Diphtheria toxin-induced channels in Vero cells selective for monovalent cations

Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of 45Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed.     

Arachidonic acid-induced calcium influx in human platelets. Comparison with the effect of thrombin.

The effects of arachidonic acid and thrombin on calcium movements have been studied in fura-2-loaded platelets by a procedure which allows simultaneous monitoring of the uptake of manganese, a calcium surrogate for Ca2+ channels, and the release of Ca2+ from intracellular stores. Arachidonic acid induced both Ca2+ (Mn2+) entry through the plasma membrane and Ca2+ release from the intracellular stores. The release of Ca2+ was prevented by cyclo-oxygenase inhibitors and mimicked by the prostaglandin H2/thromboxane A2 receptor agonist U46619. Ca2+ (Mn2+) entry required higher concentrations of arachidonic acid and was not prevented by either cyclo-oxygenase or lipoxygenase inhibitors. Several polyunsaturated fatty acids reproduced the effect of arachidonic acid on Ca2+ (Mn2+) entry, but higher concentrations were required. The effects of maximal concentrations of arachidonic acid and thrombin on the uptake of Mn2+ were not additive. Both agonists induced the entry of Ca2+, Mn2+, Co2+ and Ba2+, but not Ni2+, which, in addition, blocked the entry of the other divalent cations. However, arachidonic acid, but not thrombin, increased a Ni2(+)-sensitive permeability to Mg2+. The effect of thrombin but not that of arachidonic acid was prevented either by pretreatment with phorbol ester or by an increase in cyclic-AMP levels. Arachidonic acid also accelerated the uptake of Mn2+ by human neutrophils, rat thymocytes and Ehrlich ascites-tumour cells.     

The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.     

Regulation of intracellular magnesium by Mg2+ efflux

Chicken erythrocytes were loaded with Mg2+ by incubation with the cation ionophore A 23187 in the presence of Mg2+. After removing A 23187 by intensive washing with serum albumin and reincubating the Mg2+-loaded cells, Mg2+ was transported out of the cells until the original Mg2+ content was achieved. The net Mg2+ efflux followed Michaelis-Menten-kinetics and was independent of extracellular and intracellular Ca2+ and calmodulin. The net Mg2+ efflux was not affected by adrenalin, isoproterenol, p-chloromercuribenzenesulfonate, ouabain and tetrodotoxin, but was inhibited by dicyclohexylcarbodiimide, KCN, iodoacetate, high extracellular concentrations of Mg2+, Mn2+ and when extracellular Na+ was substituted by choline or K+. The efflux of 1 Mg2+ was coupled with the uptake of 2 Na+. It is concluded that there exists an additional gating process at the inner cell surface becoming active only at increased concentrations of intracellular free Mg2+ regulating the exit of Mg2+ by the efflux system.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.     

Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.     

Epoxyeicosatrienoic acids inhibit Ca2+ entry into platelets stimulated by thapsigargin and thrombin.

The epoxyeicosatrienoic acids derived from the cytochrome P-450 pathway of arachidonic acid metabolism have a unique platelet antiaggregatory profile. This prompted us to examine their influence on cellular Ca2+ mobilization. 14,15-cis-Epoxyeicosatrienoic acid and related compounds inhibited the rise in cytosolic Ca2+ following agonist stimulation of platelets by thapsigargin, a receptor-independent agonist, and thrombin, a receptor-dependent agonist. The epoxyeicosatrienoic acids selectively inhibited the entry of Ca2+ from the exterior of the platelets but did not alter Ca2+ discharge from intracellular pools. The magnitude of inhibition by 14,15-cis-epoxyeicosatrienoic acid was proportional to the rate of Ca2+ entry. 14,15-cis-Epoxyeicosatrienoic acid also inhibited the rate of influx of Mn2+, a cation which enters platelets via pathways similar to Ca2+. The magnitude of inhibition was proportional to the rate of Mn2+ entry, suggesting that epoxyeicosatrienoic acids act on divalent cation channels in a fashion which depends on the state of opening of the channel. Selective inhibition of Ca2+ entry into platelets may account for the antiaggregatory effects of the epoxyeicosatrienoic acids. We are unaware of other endogenous compounds exhibiting this property, suggesting that epoxyeicosatrienoic acids may be useful to probe agonist-stimulated Ca2+ mobilization in nonexcitable cells.     

Calcium antagonists and lipolysis in isolated rat epididymal adipocytes: effects of tetracaine, manganese, cobaltous and lanthanum ions and D600.

