Effects of divalent cations on single-channel conduction properties of Xenopus IP3 receptor

The effects ofMg²⁺ andBa²⁺ on single-channel propertiesof the inositol 1,4,5-trisphosphate receptor(IP₃R) were studied by patch clampof isolated nuclei from Xenopusoocytes. In 140 mM K⁺ theIP₃R channel kinetics and presenceof conductance substates were similar over a range (0-9.5 mM) offree Mg²⁺. In 0 mMMg²⁺ the channel current-voltage(I-V) relation was linear withconductance of ~320 pS. Conductance varied slowly and continuouslyover a wide range (SD  60 pS) and sometimes fluctuated during single openings. The presence of Mg²⁺ oneither or both sides of the channel reduced the current (blocking constant ~0.6 mM in symmetricalMg²⁺), as well as the range ofconductances observed, and made the I-V relation nonlinear (slopeconductance ~120 pS near 0 mV and ~360 pS at ±70 mV insymmetrical 2.5 mM Mg²⁺).Ba²⁺ exhibited similar effects onchannel conductance. Mg²⁺ andBa²⁺ permeated the channel with aratio of permeability of Ba²⁺ toMg²⁺ toK⁺ of 3.5:2.6:1. These resultsindicate that divalent cations induce nonlinearity in theI-V relation and reduce current by amechanism involving permeation block of theIP₃R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization ofconductance by divalent cations reveals a novel interaction between thecations and the IP₃R.

     

Taste responses to divalent cations in the frog glossopharyngeal nerve: competitive inhibition of the Mg2+ response by Ca2+

In the frog glossopharyngeal nerve, single water fibers respondto low CaCl₂ (1–2 mM) and relatively high MgCl₂ (100 mM).In the present study, it was found that stimulation by a mixtureof low CaCl₂ and relatively high MgCl₂ led to a small response.This suggests that the Ca⁺ response is inhibited by the presenceof Mg²⁺ and the Mg²⁺ response is inhibited by the presence ofCa²⁺. Hence, it is suggested that there are different receptorsites for divalent cations in single water fibers of the frogglossopharyngeal nerve, a calcium receptor site (X_Ca) responsiblefor the Ca²⁺ response and a magnesium receptor site (X_Mg) responsiblefor the Mg²⁺ response. It has been reported that Mg²⁺ inhibitsthe Ca²⁺ response by competing with Ca²⁺ for X_Ca (Kitada andShimada, 1980). In the present study, the inhibition of theMg²⁺ response by Ca²⁺ was examined quantitatively under theassumption that the magnitude of the neural response is proportionalto the amount of MgX_Mg complex minus a constant (the thresholdconcentration of the MgX_Mg complex). The results obtained indicatethat Ca²⁺ competes with Mg²⁺ for X^Mg. The apparent dissociationconstants for MgX_Mg complex and CaX_Mg complex, which were obtainedfrom the present study, were 8.0 x 10^–2 M and 7.2 x 10^–4M, respectively. Thus, competition between Ca⁺ and Mg²⁺ forthe distinct receptor sites involved in taste reception wasdemonstrated by the results described in this paper. Since thedivalent cations do not always bring about activation of tastereceptors, the responses to salts in the frog glossopharyngealnerve cannot be explained in terms of changes in the surfacepotential outside the taste cells. The present results suggestthat there exist multiple specific receptor sites for cationsinvolved in salt taste responses, and only the binding of eachseparate cation to its appropriate receptor sites leads to activationof the receptor and the initiation of impulses in sensory nerveendings.     

Passive Interactions of Ca2+ and other Multivalent Cations with the Membranes of Isolated Corn Mitochondria

The presence of Ca²⁺ in a hypo-osmotic reaction medium reducessuccinate: cytochrome c reductase activity and the release ofouter membrane-specific antimycin A-insensitive NADH: cytochromec reductase. The action of Ca²⁺ is non-competitive and approximately30 mmol m^–3 Ca²⁺ affords half-maximal (I₅₀) protection.The effect of a range of inorganic and organic multivalent cationson succinate: cytochrome c reductase activity suggests thatthe action of Ca²⁺ is non-specific and probably involves Ca²⁺binding to outer membrane component(s) which may be proteins. Valinomycin- or gramicidin-induced passive swelling of isolatedcorn mitochondria in isotonic K.C1 is also non-competitivelyinhibited by up to 50% with Ca²⁺. Half-maximal inhibition (I₅₀)occurs at 0-35 mol m^–3 Ca²⁺ for valinomycin and 1-0 molm^–3 Ca²⁺ for gramicidin. Other divalent cations, Mg²⁺,Sr²⁺ and Ba²⁺, seem to inhibit similarly while the trivalentcations La³⁺ and Ho³⁺ show a maximum inhibition of up to 85%,with an I₅₀ of 0.1 mol m^–3 for valinomycin. It is suggestedthat non-specific cation binding may reduce membrane fluiditythereby slowing down the rate of ionophore penetration throughthe inner membrane. Key words: Calcium, Mitochondria, Membranes     

