Ethanol sensitivity of BK(Ca) channels from arterial smooth muscle does not require the presence of the beta 1-subunit

Ethanol inhibition of large-conductance,Ca²⁺-activated K⁺ (BK_Ca) channelsin aortic myocytes may contribute to the direct contraction of aorticsmooth muscle produced by acute alcohol exposure. In this tissue,BK_Ca channels consist of pore-forming (bslo) and modulatory () subunits. Here, modulation of aortic myocyteBK_Ca channels by acute alcohol was explored by expressingbslo subunits in Xenopus oocytes, in the absenceand presence of ₁-subunits, and studying channelresponses to clinically relevant concentrations of ethanol in excisedmembrane patches. Overall, average values of bslo channelactivity (NP_o, with N = no. ofchannels present in the patch; P_o = probability of a single channel being open) in response to ethanol(3-200 mM) mildly decrease when compared with pre-ethanol,isosmotic controls. However, channel responses show qualitativeheterogeneity at all ethanol concentrations. In the majority of patches(42/71 patches, i.e., 59%), a reversible reduction inNP_o is observed. In this subset, the maximaleffect is obtained with 100 mM ethanol, at whichNP_o reaches 46.2 ± 9% of control. Thepresence of ₁-subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17-estradiol, fails to modifyethanol action on bslo channels. Ethanol inhibition of bslo channels results from a marked increase in the meanclosed time. Although the voltage dependence of gating remainsunaffected, the apparent effectiveness of Ca²⁺ to gate thechannel is decreased by ethanol. These changes occur withoutmodifications of channel conduction. In conclusion, a new molecularmechanism that may contribute to ethanol-induced aortic smooth musclecontraction has been identified and characterized: a functionalinteraction between ethanol and the bslo subunit and/or itslipid microenvironment, which leads to a decrease in BK_Cachannel activity.

     

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.

     

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II

The effect ofCa²⁺/calmodulin-dependent protein kinase II (CaMKII)stimulation on unitary low voltage-activated (LVA) T-type Ca²⁺ channel currents in isolated bovine adrenalglomerulosa (AG) cells was measured using the patch-clamp technique. Incell-attached and inside-out patches, LVA channel activity wasidentified by voltage-dependent inactivation and a single-channelconductance of ~9 pS in 110 mM BaCl₂ orCaCl₂. In the cell-attached patch, elevation of bathCa²⁺ from 150 nM to 1 µM raised intracellularCa²⁺ in K⁺-depolarized (140 mM) cells andevoked an increase in the LVA Ca²⁺ channel probability ofopening (NP_o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblanksweeps, a rise in the frequency of channel opening in nonblank sweeps,and a 30% reduction in first latency. No apparent changes in thesingle-channel open-time distribution, burst lengths, or openings/burstwere apparent. Preincubation of AG cells with lipophilic or peptideinhibitors of CaMKII in the cell-attached or excised (inside-out)configurations prevented the rise in NP_o elicited by elevated Ca²⁺ concentration.Furthermore, administration of a mutant recombinant CaMKIIexhibiting cofactor-independent activity in the absence of elevatedCa²⁺ produced a threefold elevation in LVA channelNP_o. These data indicate that CaMKII activity isboth necessary and sufficient for LVA channel activation byCa²⁺.

     

BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy

Weinvestigated the influence of pregnancy on large-conductancecalcium-activated potassium channel (BK_Ca) activity(NP_o) and on channel expression in membranes ofisolated human myometrial smooth muscle cells.NP_o in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and thehalf-maximal activation potential was shifted by 39 mV to more negativepotentials. This effect was not due to an enhanced BK_Cachannel expression. In the presence of cAMP kinase (PKA) or cGMP kinase(PKG), NP_o increased in patches from PMbut decreased in those from NPM. Western blot analysis and use of aspecific PKG inhibitor (1 µM KT-5823) verified the existence of apartially active membrane-associated PKG. Inhibition of PKA by100 nM PKI, the inhibitory peptide of PKA, had no effect onNP_o. 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect wasabolished by iberiotoxin, a specific blocker of BK_Cachannels. It is concluded that an endogenous, membrane-bound PKG inmyometrial cells specifically enhances BK_Ca channelactivity during pregnancy and thus may contribute to uterine quiescenceduring pregnancy.

     

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.

     

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.