The present study reports the effects on lipolysis occurring in isolated rat epididymal adipocytes of several agents which have each been found to interfere with membrane calcium transport in a variety of tissues. As reported by other workers, the local tetracaine was a strong inhibitor of hormone accelerated but not of basal lipolysis. The bivalent cations Mn2+ and Co2+ were similarly found to inhibit lipolysis stimulated with either epinephrine, ACTH, theophylline or dibutyryl cyclic AMP, whereas basal lipolysis was not markedly altered. This effect of Mn2+ and Co2+ was not mimicked by either Sr2+, Ba2+, Mg2+ or Ca2+. Cyclic AMP levels in adipocytes stimulated with epinephrine or ACTH tended to be higher in the presence of Mn2+ and Co2+. It is concluded, therefore, that Mn2+ and Co2+ inhibit lipolysis by uncoupling cyclic AMP accumulation from activation of triglyceride lipase. In contrast to Mn2+ and Co2+, the calcium antagonists La3+ and D600 were without effect on lipolysis. The antilipolytic effect of tetracaine, Mn2+ and Co2+ was found to persist in the absence of extracellular calcium, suggesting therefore that the antilipolytic effect of these drugs is unrelated to inhibition of calcium influx into adipocytes. The possibility is discussed that lipolytic agents cause an intracellular redistribution of calcium ion and that local anesthetics, Mn2+ and Co2+ interfere with lipolysis by preventing this intracellular redistribution of calcium.     

Transport of Ca and Mn by mitochondria from rat liver, heart and brain

The energy-dependent, respiration-supported uptake and the uncoupler- or Na+-induced release of Ca2+ and Mn2+ by mitochondria from rat liver, heart and brain were investigated, using as indicators radioisotopes (45Ca and 54Mn), proton ejection, oxygen consumption, nicotinamide nucleotide oxidation-reduction and, in the case of Ca2+, the metallochromic dye Arsenazo III. Ca2+ uptake in the presence of Pi was rapid in mitochondria from liver and brain, and less rapid in those from heart. Mn2+ uptake was much slower than that of Ca2+ in liver and heart, but only slightly slower in brain. When added together, Ca2+ accelerated the uptake of Mn2+, and Mn2+ retarded the uptake of Ca2+, by mitochondria from all three tissues. When Mn2+ was present during Ca2+ uptake, its own uptake remained accelerated even after Ca2+ uptake was terminated. Mg2+, which was not taken up, inhibited Ca2+ uptake by mitochondria from all three tissues, and, when present during Ca2+ uptake, accelerated the subsequent uptake of Mn2+. The uncoupler CCCP induced a release of both Ca2+ and Mn2+ from all three sources of mitochondria; yet, release of Mn2+ took place only in the absence of Pi. The release followed the same pattern as the uptake, i.e., Ca2+ accelerated the release of Mn2+ and Mn2+ retarded the release of Ca2+. Na+ induced a release of both Ca2+ and Mn2+ from heart and brain but not from liver mitochondria; again, Mn2+ release occurred only in the absence of Pi. The Na+-induced release of Ca2+ was inhibited by Mn2+, but the Na+-induced release of Mn2+ was not accelerated by Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool

The abilities of various divalent cations to enter the cytoplasm of mouse lacrimal acinar cells was examined under resting and agonist-stimulated conditions, by monitoring their effects on the fluorescence of cytosolic fura-2. In vitro, Ni2+, Co2+, and Mn2+ quenched the fura-2 fluorescence, whereas Sr2+, Ba2+, and La3+ produced an excitation spectrum and maximum brightness similar to Ca2+. Stimulation of mouse lacrimal acinar cells with methacholine (MeCh) caused a biphasic elevation of intracellular Ca2+ concentration [( Ca2+]i) resulting from a release of Ca2+ from intracellular pools followed by a sustained entry of extracellular Ca2+. Neither La3+ nor Ni2+ entered the cells under resting or stimulated conditions, but both blocked Ca2+ entry. Although both Co2+ and Mn2+ entered unstimulated cells, this process was not increased by MeCh. Both Sr2+ and Ba2+ were capable of supporting a sustained increase in fura-2 fluorescence in response to MeCh, indicating that these cations can enter the cells through the agonist-regulated channels. However, Sr2+, but not Ba2+, was capable of refilling the agonist-sensitive intracellular stores. These findings demonstrate dissociation of agonist-induced Ca2+ entry from intracellular Ca2+ pool refilling and thereby provide strong support for the recently modified version of the capacitative Ca2+ entry model according to which influx into the cytoplasm occurs directly across the plasma membrane and does not require a specialized cation channel directly linking the extracellular space and the intracellular Ca2+ stores.     

Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.     

Modulation of Na+/alanine cotransport in liver sinusoidal membrane vesicles by internal divalent cations

Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane.     