Competitive inhibition of the response to Na+ by Ca2+ in water fibers of the frog glossopharyngeal nerve

Single water fibers of the frog glossopharyngeal nerve respondto low concentrations of CaCl₂ (1–2 mM) and to relativelyhigh concentrations of NaCl(>80 mM). However, stimulationby a mixture with a low concentration of CaCl₂ and relativelyhigh concentration of NaCl gives rise to only a small response,suggesting that the effects of Ca²⁺ and Na⁺ are mutually antagonistic.It has been reported that Na⁺ inhibits the response to Ca²⁺by competing with Ca²⁺ for a calcium receptor site (X_Ca; Kitadaand Shimada, 1980). In the present study, it was found tha Ca²⁺inhibited the response to Na⁺. Therefore, the sodium receptorsite (X_Na) responsible for the response to Na is different fromX_Ca. The inhibition of the response to Na⁺ by Ca²⁺ was examinedquantitatively on the assumption that the magnitude of the neuralresponse is proportinal to the amount of NaX_Na complex minusa constant (the threshold concentration of the NaX_Na complex).The results obtained indicate that Ca²⁺ competes with Na₊ forX_Na. The apparent dissociation constants for the NaX_Na complexand the CaX_Na complex obtained from the present study were 1.0M and 1.2 x 10^-3 M, respectively, X_Na as proposed here, doesnot represent simply a binding site for cations since therecan be competition for X_Na by an antagonistie cation. The highaffinity of X_Na for Ca²⁺ suggests that X_Na is a specific receptorsite involved in salt-taste reception. Since Mg²⁺ did not affectthe response to Na⁺, the affinity of X_Na for cations is notcharge-specific but is, rather, chemically specific. The presentresults indicate that both Ca²⁺ and Na⁺ have a dual action,being involved both in excitation and in inhibition, in waterfibers of the frog glossopharyngeal nerve.     

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger

We investigated the role of intracellular Mg²⁺(Mg_i²⁺) on the ATP regulation ofNa⁺/Ca²⁺ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg²⁺, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMg_i²⁺. At constant ATP concentration, the effectof intracellular Mg²⁺ concentration([Mg²⁺]_i) on the ATP regulation of exchangeris biphasic: activation at low [Mg²⁺]_i,followed by deactivation as [Mg²⁺]_i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P₂ into membrane vesicles activates Na⁺/Ca²⁺ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mg_i²⁺ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa⁺/Ca²⁺ exchanger is different in mammalianheart and squid nerves.

     

Permeant but not impermeant divalent cations enhance activation of nondesensitizing alpha(7) nicotinic receptors

Neuronal₇ nicotinic acetylcholine receptors (nAChRs) arepermeable to Ca²⁺ and other divalent cations. Wecharacterized the modulation of the pharmacological properties ofnondesensitizing mutant (L²⁴⁷T andS²⁴⁰T/L²⁴⁷T) ₇ nAChRs bypermeant (Ca²⁺, Ba²⁺, and Sr²⁺) andimpermeant (Cd²⁺ and Zn²⁺) divalent cations.₇ receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,whereas impermeant divalent cations decreased potency and maximalefficacy. The antagonist dihydro--erythroidine (DHE) was a strongpartial agonist of L²⁴⁷T andS²⁴⁰T/L²⁴⁷T ₇ receptors in thepresence of divalent cations but was a weak partial agonist in thepresence of impermeant divalent cations. Mutation of the"intermediate ring" glutamates (E²³⁷A) inL²⁴⁷T ₇ nAChRs eliminated Ca²⁺conductance but did not alter the Ca²⁺-dependent increasein ACh potency, suggesting that site(s) required for modulation are onthe extracellular side of the intermediate ring. The difference betweenpermeant and impermeant divalent cations suggests that sites within thepore are important for modulation by divalent cations.