     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

Secretory modulation of basolateral membrane inwardly rectified K(+) channel in guinea pig distal colonic crypts

Cell-attached recordings revealedK⁺ channel activity in basolateral membranes ofguinea pig distal colonic crypts. Inwardly rectified currents wereapparent with a pipette solution containing 140 mM K⁺.Single-channel conductance () was 9 pS at the resting membrane potential. Another inward rectifier with  of 19 pS was observed occasionally. At a holding potential of 80 mV,  was 21 and 41 pS,respectively. Identity as K⁺ channels was confirmed afterpatch excision by changing the bath ion composition. From reversalpotentials, relative permeability of Na⁺ overK⁺ (P_Na/P_K)was 0.02 ± 0.02, withP_Rb/P_K = 1.1 andP_Cl/P_K < 0.03. Spontaneous open probability (P_o) of the 9-pSinward rectifier (^gpK_ir) was voltageindependent in cell-attached patches. Both a low(P_o = 0.09 ± 0.01) and a moderate(P_o = 0.41 ± 0.01) activity mode wereobserved. Excision moved ^gpK_ir to the mediumactivity mode; P_o of^gpK_ir was independent of bath Ca²⁺activity and bath acidification. Addition of Cl andK⁺ secretagogues altered P_o of^gpK_ir. Forskolin or carbachol (10 µM)activated the small-conductance ^gpK_ir inquiescent patches and increased P_o inlow-activity patches. K⁺ secretagogues, either epinephrine(5 µM) or prostaglandin E₂ (100 nM), decreasedP_o of ^gpK_ir in activepatches. This ^gpK_ir may be involved inelectrogenic secretion of Cl and K⁺ acrossthe colonic epithelium, which requires a large basolateral membraneK⁺ conductance during maximal Cl secretionand, presumably, a lower K⁺ conductance during primaryelectrogenic K⁺ secretion.

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells

Fattyacids and other negatively charged single-chain lipids increaselarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, whereas sphingosine and otherpositively charged single-chain lipids suppress activity. Because thesemolecules are effective on both inside-out and outside-out patches andbecause they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids onthis channel, we used two compounds that are unlikely to flip acrossthe lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify thesite of action of negatively charged lipids, and a positively chargedmyristoylated pentapeptide (myr-KPRPK) was used to investigate the siteof action of positively charged lipids. The effect of these compoundson channel activity was studied in excised patches using patch-clamptechniques. In "normal" ionic strength solutions and in experimentswhere high-ionic strength solutions were used to shield membranesurface charge, PCoA increased channel activity only when applied tooutside-out patches, suggesting that the site of action of negativelycharged lipids is located on the outer surface of the membrane. Adecrease in activity, similar to that of other positively chargedlipids, was observed only when myr-KPRPK was applied to outside-outpatches, suggesting that positively charged lipids suppress activity byalso acting on the outer membrane surface. Some channel blockadeeffects of myr-KPRPK and KPRPK are also described. The sidedness ofaction suggests that modulation of channel activity by single-chainlipids can occur by their interaction with the channel protein.

     

Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells

The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca²⁺]_i. [Ca²⁺]_i and membrane potentials were strongly correlated. In whole cell clamped cells, BK_Ca currents were activated by increasing [Ca²⁺]_i via cell dialysis with pipette solution, and the activated BK_Ca currents were further enhanced by S1P. When [Ca²⁺]_i was buffered at 1 µM, the S1P concentration required to evoke half-maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BK_Ca channel activity in a reversible manner and shifted the relationship between Ca²⁺ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPS; 1 mM) using a patch pipette, GDPS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK_Ca current and channel activation. These results suggest that S1P enhances BK_Ca channel activity by increasing Ca²⁺ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca²⁺ influx through Ca²⁺ entry channels. Inasmuch as S1P activates BK_Ca channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca²⁺ mobilization in human endothelial cells. sphingolipid metabolites; intracellular second messenger; Ca²⁺ mobilization     

Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells

Because the activity of thesodium pump (Na-K-ATPase) influences the secretion of aldosterone, wedetermined how extracellular potassium (K_o) and calciumaffect sodium pump activity in rat adrenal glomerulosa cells. Sodiumpump activity was measured as ouabain-sensitive ⁸⁶Rb uptakein freshly dispersed cells containing 20 mM sodium as measured withsodium-binding benzofluran isophthalate. Increasing K_o from4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca_o) stimulated sodium pump activity up to 165% andincreased intracellular free calcium as measured with fura 2. Increasing K_o from 4 to 10 mM in the absence ofCa_o stimulated the sodium pump ~30% and did not increaseintracellular free calcium concentration ([Ca²⁺]_i). In some experiments, addition of1.8 mM Ca_o in the presence of 4 mM K_o increased[Ca²⁺]_i above the levels observed in theabsence of Ca_o and stimulated the sodium pump up to 100%.Ca-dependent stimulation of the sodium pump by K_o andCa_o was inhibited by isradipine (10 µM), a blocker of L-and T-type calcium channels, by compound 48/80 (40 µg/ml) andcalmidizolium (10 µM), which inhibits calmodulin (CaM), and by KN-62(10 µM), which blocks some forms of Ca/CaM kinase II (CaMKII).Staurosporine (1 µM), which effectively blocks most forms of proteinkinase C, had no effect. In the presence of A-23187, a calciumionophore, the addition of 0.1 mM Ca_o increased[Ca²⁺]_i to the level observed in the presenceof 10 mM K_o and 1.8 mM Ca_o and stimulated thesodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mMCa_o was not reduced by isradipine but was blocked by KN-62.Thus, under the conditions that K_o stimulates aldosteronesecretion, it stimulates the sodium pump by two mechanisms: directbinding to the pump and by increasing calcium influx, which isdependent on Ca_o. The resulting increase in[Ca²⁺]_i may stimulate the sodium pump byactivating CaM and/or CaMKII.

     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

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.

     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca²⁺ concentration ([Ca²⁺]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263–1277, 1998), with low, moderate, and high maximal fractional open times (P_o). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 µM cytoplasmic [Ca²⁺]. At 10 µM [Ca²⁺], low-P_o channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (K_aATP) = 422 µM] than moderate-P_o channels (K_aATP = 82 µM). Oxidation of low-P_o channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-P_o channels and decreased K_aATP from 422 to 82 µM. At 0.1 µM cytoplasmic [Ca²⁺], ATP induced an almost negligible activation of low-P_o channels. After oxidation to high-P_o behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-P_o channels at 10 µM [Ca²⁺] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode. subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine     

Functional reconstitution of an eicosanoid-modulated Cl- channel from bovine tracheal smooth muscle

We describe thebiochemical properties of an eicosanoid-modulated Clchannel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its openprobability (P_o). After a purification protocolinvolving wheat-germ agglutinin affinity and anion-exchangechromatography, the proteins were sequentially inserted into liposomes,which were then fused into PLBs. Functional and biochemicalcharacterization tests confirm that the Cl channel is a55-kDa glycosylated monomer with voltage- and Ca²⁺concentration-independent activity. 5,6- and 8,9-EET decreased theconductance of the native channel (control conductance: 70 ± 5 pSin asymmetrical 50 mM trans/250 mM cis CsCl) in aconcentration-dependent manner, with respective 50% inhibitoryconcentration values of 0.31 and 0.42 µM. These regioisomerssimilarly decreased the conductance of the purified channel (controlconductance value: 75 ± 5 pS in asymmetrical 50 mMtrans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P_o of the nativechannel with respective 50% inhibitory concentration values of 0.27 and 0.30 µM but failed to alter the P_o of thepurified protein. Thus we suggest that the effects of these EETs onchannel conductance likely result from direct interactions ofEET anions with the channel pore, whereas the alterationof P_o requires a lipid environment of specificcomposition that is lost on solubilization and purification of the protein.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

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.

     

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells

Rapamycin and FK-506 are immunosuppressive drugs thatbind a ubiquitous immunophilin, FKBP12, but immunosuppressivemechanisms and side effects appear to be different. Rapamycin bindsrenal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na⁺ channels in A6cells. Channel NP_o was 0.5 ± 0.08 (n = 6)during the first 5 min but fell close to zero after 20 min. Application of 1 µM rapamycin reactivated Na⁺ channels(NP_o = 0.47 ± 0.1; n=6), but 1 µMFK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor,mimicked the rapamycin-induced reactivation in a nonadditive manner.However, rapamycin did not reactivate Na⁺ channels if cellswere exposed to 1 µM FK-506 before rapamycin. In PKC assays,rapamycin was as effective as the PKC inhibitor; however, epithelialNa⁺ channel (ENaC) phosphorylation was low under baselineconditions and was not altered by PKC inhibitors or activators. Theseresults suggest that rapamycin activates Na⁺ channels bybinding FKBP12 and inhibiting PKC, and, in renal cells, despite bindingthe same immunophilin, rapamycin and FK-506 activate differentintracellular signaling pathways.