Significance of Ca2+ and K+ in Micrasterias Growth and Morphogenesis

Significance of Ca²⁺ and K⁺ for the complex morphogenesis ofMicrasterias, which takes place through multipolar tip growth,was investigated. Studies with different external Ca²⁺ concentrationsand Ca²⁺ channel inhibitors LaCl₃ and verapamil indicate thatCa²⁺ and Ca²⁺ channels are essential in the development, whiletreatments with different K⁺ concentrations and K⁺ channel inhibitorTEA demonstrate that potassium or K⁺ channels are not neededin the process, albeit the existence of K⁺ channels. K⁺ is notneeded even for the regulation of turgor pressure, which wasfound to decrease clearly during cell development. The plasmamembrane ATPase inhibitors diethylstilbesterol (DES) and Na-orthovanadatestop morphogenesis and indicate the importance of ion pumpsin the developmental process. Both supraoptimal, external K⁺and Ca²⁺ cause abundant Ca²⁺ precipitate formation in chloroplasts,which shows that chloroplasts are important in regulation ofcytoplasmic Ca²⁺ metabolism and that K⁺ activates the uptakeof Ca²⁺ through Ca²⁺ channels. (Received June 13, 1995; Accepted September 13, 1996)     

Activation of K+-Channel in Membrane Excitation of Nitella axilliformis

Two processes of the K⁺ channel activation in plasma membraneexcitation are suggested for Nitella axilliformis. One is relatedto the repolarizing process in the action potential and theother to the after-hyperpolarization (AH). Extra- and intracellulartetraethylammonium (TEA⁺) and extracellular Co²⁺ prolonged theaction potential, indicating involvement of K⁺ channel activationin the repolarizing process of the action potential. The following findings showed that AH is caused by K⁺ channelactivation. First, AH was inhibited by extracellular K⁺ andRb⁺ but not by Na⁺ and Li⁺. Second, it was not inhibited byintracellular TEA⁺ but by extracellular TEA⁺. Third, the membraneconductance increased during AH. Generation of AH was dependenton the level of the resting membrane potential [(E_m)_rest] whichis affected by the activity of the electrogenic H⁺ pump. AHwas generated, when (E_m)_rest was more positive than a criticalvalue, which was supposed to be the equilibrium potential forK⁺ across the plasma membrane. Since extracellular Ca²⁺ competed with extracellular TEA⁺ andCo²⁺ in prolonging the action potential, and sometimes in inhibitingAH, Ca²⁺ may be involved in the K⁺ channel activation. (Received June 11, 1983; Accepted September 21, 1983)     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

K+ Channel in the Chora Plasmalemma: Estimations of K+ Channel Density and Single K+ Channel Conductance

The electromotive force E and the conductance G of the Characorallina plasmalemma were measured under voltage clamp conditions.In the depolarized voltage range less negative than –60mV, E changed according to the Nerhst equation for K⁺, and Gincreased with the external K⁺ concentration [K⁺]_o and alsowith the depolarization of the membrane potential. This is attributedto the voltage-dependent opening of the K⁺ channels in the largelydepolarized voltage region. The voltage-dependent increase ofG was due to the increase of the number of open K⁺ channelsper unit area. The density of the total K⁺ channels in the C. corallina plasmalemmawas estimated to be about 6.50/(10 µm)2. The single K⁺channel conductance _K changed with the external [K⁺]_o; it was79.3, 86.1, 105.9, 119.0 pS for external [K⁺]_o of 0.2, 0.5,2.0 and 5.0 mu respectively. (Received May 22, 1986; Accepted August 22, 1986)     

Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes

Two populations,Ca²⁺-dependent(BK_Ca) andCa²⁺-independentK⁺ (BK) channels of largeconductance were identified in inside-out patches of nonlabor and laborfreshly dispersed human pregnant myometrial cells, respectively.Cell-attached recordings from nonlabor myometrial cells frequentlydisplayed BK_Ca channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that oftenexhibited clear, oscillatory activity. In inside-out patch recordings,Ba²⁺ (2-10 mM),4-aminopyridine (0.1-1 mM), andShaker B inactivating peptide("ball peptide") blocked theBK_Ca channel but were much lesseffective on BK channels. Application of tetraethylammonium toinside-out membrane patches reduced unitary current amplitude ofBK_Ca and BK channels, withdissociation constants of 46 mM and 53 µM, respectively.Tetraethylammonium applied to outside-out patches decreased the unitaryconductance of BK_Ca and BKchannels, with dissociation constants of 423 and 395 µM,respectively. These results demonstrate that the properties of humanmyometrial large-conductance K⁺channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.