The interaction between manganese and calcium fluxes in pancreatic beta-cells.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets isolated from ob/ob mice. The efflux from preloaded islets was estimated from the amounts remaining after 30 min of subsequent test incubations in the absence of Mn2+. An increase in the extracellular Mg2+ concentration promoted the Mn2+ efflux and removal of Na+ from a Ca2+-deficient medium had the opposite effect. Addition of 25 mM-K+ failed to affect Mn2+ outflow as did 3-isobutyl-1-methylxanthine and dibutyryl cyclic AMP. Whereas tolbutamide caused retention of manganese, the ionophore Br-X537A promoted an efflux. D-Glucose was equally potent in retaining the islet manganese when the external Ca2+ concentration ranged from 15 microM to 6.30 mM. Subcellular-fractionation experiments indicated a glucose-stimulated incorporation of manganese into all fractions except the microsomes. The effect was most pronounced in the mitochondrial fraction, being as high as 164%. The glucose-induced uptake of intracellular 45Ca was abolished in the presence of 0.25 mM-Mn2+. When added to medium containing 2.5 mM-Mn2+, glucose even tended to decrease 45Ca2+ uptake. The inhibitory effect of Mn2+ was apparent also from a diminished uptake of 45Ca into all subcellular fractions. The efflux of 45Ca2+ was markedly influenced by Mn2+ as manifested in a prominent stimulation followed by inhibition. In addition to demonstrating marked interactions between fluxes of Mn2+ and Ca2+, the present studies support the view that the glucose inhibition of the efflux of bivalent cations from pancreatic beta-cells is accounted for by their accumulation in the mitochondria.     

Block by calcium of ATP-activated channels in pheochromocytoma cells

We have investigated the effects of Ca2+ on Na+ influx through ATP- activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1- 21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.     

Artificial cell based on lipid hollow polyelectrolyte microcapsules: channel reconstruction and membrane potential measurement

The CitM transporter from Bacillus subtilis transports citrate as a complex with Mg2+. In this study, CitM was functionally expressed and characterized in E. coli DH5a cells. In the presence of saturating Mg2+ concentrations, the Km for citrate in CitM was 274 mM, similar to previous studies using whole cells of B. subtilis. CitM has a high substrate specificity for citrate. Other di- and tricarboxylic acids including succinate, isocitrate, cis-aconitate and tricarballylic acid did not significantly inhibit the uptake of citrate in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The highest rate of citrate transport was seen in the presence of Mg2+, followed in order of preference by Mn2+, Ba2+, Ni2+, Co2+ and Ca2+. Citrate transport by CitM appears to be proton coupled. The transport was inhibited in transport buffers more alkaline than pH 7.5 and not affected by pH at acidic values. Transport was also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin. Valinomycin did not affect the uptake by CitM, suggesting that transport is electroneutral. In conclusion, the cloned CitM transporter from B. subtilis expressed in E. coli has properties similar to the transporter in intact B. subtilis cells. The results support a transport model with a coupling stoichiometry of one proton coupled to the uptake of one complex of (Mg2+-citrate)1-.     

Divalent cation transport systems of Rhodopseudomonas capsulata.

Separate divalent cation transport systems for energy-dependent uptake of Mg2+ and Mn2+ were found both with aerobically and heterotrophically grown and with photosynthetically grown cells of Rhodopseudomonas capsulata. The maximum rate of Mg2+ uptake differed between photosynthetic and aerobic cells, while the Km for the Mg2+ transport system was constant. Photosynthetic midlog-phase cells exhibited Km's for uptake of about 55 micrometer Mg2+ and 0.5 micrometer Mn2+. The Vmax's also differed between the two systems: 0.6 to 1.8 mumol/min per g (dry weight) of cells for Mg2+, but only 0.020 mumol/min per g for Mn2+, making the distinction between a "macro-requirement" system and a system functioning at trace nutrient levels. Calcium was not normally taken up by intact cells of R. capsulata. However, chromatophore membranes isolated from photosynthetic cells took up Ca2+ by an energy-dependent process.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.     

Potassium current inhibition by nonselective cation channel-mediated sodium entry in rat pheochromocytoma (PC-12) cells.

Under physiological conditions, nonselective cation (NSC) channels mediate the entry of cations into cells, the most important being Na+ and Ca2+. In contrast to the Ca(2+)-dependent signaling mechanisms, little is known about the consequences and the spatial distribution of intracellular [Na+] elevation. In this study we demonstrate that Na+ entry, during the opening of ATP-activated NSC channels, leads to an inhibition of voltage-dependent K+ currents (IK) in cromaffin-like undifferentiated PC-12 cells. The effect was dependent on the charge carrier as well as on the density of the ATP-activated current. Extracellular alkali cations (Na+, Li+) were more efficient than NH4+ in suppressing IK. Intracellular infusion of Na+ had the same effect as Na+ influx through ATP-activated NSC channels. The inhibition of IK persisted when the total ATP-induced Na+ entry was reduced by membrane depolarization, suggesting a spatial restriction of the required Na+ accumulation. Our results indicate that NSC channels influence the function of other ion channels by changing local intracellular ion concentrations.