     

Probing the extracellular release site of the plasma membrane calcium pump

Theplasma membrane Ca²⁺ pump is known to mediateCa²⁺/H⁺ exchange. Extracellular protonsactivated ⁴⁵Ca²⁺ efflux from human red bloodcells with a half-maximal inhibition constant of 2 nM when theintracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreasedthe IC₅₀ for extracellular Ca²⁺ from ~33 to~6 mM. Changing the membrane potential by >54 mV had no effect onthe IC₅₀ for extracellular Ca²⁺. This arguesagainst Ca²⁺ release through a high-field access channel.Extracellular Ni²⁺ inhibited Ca²⁺ efflux withan IC₅₀ of 11 mM. Extracellular Cd²⁺ inhibitedwith an IC₅₀ of 1.5 mM, >10 times better thanCa²⁺. The Cd²⁺ IC₅₀ also decreasedwhen the pH was raised from 7.1 to 8.2, consistent withCa²⁺, Cd²⁺, and H⁺ competing forthe same site. The higher affinity for inhibition by Ni²⁺and Cd²⁺ is consistent with a histidine or cysteine as partof the release site. The cysteine reagent 2-(trimethylammonium)ethylmethanethiosulfonate did not inhibit Ca²⁺ efflux. Ourresults are consistent with the notion that the release site contains a histidine.

     

An Inwardly Rectifying Cation Current across the Plasma Membrane of the Green Alga Eremosphaera viridis

Ion currents across the plasma membrane of the unicellular greenalga Eremosphaera viridis were characterized with electrophysiologicalmethods, especially the two electrode voltage-clamp-technique.Under different conditions, at increased external Cl^–concentrations or after perfusion of different anion channelblockers (A9C (anthracen-9-carboxylic acid), NPPB ((5-nitro-2-3-phenylpropylamino)benzoic acid) and ZnCl₂), increased instantaneous negative currentswere observed. The negative currents were carried by cationfluxes into the cell. The transporter responsible had low selectivityamong potassium and sodium. Additionally also divalent cationswere transported. The cation influx was not affected by thepotassium channel inhibitors TEA (tetraethylammoniumsulfate),Ba²⁺ or Cs⁺ at concentrations of 1 mM, but was strongly reducedby 100 µM AlCl₃. Our results with E. viridis demonstrate,for the first time for an unicellular alga, the existence ofan inwardly rectifying cation current across the plasma membrane.Parallels and differences to inwardly rectifying cation currentsand channels described in plasma membranes of other plant cellsare discussed. (Received May 10, 1993; Accepted September 13, 1993)     

ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes

The Mg²⁺-inhibited cation (MIC) current (I_MIC) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg²⁺ homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I_MIC, studied in the presence or absence of extracellular divalent cations, was sustained for 50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I_MIC exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(,-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I_MIC rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 µM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) by prevention of its resynthesis (10 µM wortmannin or 15 µM phenylarsine oxide) induced rundown of I_MIC. Finally, loading ATP-depleted cells with exogenous PIP₂ (10 µM) prevented rundown. These results suggest that PIP₂, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity. cation channels; hydrolysis; phosphoinositides; rundown     

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes

Store-operated Ca²⁺ entry (SOCE), which is Ca²⁺ entry triggered by the depletion of intracellular Ca²⁺ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca²⁺ loading (load_SR) that was not altered by inhibition of L-type Ca²⁺ channels, reverse mode Na⁺/Ca²⁺ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca²⁺] to 0 mM or adding either 0.5 mM Zn²⁺ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 µM) inhibited load_SR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced load_SR. In addition, manipulation of the extracellular and intracellular Na⁺ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca²⁺ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca²⁺ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca²⁺ overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis     

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca²⁺ stores opens store-operated Ca²⁺ channels (SOC) and causes Ca²⁺ entry. Transient receptor potential (TRP) cation channels that are permeable to Na⁺ and Ca²⁺ are believed to form functional SOC. Because sarcolemmal Na⁺/Ca²⁺ exchanger has also been implicated in regulating [Ca²⁺]_cyt, this study was designed to test the hypothesis that the Na⁺/Ca²⁺ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca²⁺]_cyt by contributing to store depletion-mediated Ca²⁺ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na⁺, which switches the Na⁺/Ca²⁺ exchanger from the forward (Ca²⁺ exit) to reverse (Ca²⁺ entry) mode, significantly increased [Ca²⁺]_cyt, whereas inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Store depletion also induced a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Removal of extracellular Na⁺ or inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 attenuated the store depletion-mediated Ca²⁺ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange contributes to store depletion-mediated increase in [Ca²⁺]_cyt, and that blockade of the Na⁺/Ca²⁺ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle     