     

Measurement of Changes in Cytosolic Ca2+ in Arabidopsis Guard Cells and Mesophyll Cells in Response to Blue Light

Phototropins (phot1 and phot2) are blue light (BL) receptorsthat mediate responses including phototropism, chloroplast movementand stomatal opening, and increased cytosolic Ca²⁺. BL absorbedby phototropins activates plasma membrane H⁺-ATPase in guardcells, resulting in membrane hyperpolarization, and drives K⁺uptake and stomatal opening. However, it is unclear whetherthe phototropin-mediated Ca²⁺ increase activates the H⁺-ATPase.Here, we determined cytosolic Ca²⁺ concentrations in guard cellprotoplasts (GCPs) from Arabidopsis transformed with aequorin.Cytosolic Ca²⁺ increased rapidly in response to BL in GCPs fromboth the wild type and phot1 phot2 double mutants, but was mostlysuppressed by an inhibitor of photosynthetic electron flow (DCMU).With depleted external K⁺, we observed another slower Ca²⁺ increase,which was phototropin- dependent. Fusicoccin, a H⁺-ATPase activator,mimicked the effect of BL. The slow Ca²⁺ increase thus appearsto result from membrane hyperpolarization. The slow Ca²⁺ increasewas suppressed by external K⁺ and was restored by blockers ofinward-rectifying K⁺ channels, CsCl and tetraethylammonium,suggesting the preferential uptake of K⁺ over Ca²⁺. Such efficientK⁺ uptake in response to BL was not found in mesophyll cells.Both the fast and the slow Ca²⁺ increases were inhibited byCa²⁺ channel blockers (CoCl₂ and LaCl₃) and a chelating agent(EGTA). These results indicate that the phototropin-mediatedCa²⁺ increase was not observed prior to H⁺-ATPase activationin guard cells and that Ca²⁺ entered guard cells via Ca²⁺ channelsthrough photosynthesis and phototropin-mediated membrane hyperpolarization.     

Patch-Clamp Study on a Ca2+-Regulated K+ Channel in the Tonoplast of the Brackish Characeae Lamprothamnium succinctum

Cytoplasmic drops were prepared from internodal cells of thebrackish Characeae Lamprothamnium succinctum. Applying the patch-clamptechnique to single drops covered with tonoplast, we demonstratedthe presence of Ca²⁺-regulated K⁺ channels in the tonoplast.In a cell-attached mode, the selectivity of such channels forK⁺ was about 50 times that for Na⁺. This channel showed a tendencyto rectify in an outward direction. In the negative region ofthe pipette voltage, the conductance of this channel was 50pS, while it was 100 pS in the positive voltage region. Whenthe pipette voltage was increased above 50 mV, two conductancelevels were found in the cell-attached mode as well as in theexcised patch (cytoplasmic-side-out patch), which was obtainedby pulling the patch pipette from the cytoplasmic drop underconditions of low levels of Ca²⁺. Using the excised patch, wecontrolled the level of Ca²⁺ on the cytoplasmic side of thechannels. At a low level of Ca²⁺ (pCa=8) on the cytoplasmicside, the open frequency was very low and the opening time wasshort. An increase in Ca²⁺ on the cytoplasmic side (pCa = 5)increased both the frequency and the duration of opening. However,the conductance of the channels did not change. This regulationby Ca²⁺ of the K⁺ channels was reversible, that is, additionof EGTA on the cytoplasmic side inactivated the channels. Thepresent study demonstrates a direct action of Ca²⁺ on the K⁺channels. The physiological role of the K⁺ channel in the regulationof turgor in Lamprothamnium is discussed. (Received January 9, 1989; Accepted March 8, 1989)     

Modulation of K+ channels by arachidonic acid in T84 cells. I.Inhibition of the Ca2+-dependent K+ channel

