive Na⁺ channels seem to be used preferentially in differentiated cells and in higher animals instead of TTX-resistant Na⁺ channels for rapid and effective processing of information.8. Possible evolution courses for Na⁺ and Ca²⁺ channels are discussed with regard to ontogenesis and phylogenesis.     

Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca²⁺-activated K⁺ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca²⁺-activated K⁺ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca²⁺ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K _0.5 for Ca²⁺ was raised from 20nm at a potential of 0 mV to 300nm at –40 mV. A decrease in pH at the cytoplasmic face of the K⁺ channel reduced the Ca²⁺ sensitivity dramatically. A loss of the high sensitivity to Ca²⁺ was also observed after incubation with MgCl₂, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca²⁺ sensitivity between studies on K⁺ channels from the same tissue. High affinity inhibition (K _0.5=10nm) by charybdotoxin of the Ca²⁺-activated K⁺ channel from the extracellular face could be lifted by an outward flux of K⁺ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K⁺ channels. The high sensitivity for Ca²⁺ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K⁺ channel in the epithelial cells.Dr. E. Moczydlowsky, Yale University School of Medicine, New Haven, CT, and Dr. Per Stampe, Brandeis University, Waltham, MA, are thanked for introduction to the bilayer technique. Tove Soland is thanked for excellent technical assistance. This work was supported by the Novo Nordisk Foundation, the Carlsberg Foundation, the Danish Medical Research Council, and the Austrian Research Council.     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

MthK is a Ca²⁺-gated K⁺ channel whose activity is inhibited by cytoplasmic H⁺. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H⁺ inhibition and Ca²⁺-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca²⁺] (ranging from nominally 0 to 30 mM) at a given [H⁺], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca²⁺] and open probability (Po), with a mean Hill coefficient (n_H) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor n_H changed significantly as a function of [H⁺] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca²⁺ was not H⁺ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H⁺] raised the EC₅₀ for Ca²⁺ activation by ∼4.7-fold per tenfold increase in [H⁺], displaying a linear relation between log(EC₅₀) and log([H⁺]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H⁺ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca²⁺ binding and channel opening. We can account for the Ca²⁺ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca²⁺ allosterically activates MthK, whereas H⁺ opposes activation by destabilizing the binding of Ca²⁺.     

Temporal switching and cell‐to‐cell variability in Ca2+ release activity in mammalian cells

Genetically identical cells in a uniform external environment can exhibit different phenotypes, which are often masked by conventional measurements that average over cell populations. Although most studies on this topic have used microorganisms, differentiated mammalian cells have rarely been explored. Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca²⁺ increase when ryanodine receptor Ca²⁺ release channels in the endoplasmic reticulum are maximally activated by caffeine. On the other hand, the expression levels of ryanodine receptor showed a unimodal distribution. We showed that the difference in the caffeine sensitivity depends on a critical balance between Ca²⁺ release and Ca²⁺ uptake activities, which is amplified by the regenerative nature of the Ca²⁺ release mechanism. Furthermore, individual cells switched between the caffeine‐sensitive and caffeine‐insensitive states with an average transition time of approximately 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response. These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell‐to‐cell phenotypic variation in mammalian cells.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide

Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na⁺+K⁺)-ATPase and K⁺-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na⁺+K⁺)-ATPase is impeded by either Na⁺ or K⁺ whereas only K⁺ impedes protection by ATP of K⁺-activated phosphatase. On the other hand, Na⁺ or K⁺ slightly protects Ca²⁺-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca²⁺. This effect is half-maximal at less than 1 m Ca²⁺ and does not require ATP, which suggests that sites with high affinity for Ca²⁺ exist in the Ca²⁺-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K⁺ or Na⁺ in the presence of Ca²⁺ parallels that of the Ca²⁺-dependent activities, suggesting that the Ca²⁺-ATPase system possesses sites at which monovalent cations bind to increase its activity.     

Emerging Role of the Calcium-Activated,Small Conductance,SK3 K+ Channel in Distal Tubule Function: Regulation by TRPV4

The Ca²⁺-activated, maxi-K (BK) K⁺ channel, with low Ca²⁺-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K⁺ secretion. In the present study we demonstrate that the Ca²⁺-activated, SK3 (K_Ca2.3) K⁺ channel, with high Ca²⁺-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K⁺ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca²⁺-dependent K⁺ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca²⁺-permeable TRPV4 channel, thereby inducing Ca²⁺ influx and elevating intracellular Ca²⁺ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca²⁺ influx, leading to elevated intracellular Ca²⁺ levels, activates this high Ca²⁺-affinity K⁺ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca²⁺-dependent regulation of membrane potential and K⁺ secretion.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.     

CALCIUM UPTAKE INTO RAT PHEOCHROMOCYTOMA CELLS1

Abstract– The uptake of ⁴⁵Ca²⁺ into cell suspensions prepared from a transplantable rat pheochromocytoma was measured. The uptake of Ca²⁺ into these cells is biphasic; there is a rapid, initial uptake of Ca⁺, followed by a slower uptake that proceeds at a linear rate for at least 10min at 37°C. The uptake of Ca²⁺ is a linear function of the external Ca²⁺ concentration over the range of 0.13-2.5 mm -Ca²⁺ Incubation of the cells in a medium containing 56mm -K⁺ results in a 2-3 fold increase in the uptake of Ca²⁺ into the cells; 56mm -K⁺ increases both phases of Ca²⁺ uptake. The cells apparently lack a mechanism to inactivate this 56 mm -K⁺-induced increase-in Ca²⁺ permeability. Two inhibitors of K⁺ stimulated catecholamine secretion, diphenylhydantoin and verapamil, both inhibit K ⁺-stimulated Ca²⁺ uptake. These results provide a direct demonstration of the stimulus-coupled uptake of Ca²⁺ into chromaffin cells, and provide additional evidence for the correlation of Ca²⁺ uptake with catecholamine secretion by these cells.