Studies of Interaction between Chloroplast Coupling Factor 1 and Nucleotides by Circular Dichroism and Ultra-Violet Spectroscopies

When ADP, CDP, GDP, IDP or UDP was mixed with chloroplast couplingfactor 1 (CF₁) in the presence of MgCl₂, changes were inducedin the ultraviolet absorption spectrum as well as the circulardichroism spectrum. These changes were about 70% complete inone minute. Also, these two changes were similar in the concentrationcurves of nucleotides, the competition between ADP with CDP,the inhibition by PP₁, and the requirement for divalent cations.A minor difference was also noted in the requirement for divalentcations by some of the nucleotides. The order of the affinitiesof these nucleotides for CF₁ was: ADP>CDP>UDP>IDP>GDP.The UV spectral changes induced by these nucleotides are interpretedas shifts of the absorption spectra of bound nucleotides bysome 10 nm to longer wavelengths accompanied by decrease inabsorbance. In difference CD spectra, negative peaks were foundat about the same wavelengths of the absorption peaks of therespective nucleotides. Ca²⁺ was as effective as Mg²⁺ to these changes induced by ADPor GDP, but less effective than Mg²⁺ for those induced by CDP,UDP or IDP. In the presence of EDTA, the changes induced byADP were somewhat lower and those induced by CDP, UDP or IDPbecame almost zero. However, the UV absorbance change inducedby GDP was larger in the presence of EDTA than in the presenceof Mg²⁺ or Ca²⁺. (Received October 27, 1980; Accepted February 27, 1981)     

Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes

We examined the effectsof metabolic inhibition on intracellular Ca²⁺ release insingle pulmonary arterial smooth muscle cells (PASMCs). Severemetabolic inhibition with cyanide (CN, 10 mM) increased intracellularcalcium concentration ([Ca²⁺]_i) and activatedCa²⁺-activated Cl currents[I_Cl(Ca)] in PASMCs, responses that were greatlyinhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents andCa²⁺ sparks in PASMCs. In Xenopus oocytes, CNalso induced Ca²⁺ release and activatedI_Cl(Ca), and these responses were inhibited by thapsigarginand cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)Ca²⁺, whereas neither heparin nor anti-inositol1,4,5-trisphosphate receptor (IP₃R) antibodies affected CNresponses. In both PASMCs and oocytes, CN-evoked Ca²⁺release was inhibited by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin or CCCP andthapsigargin. Whereas hypoxic stimuli resulted in Ca²⁺release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition withCN increases [Ca²⁺]_i in both pulmonary andsystemic artery myocytes by stimulating Ca²⁺ release fromthe SR and mitochondria.

     

Decreased cation channel activity and blunted channel-dependent eryptosis in neonatal erythrocytes

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca²⁺-permeable cation channels with subsequent entry of Ca²⁺ and stimulation of Ca²⁺-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl^– removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca²⁺ activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E₂ (PGE₂) formation, cation channel activity, Ca²⁺ entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl^– in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE_2, and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca²⁺ leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes. annexin V; osmotic cell shrinkage; calcium; apoptosis     

Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells

The role of glycolytically generated ATP in Ca²⁺/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca²⁺ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i, suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg²⁺ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R. Ca²⁺/calmodulin-dependent kinase II; glycolysis; calcium regulation     

Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca²⁺ (Ca_i²⁺) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Ca_i²⁺ (to micromolar concentrations) observed after the addition of ionophore plus Ca²⁺ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Ca_i²⁺, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Ca_i²⁺ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Ca_i²⁺ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Ca_i²⁺. calcium; connexin43; lens gap junctions     

SEA-0400, a potent inhibitor of the Na+/Ca2+ exchanger, as a tool to study exchanger ionic and metabolic regulation