TheCl secretory response ofcolonic cells to Ca²⁺-mediatedagonists is transient despite a sustained elevation of intracellular Ca²⁺. We evaluated the effects ofsecond messengers proposed to limit Ca²⁺-mediatedCl secretion on thebasolateral membrane,Ca²⁺-dependentK⁺ channel(K_Ca) in colonic secretorycells, T84. Neither protein kinase C (PKC) nor inositoltetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affectedK_Ca in excised inside-out patches.In contrast, arachidonic acid (AA; 3 µM) potently inhibitedK_Ca, reducingNP_o, the productof number of channels and channel open probability, by 95%. Theapparent inhibition constant for this AA effect was 425 nM. AAinhibited K_Ca in the presence ofboth indomethacin and nordihydroguaiaretic acid, blockers of thecyclooxygenase and lipoxygenase pathways. In the presence of albumin,the effect of AA on K_Ca wasreversed. A similar effect of AA was observed onK_Ca during outside-out recording.We determined also the effect of thecis-unsaturated fatty acid linoleate,the trans-unsaturated fatty acidelaidate, and the saturated fatty acid myristate. At 3 µM, all ofthese fatty acids inhibited K_Ca,reducing NP_o by 72-86%. Finally, the effect of the cytosolic phospholipaseA₂ inhibitorarachidonyltrifluoromethyl ketone(AACOCF₃) on thecarbachol-induced short-circuit current(I_sc) responsewas determined. In the presence ofAACOCF₃, the peakcarbachol-inducedI_sc response wasincreased ~2.5-fold. Our results suggest that AA generation inducedby Ca²⁺-mediated agonists maycontribute to the dissociation observed between the rise inintracellular Ca²⁺ evoked by theseagonists and the associatedCl secretory response.

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K+ and Ca2+ channels

Westudied chemosensitive signaling in locus coeruleus (LC) neurons usingboth perforated and whole cell patch techniques. Upon inhibition offast Na⁺ spikes by tetrodotoxin (TTX), hypercapnic acidosis[HA; 15% CO₂, extracellular pH (pH_o) 6.8]induced small, slow spikes. These spikes were inhibited byCo²⁺ or nifedipine and were attributed to activation ofL-type Ca²⁺ channels by HA. Upon inhibition of bothNa⁺ and Ca²⁺ spikes, HA resulted in a membranedepolarization of 3.52 ± 0.61 mV (n = 17) thatwas reduced by tetraethylammonium (TEA) (1.49 ± 0.70 mV,n = 7; P < 0.05) and absent(0.97 ± 0.73 mV, n = 7; P < 0.001) upon exposure to isohydric hypercapnia (IH; 15%CO₂, 77 mM HCO, pH_o 7.45).Either HA or IH, but not 50 mM Na-propionate, activatedCa²⁺ channels. Inhibition of L-type Ca²⁺channels by nifedipine reduced HA-induced increased firing rate andeliminated IH-induced increased firing rate. We conclude that chemosensitive signals (e.g., HA or IH) have multiple targets in LCneurons, including TEA-sensitive K⁺ channels andTWIK-related acid-sensitive K⁺ (TASK) channels.Furthermore, HA and IH activate L-type Ca²⁺ channels, andthis activation is part of chemosensitive signaling in LC neurons.

     

Permeation of Ca2+ and monovalent cations through an outwardly rectifying channel in maize root stelar cells

A depolarization-activated outwardly-rectifying channel (OR),most likely involved in the passive release of K⁺ from the rootsymplasm into the stelar apoplast (for subsequent transportto the shoot via the xylem vessels), has been characterizedin the plasma membrane of maize root stelar cells (Roberts andTester, 1995). In the present study, the selectivity of thischannel was further characterized using single channel current-voltagecurves generated using a voltage ramp protocol. This protocolpermitted the accurate and unambiguous measurement of the reversalpotentials of currents resulting from single channel openings.Using the voltage ramp protocol, it was shown that the OR allowsboth K⁺ efflux and Ca²⁺ influx at potentials positive of E_Kand negative of E_Ca. The OR had a P_Ca/P_K of 1.72–0.21decreasing as extracellular Ca²⁺ was increased. The permeabilityof the OR for monovalent cations other than K⁺ was also investigated.In biionic conditions, a relative permeability sequence of was determined (i.e. Eisenman sequenceIV). The physiological implications of the selectivity of theOR are discussed. Key words: Maize roots, K⁺ channel selectivity, Ca²⁺ permeation     

Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

The contribution of small-conductance (SK_Ca) and intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels to the generation of nitric oxide (NO) by Ca²⁺-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca²⁺ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca²⁺, as judged by 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl₂ (50 µmol/l) + ouabain (100 µmol/l), depleting intracellular Ca²⁺ stores by thapsigargin or removing external Ca²⁺ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SK_Ca and IK_Ca channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca²⁺ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SK_Ca and IK_Ca channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SK_Ca and IK_Ca channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca²⁺ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca²⁺ stores is to trigger the opening of both K_Ca channels along with Ca²⁺ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SK_Ca and IK_Ca channels are essential for NO-mediated vasorelaxation. calcium; endothelium; hyperpolarization; small-conductance calcium-activated potassium channel; intermediate-conductance calcium-activated potassium channel channel     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes

Hyperpolarization in human leukemia THP-1 monocytes adherent tovascular cell adhesion molecule (VCAM)-1 is due to an induction ofinwardly rectifying K⁺ currents(I_ir) (Colden-Stanfield M and Gallin EK,Am J Physiol Cell Physiol 275: C267-C277, 1998).We determined whether the VCAM-1-induced hyperpolarization issufficient to augment the increase in intracellular free calcium([Ca²⁺]_i) produced by Ca²⁺ storedepletion with thapsigargin (TG) and readdition of external CaCl₂ in fura 2-loaded THP-1 monocytes. Whereas there was a2.1-fold increase in [Ca²⁺]_i in monocytesbound to glass for 5 h in response to TG and CaCl₂ addition, adherence to VCAM-1 produced a 5-fold increase in[Ca²⁺]_i. Depolarization of monocytes adherentto VCAM-1 by I_ir blockade or exposure to high[K⁺] abolished the enhancement of the peak[Ca²⁺]_i response. In monocytes bound toglass, hyperpolarization of the membrane potential with valinomycin, aK⁺ ionophore, to the level of hyperpolarization seen incells adherent to VCAM-1 produced similar changes in peak[Ca²⁺]_i. Adherence of monocytes to E-selectinproduced a similar peak [Ca²⁺]_i to cellsbound to glass. Thus monocyte adherence to the physiological substrateVCAM-1 produces a hyperpolarization that is sufficient to enhanceCa²⁺ entry and may impact Ca²⁺-dependentmonocyte function.

     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium

Secretion of Cl^– and K⁺ in the colonic epithelium operates through a cellular mechanism requiring K⁺ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (I_sc)] and conductance (G_t) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE₂ and synergistically by PGE₂ and carbachol (PGE₂ + CCh). TRAM-34 at 0.5 µM, an inhibitor of K_Ca3.1 (IK, Kcnn4) K⁺ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory I_sc or G_t in guinea pig or rat colon. The presence of K_Ca3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited I_sc and G_t activated by Epi (4%), PGE₂ (30%) and PGE₂ + CCh (60%). The IC₅₀ of 4.0 µM implicated involvement of K⁺ channels other than K_Ca3.1. The secretory responses augmented by the K⁺ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that K_Ca3.1 was not involved. Sensitivity of the synergistic response (PGE₂ + CCh) to a high concentration TRAM-34 supported a requirement for multiple K⁺ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl^– secretory I_sc and G_t activated by PGE₂ (20%) but not K⁺ secretion activated by Epi. Thus Cl^– secretion activated by physiological secretagogues occurred without apparent activity of K_Ca3.1 channels but was dependent on other types of K⁺ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E₂; cholinergic; Kcnn4; TRAM-34; clofilium     

Studies on Mechano-Perception in Characeae: Effects of External Ca2+ and Cl-

The effects of modification of extracellular concentrationsof Ca²⁺ and C^– on mechano-perception were studied in internodalcells of Chara corallina. Cells were stimulated by droppinga piece of glass tubing on them, and the resulting receptorpotentials and action potentials were analyzed. When the Ca²⁺concentration was extremely lowered by adding EGTA, the amplitudesof both receptor potentials and action potentials were attenuated,suggesting the involvement of Ca²⁺ channels. However, the possibilityremained that attenuation of the amplitude of the receptor potentialwas caused by modification of membrane characteristics by extremelowering of [Ca²⁺]_o. When the plasma membrane was depolarizedto about 0 mV by adding 100 mM KC1, responses in the negativedirection were induced upon mechanical stimulation. When theplasma membrane was depolarized by adding 50 mM K²SO⁴, responsesin the positive direction were induced. Thus, Cl^– channelsmay be involved in responses induced by mechanical stimulationunder K+-induced depolarization. (Received January 16, 1996; Accepted March 25, 1997)     

Role of Ca2+-activated K+ channels in human erythrocyte apoptosis

Exposure of erythrocytes to the Ca²⁺ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca²⁺-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca²⁺-sensitive Gardos K⁺ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 µM) induces activation of inwardly rectifying K⁺-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K⁺ concentration to 25 mM and exposure to the Gardos K⁺ channel blockers charybdotoxin (230 nM) and clotrimazole (5 µM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K⁺ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 µM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K⁺ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca²⁺-activated K⁺ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca²⁺-sensitive K⁺ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis. cell volume; charybdotoxin; osmolarity; phosphatidylserine; annexin