The effects of a new, potent, and selective inhibitor of the Na⁺/Ca²⁺ exchange, SEA-0400 (SEA), on steady-state outward (forward exchange), inward (reverse exchange), and Ca²⁺/Ca²⁺ transport exchange modes were studied in internally dialyzed squid giant axons from both the extra- and intracellular sides. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: it increases synergic intracellular Na⁺ (Na_i⁺) + intracellular H⁺ (H_i⁺) inactivation, is antagonized by ATP and intracellular alkalinization, and is enhanced by intracellular acidification even in the absence of Na⁺. Inhibition by SEA is still present even after 1 h of its removal from the experimental solutions, whereas removal of the cointeracting agents of inhibition, Na_i⁺ and H_i⁺, even in the continuous presence of SEA, releases inhibition, indicating that SEA facilitates the reversible attachment of the natural H_i⁺ and Na_i⁺ synergic inhibitors. On the basis of a recent model of squid Na⁺/Ca²⁺ exchange regulation (DiPolo R and Beaugé L. J Physiol 539: 791–803, 2002), we suggest that SEA acts on the H_i⁺ + Na_i⁺ inactivation process and can interact with the Na⁺-free and Na⁺-bound protonized carrier. Protection by ATP concurs with the antagonism of the nucleotide by H_i⁺ + Na_i⁺ synergic inhibition. ionic-metabolic interactions     

Phytotoxicity of Phthalate Plasticisers: 2. SITE AND MODE OF ACTION

The effects of phthalate esters on chlorophyll a₂ fluorescencein radish plants (Raphanus sativus L. cv. Cherry Belle) wereexamined Fluorescence yield was increased in those plants exposedto an aerial concentration of 120 ng dm^–3 dibutyl phthaiatc(DBP) at a rate of 3.0 dm³ min^–1 for 13 d. Comparisonof fluorescence enhancement ratios and F_red/F_ox, suggests thatDBP inhibits photosynthesis in radish plants at a site afterQ_A. Both DBP and diisobutyl phthalate (DIBP) strongly inhibiteduncoupled (PS2+PS1) electron transport rates in thylakoids isolatedfrom spinach. At a chlorophyll concentration of 10 µgcm^–3 the concentrations of DBP and DIBP exhibiting 50%inhibition were 44 mmol m^–3 and 42 mmol m^–3 respectively.Basal electron transport rates were also inhibited, with 87mmol m^–3 of DBP or DIBP producing 50% inhibition. Measurementof photosystcm 1 activity suggested that the main site of actionof these phthalates was localized at a site near the reducingside of photosystem 2. Key words: Phthalate, plasticiser, chlorophyll, fluorescence, photosynthesis, inhibition     

Biochemical properties of 1-aminocyclopropane-1-carboxylate N-malonyltransferase activity from early growing embryonic axes of chick-pea (Cicer arietinum L.) seeds

In the present work, certain biochemical characteristics ofthe enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase(ACC N-MTase) which is responsible for the malonylation of 1-aminocyclopropane-1-carboxylate(ACC) in chickpea (Cicer arietinum) are described. Phosphatebuffer was the most appropriate buffer with regard to enzymestability and, therefore, ACC N-MTase was extracted, assayedand purified in the presence of this buffer. ACC N-MTase waspartially purified approximately 900-fold from embryonic axesof chick-pea seeds using ammonium sulphate precipitation, hydrophobicinteraction and molecular filtration chromatography. By gelfiltration chromatography on Superose-12, the molecular massof the enzyme was estimated to be 54 4 kDa. ACC N-MTase hadan optimal pH and temperature of 7.5 and 40C, respectively,as well as a K_m for ACC and malonyl-CoA of 400 M and 90 M,respectively. D-Phenylalanine was a competitive inhibitor ofACC N-MTase with respect to ACC (K_i of 720 M), whereas co-enzymeA was a competitive product inhibitor with respect to malonyl-CoA(K_i of 300 M) and a non-competitive inhibitor with respectto ACC (K_i of 600 M). Under optimal assay conditions, ACC N-MTasewas strongly inhibited by (a)divalent [Zn²⁺>Mg²⁺>>Co²⁺>Co²⁺>(NH₄)²⁺>Fe²⁺]and monovalent metal cations (Li⁺>Na⁺>K⁺), without activitybeing detected in the presence of Hg²⁺, and (b) PCMB or mersalicacid, suggesting that sulphydryl group(s) are involved at theactive site of the enzyme. Key words: ACC-N-malonyltransferase, Cicer arietinum, embryonic axes, ethylene, germination, seeds